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Nucleosome Structure and FunctionClick to copy article linkArticle link copied!
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Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
*E-mail: [email protected]
Copyright © 2014 American Chemical Society
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1 Introduction
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When fully extended, one copy of the three billion base pair human genome reaches a length of over two meters. Yet, it must be packaged into the nucleus of a cell with an average diameter of less than 10 μm. Within this context, specific segments of the genome must be transcriptionally active or repressed in a coordinated fashion to allow a cell to react to its ever-changing environment. This is akin to arranging 30 miles of thread inside a basketball such that at moment’s notice key segments can be accessed. To establish such compaction while maintaining coordinated accessibility, organisms ranging from yeast to man organize their genomes in a polymeric complex called chromatin. The fundamental unit of the chromatin polymer is the nucleosome, which repeats every 160 to 240 bp across the genome. (1) Each nucleosome contains a nucleosome core, composed of an octameric complex of the core histone proteins, which forms a spool to wrap 145 to 147 bp of DNA. The nucleosome core is connected to the adjacent nucleosome core through a segment of linker DNA, which often associates with the linker histone protein (H1 or H5). The nucleosome core with ∼165 bp of DNA together with the linker histone is called the chromatosome. (2) The chromatosome and the additional linker DNA constitutes a nucleosome. (2) Despite these technical definitions, the nucleosome core particle is often colloquially referred to as the nucleosome.
The nucleosome serves three primary functions. First, it brings about the first level of genomic compaction, organizing ∼200 bp of DNA. Second, the nucleosome acts as a signaling hub for chromatin-templated processes by providing a scaffold for the binding of chromatin enzymes and displaying a combinatorial array of post-translational modifications (PTMs). This array of PTMs further regulates the recruitment of chromatin enzymes (3) and tunes both nucleosome stability (4) and the higher-order compaction of chromatin. (5-7) Third, the nucleosome can self-assemble into higher-order chromatin structures, allowing for further compaction of the genome. The first level of higher-order compaction is the 30 nm chromatin fiber for which several models have been created based on experimental data using cryogenic-electron microscopy (cryo-EM) and X-ray crystallography. (8)
This review focuses on recent advances in our understanding of the structure and function of the nucleosome and is divided into four parts. First, we present a primer covering the fundamentals of the nucleosome core particle structure determined at atomic scale by X-ray crystallography in 1997. (9) Next we discuss recent insights into the role of DNA sequence in the structure of nucleosomal DNA based on structure–function studies of nucleosome core particles containing derivatives of the Widom 601 nucleosome positioning sequence. (10) We then introduce patterns of nucleosome recognition by chromatin factors using recent crystal structures and NMR and cryo-EM models of peptide and protein macromolecular chromatin factors bound to the nucleosome core particle. Finally, we will compare a recent cryo-EM model for the 30 nm chromatin fiber (11) to two previous models based on crystallographic and cryo-EM data. (12, 13)
2 Fundamentals of the Nucleosome Core Particle Structure
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While the composition of the nucleosome had long since been realized, the 1997 2.8 Å crystal structure of the nucleosome core particle (NCP) solved by Luger et al. afforded the first atomic depiction of this fundamental genomic unit. (9) This structure showed 146 bp of the human alpha-satellite sequence wrapped 1.65 times around an octameric scaffold of Xenopus laevis histone proteins in a left-handed superhelix (Figure 1a). A single base pair is centered on the nucleosome dyad, (14) which defines the pseudo-2-fold symmetry axis of the NCP. DNA locations are designated by superhelical locations (SHL) representing superhelical turns from the dyad (SHL 0) and ranging from SHL −7 to SHL 7. The central histone octamer contains two copies of each of the core histone proteins, H2A, H2B, H3, and H4 as established by Arents and Moudrianakis in the 1991 3.1 Å crystal structure of the histone octamer. (15) The core histones are assembled into four histone-fold heterodimers (two each of H2A/H2B and H3/H4). Ten flexible tails protrude from the NCP at defined locations, one N-terminal tail from each of the eight core histone proteins and two additional C-terminal tails contributed by H2A.
Figure 1
Figure 1. Nucleosome core particle structure and the histone-fold heterodimers. (a) Nucleosome core particle structure (PDB ID 1KX5). Histones and DNA are depicted in cartoon and sticks representations, respectively, and colored as indicated. (b) H3/H4 histone-fold heterodimer. (c) H2A/H2B histone-fold heterodimer. Structures (top) and schemes (bottom) with secondary structure elements indicated. All molecular graphics in this review were prepared using PyMOL software (The PyMOL Molecular Graphics System, version 1.6, Schrodinger, LLC). All structures of NCP using high-resolution structure (17) (PDB ID 1KX5) unless indicated otherwise.
2.1 Histone-Fold Heterodimers
Each of the core histones contains a central α-helical region that forms a histone-fold motif, flanked by N- and C-terminal extensions. The histone-fold is constructed from three α helices connected by two intervening loops specified as α1-L1-α2-L2-α3 (Figure 1b,c). (9, 15, 16) The two shorter α1 and α3 helices loop back to pack against the longer central α2 helix. Each histone-fold pairs with a complementary histone-fold, H3 pairs with H4 and H2A pairs with H2B, to form a histone-fold heterodimer handshake motif. The antiparallel arrangement of this heterodimer approximates the L1 loop from one histone-fold and the L2 loop of the complementary histone-fold, placing one L1L2 pair at each end of the heterodimer. The result is a crescent-shaped heterodimer with the convex surface including the L1L2 loops and the α1 helices and the concave surface including the α3 and central α2 helices. The convex surface of the H2A/H2B and H3/H4 heterodimers carries a strong positive charge and constitutes the primary DNA binding element of each histone-fold heterodimer.
2.2 Histone Octamer Architecture and DNA Binding
The histone octamer forms a spool for wrapping nucleosomal DNA. It is assembled from two H3/H4 and two H2A/H2B histone-fold heterodimers using a single structural motif, the four-helix bundle. Each four-helix bundle is formed by the α2 and α3 helices from the adjacent histone-folds. Two H3/H4 dimers interact in a head to head arrangement through an H3/H3 four helix bundle to form an (H3/H4)2 tetramer (Figure 2a). An H2A/H2B dimer binds to each half of the (H3/H4)2 tetramer using a four-helix bundle formed by the H4 and H2B histone folds (Figure 2b). Several structured N- and C-terminal extensions to the histone-fold regions also contribute to the histone octamer architecture. The αN helix between the N-terminal tail and histone-fold of H3 rests on top of the H4 histone-fold and organizes DNA at the entry/exit site of the NCP (Figure 3a). H2A and H2B also contain C-terminal extensions that contribute to the nucleosome core surface. The H2B αC helix extends from the center of the nucleosome disk to the DNA edge opposite the nucleosome dyad, packing against the underlying H2A/H2B histone-fold helices (Figure 3b). The H2A C-terminal extension includes a docking domain that interacts with the H2A/H2B histone-fold dimer after which it traverses the nucleosome surface toward the dyad resting on a platform generated by the underlying H3/H4 heterodimer from the opposite side of the octamer (Figure 3b).
Figure 2
Figure 2. Histone octamer constructed with four helix bundles. (a) Nucleosome core particle structure highlighting H3–H3 four helix bundle (blue). Remainder of H3 and H4 are shown in light blue and light green, respectively. (b) Nucleosome core particle structure highlighting one H4–H2B four helix bundle (green for H4 and red for H2B). Remainder of H4 and H2B are shown in light green and pink, respectively.
Figure 3
Figure 3. Histone-fold heterodimers in the nucleosome core particle structure. (a) Nucleosome core particle structure with central H3/H4 histone-fold tetramer shown in blue (H3) and green (H4). H3 and H4 extensions are shown in light blue and light green, respectively. (b) Nucleosome core particle structure with one H2A/H2B histone-fold dimer shown in yellow (H2A) and red (H2B). H2A and H2B extensions are shown in light yellow and pink, respectively.
The H2A–H2B–H4–H3–H3–H4–H2B–H2A octamer designates a nonuniform path of the nucleosomal DNA. The H2A/H2B dimers bind DNA in two planes perpendicular to the DNA superhelical axis, while the central H3/H4 tetramer forms a diagonal ramp through the nucleosomal dyad, connecting these two planes (Figure 4). Notably, the DNA gyres in neighboring planes align their major and minor grooves, respectively, as they track along the octamer surface. The histone-fold regions of the octamer bind the central ∼121 bp of nucleosomal DNA. The remaining ∼13 bp at each of the DNA ends is organized by the histone-fold extensions, especially the H3 αN helices. The histone octamer contacts the DNA superhelix at regular intervals projecting an arginine into the minor groove of the neighboring DNA segment. Histone-DNA interfaces are mediated by extensive direct and water-mediate hydrogen bonds, ionic interactions, nonpolar contacts, and the alignment of helix dipoles relative to phosphate backbone ions. (9, 17, 18)
Figure 4
Figure 4. Histone-fold heterodimers form a ramp for nucleosomal DNA. (a) H2A/H2B histone-fold heterodimers interact with DNA in two different parallel planes. Structure of NCP viewed from opposite dyad, highlighting H2A and H2B in yellow and red, respectively (left) and scheme of DNA planes (right). (b) H3/H4 tetramer forms a diagonal ramp for DNA connecting two parallel planes. Structure of NCP view from dyad (black oval and orange base pair) with H3 and H4 in blue and green, respectively, (left) and scheme of diagonal DNA ramp (right). Arrows point away from central dyad base pair.
2.3 Nucleosome Topology
The ∼200 kDa disk-shaped nucleosome core particle has a diameter of approximately 100 Å and height ranging from ∼25 Å at the dyad to ∼60 Å at the H2B αC helices. It has a multifaceted, solvent accessible surface of about 74 000 Å2 (Figure 5a). The disk face furthest from the dyad is lined by three parallel ridges, formed centrally by the H2B αC helix and on the sides by the H2B α1 helix and H3 α1 helix together with the H4 N-terminal tail, respectively. These ridges create two intervening grooves, one containing the H2A/H2B acidic patch (more details below). The disk face near the dyad contains a central depression overlaying the H3–H3 interface. The complexity of the NCP surface is furthered by the histone N-terminal tails that protrude from the nucleosome surface either outside (H4 and H2A) or between (H3 and H2B) the DNA gyres (Figure 4). These tails, ranging in length from 15 to 36 amino acids, can extend great distances from the NCP, adopt flexible structures, and bind intranucleosomal DNA and/or DNA and histone surfaces in neighboring nucleosomes. (19-22) The DNA phosphodiester backbone at the perimeter of the NCP presents a highly negative electrostatic surface (Figure 5b). An additional negatively charged surface is found in a groove on the H2A/H2B dimer surface that is often referred to as the nucleosome or H2A/H2B acidic patch. Eight acidic residues contribute to the acidic patch, six from H2A (E56, E61, E64, D90, E91, and E92) and two from H2B (E102 and E110). As discussed in detail below, this acidic patch may be a hot-spot for nucleosome binding by chromatin factors. In contrast to the prominent negatively charged surfaces of the NCP disk, the histone tails contain many arginine and lysine residues and carry a strong net positive charge. Overall, the topological and electrostatic complexity of the nucleosome core affords the opportunity for a diverse set of surfaces for nucleosome binding.
Figure 5
Figure 5. Surface topology and charge of the nucleosome core particle. (a) Surface of nucleosome core particle viewed down the DNA superhelical axis in space-filling representation. (b) Surface electrostatic potential of nucleosome core particle contoured from −5 to +5 kT/e calculated with ABPS. (164) Location of acidic patch is indicated.
2.4 Variability in NCP Structure
Since the solution of the core particle containing Xenopus histones was reported, crystal structures have been solved using histones from yeast, fly, and man. (23-25) While minor sequence differences result in small changes to the composition of exposed surfaces and complementary coevolution within the hydrophobic core, the architecture of the complexes remains nearly constant. A myriad of structural studies of histone variants have also revealed some variant-specific roles in the stability and exposed surfaces of the NCP. (26-39) For example, H2A.Z extends the H2A/H2B acidic patch and causes a subtle destabilization of the H2A/H2B interface with H3/H4. (39) Similar destabilization was observed recently with testes-specific variant H3T. (30) A 2011 structure of the NCP containing the centromeric H3 variant CENP-A revealed that CENP-A nucleosomes only organize the central 121 bp of nucleosomal DNA potentially due to a shortened H3 αN helix. (28) Other structural and biochemical data validate increased opening of the entry exit DNA in CENP-A nucleosomes. (39-43) This trait is not specific to CENP-A as biochemical and biophysical interrogation of H2A.Bbd nucleosomes show similar opening of the DNA ends in nucleosomes containing H2A.Bbd. (26, 31, 36, 44) Much like histone variants, histone PTMs can induce structural changes in the solvent accessible nucleosome surface (6) and interactions with nucleosomal DNA. (45-51) Variant- and PTM-specific structural changes can contribute to the ability of nucleosomes to recruit chromatin factors as discussed below. Finally, several structures of NCPs containing different nucleosome positioning sequences have revealed that DNA sequence has effects on nucleosomal DNA structure allowing the octamer to wrap 145–147 bp of DNA. (9, 17, 52, 53) This topic is reviewed in detail in the following section.
3 Structure of Nucleosomal DNA
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3.1 Nucleosome Positioning Sequences in Nucleosome Structures
Nucleosomes examined by structural studies have for the most part contained four types of DNA sequences: mixed sequence genomic DNA, the 5S RNA coding sequence, the human α-satellite repeat, and the Widom 601 nucleosome positioning sequence. Early structural studies of nucleosomes utilized nucleosomes isolated from naturally occurring sources such as beef kidney and consequently contained mixed sequence genomic DNA. (54, 55) Furthermore, the length of the nucleosomal DNA was variable at 147 ± 2 bp, the result of using micrococcal nuclease to digest long chromatin into nucleosome core particles. (56, 57) Such nucleosome core particles were used in the 7 Å low resolution crystal structure in 1984. (55) To improve the internal order and diffraction limits of nucleosome core particle crystals, Richmond and colleagues prepared defined length 146 bp nucleosomal DNA fragments containing the 5S RNA coding sequence characterized by Simpson and others. (58) This technical feat employing (now) classical recombinant DNA technology permitted the preparation of milligram quantities of defined sequence and defined length nucleosomal DNA in 1988, resulting in nucleosome core particle crystals which diffracted to ∼4.5 Å. (57) While this marked an improvement over the 7 Å diffraction observed for mixed sequence nucleosome core particles, the diffraction was not sufficient for atomic structure determination.
The crystal structure of the nucleosome core particle determined at 2.8 Å by the Richmond group in 1997 contained instead the human alpha satellite centromeric repeat. (9) Use of this nucleosome positioning sequence for reconstituting recombinant nucleosome core particles was first described by Bunick and colleagues in 1995. (59) The 2.8 Å Luger et al. structure revealed important features of how the histone octamer organizes nucleosomal DNA. One such feature was the somewhat surprising finding that the dyad of the nucleosome core particle lines with a base pair and not between base pairs. This means that a nucleosome core particle with 73 bp on either side of the dyad contains 147 and not 146 bp. Previous studies had generally assumed an even number of base pairs in the nucleosome, and this was in fact the basis for using 146 bp of nucleosomal DNA in nucleosome crystallization trials. The occurrence of a base pair at the dyad axis was actually predicted earlier by single base pair resolution mapping of nucleosome positions using site-directed hydroxyl radical footprinting. (14) This study examined recombinant nucleosome reconstituted with the 5S RNA sequence, providing evidence that the placement of the nucleosome dyad on the central base pair is independent of nucleosome positioning sequence. This conclusion has been borne out by all subsequent studies.
3.2 Widom 601 Nucleosome Positioning Sequence
Crystal structures of at least 20 nucleosome core particles incorporating variant histones or DNA sequence changes have been determined since the original 1997 structure. The large majority of these utilized the human α-satellite repeat DNA sequence. In contrast, the most popular DNA positioning sequence used in chromatin biochemical studies is the Widom 601 sequence. Lowary and Widom had performed an in vitro selection experiment to isolate synthetic random DNA sequences with high affinity for the histone octamer. (10) The Widom 601 sequence was among the tightest binding sequences found, and its strong nucleosome positioning and high yields in nucleosome reconstitution experiments have made it a favorite among chromatin researchers. Crystal structures of nucleosome core particles containing the Widom 601 sequence were determined on their own and in complex with the RCC1 chromatin factor in 2010. (52, 53) Since the nucleosome core particles pack differently in the 601 nucleosome versus the RCC1/601 nucleosome crystals, the similarity of the structures indicate that the structures are not artifacts of their crystal packing. This is not an insignificant consideration given that the same crystal packing is present in all crystals of nucleosome core particles on their own to date (see below).
The reasons why the Widom 601 sequence is such a strong nucleosome positioning sequence have intrigued many since the sequence was first characterized in 1998, and we are now beginning to understand the mechanistic basis. Nucleosomal DNA must endure an aggregate bend of about 600° in approximately 150 bp, and consequently, DNA sequences that can be bent to contour the histone octamer will be favored. By sequencing chicken nucleosomal DNA, Satchwell et al. showed in 1986 that AA/TT, TA, and AT base steps were favored where the double helix minor groove faces the histone octamer and conversely that GG, GC, and CG base steps were more likely to be found 5 bp away or where the minor groove faces away from the histone. (60) The particular significance of the TA base step in nucleosome positioning was suggested in several experiments, including the analysis of in vitro selected sequences (such as the 601 sequence) which highlighted a 10 bp sequence periodicity of the TA base step. (10) In fact, the 601 sequence contains the TA base step at 5 of the 8 central positions where the DNA minor groove faces the histone octamer (SHL ± 0.5, ± 1.5, ± 2.5, ± 3.5) (Figure 6). Crystallographic and biochemical experiments by the Davey laboratory now provide a structural explanation for the significance of this observation. Noting that 4 of the 5 TA base steps are located on the “left” half of the 601 sequence and the remaining one on the “right” half, Davey and colleagues examined the salt stability of nucleosome core particles reconstituted with symmetrized versions of the 601 sequence. (61) Nucleosomes containing the original, asymmetrical 601 sequence dissociated at a salt concentration of 1.26 M, significantly more than the 0.94 M concentration needed to dissociate nucleosome reconstituted with the human α-satellite sequence. However, nucleosome reconstituted with symmetrical DNA based on the left half of the 601 sequence (“601L”) were noticeably more stable to salt, while nucleosomes containing the right 601 sequence (“601R”) were less stable than the original 601 sequence (Figure 6). These results, as well as the fact that other high affinity in vitro selected nucleosome sequences, such as the 603 and 605 sequence also contain TA base steps in similar positions, (10, 62) provide evidence for an important role of the TA base step in nucleosome positioning of these high affinity in vitro selected nucleosomes.
Figure 6
Figure 6. Scheme of asymmetric and symmetric 601 sequences. Sequences of 601R symmetric, (canonical) 601 asymmetric, and 601L symmetric sequences with H3/H4 TA steps highlighted in red for left half and blue for right half. (61) Nucleosome salt stability values (molar monovalent salt) are listed at right and indicate stability as follows: 601L > 601 > 601R. This trend correlates with the number of H3/H4 TA steps: 601L (6), 601 (4), 601R (2). The dyad position is indicated (purple).
Crystal structures of nucleosome core particles containing the 601 and 601L sequences confirm that the TA base steps at the aforementioned central positions directly face the histone octamer. (53, 61) These TA base steps also exhibit significant distortions from ideality particularly in their propeller twist values. The crystal structures help explain the significance of this observation. Histones H3/H4 form the tetramer, which binds DNA around the nucleosomal dyad. The L1/L2 loops, α1/α1 N-terminal ends and L2/L1 loops of histone H3/H4 grip DNA phosphates groups where the minor groove faces the histone octamer and thus constrains the nucleosomal DNA path to these locations (termed “pressure points” by Davey and colleagues) (Figure 7). These pressure points occur at SHL ± 0.5, ± 1.5, and ± 2.5 (i.e., 5 bp from the nucleosomal dyad and then again at 10 bp intervals), precisely where TA base steps are located. Fixing these phosphate groups at these pressure points creates stress particularly in the base pairs between the phosphates. Base steps that are more flexible will more easily accommodate this stress through distortions such as propeller twisting within a base pair, rolling between base pairs or sliding one base pair with respect to the adjacent base pair. (63, 64) Since the TA base step is the most flexible of all base steps, (65, 66) it is most able to accommodate the stress created at the pressure points (Figure 7). Thus, we can understand why high affinity nucleosome positioning sequences such as the 601 sequence contain TA base steps where the minor groove faces the histone tetramer. Additional analyses of relevant DNA parameters for nucleosome positioning in the human α-satellite and 601 sequence have been described elsewhere. (67, 68)
Figure 7
Figure 7. Location of TA steps in 601L nucleosome core particle structure. (a) 601L NCP structure viewed down the DNA superhelical axis with TA steps interacting with H3/H4 and H2A/H2B colored red and orange, respectively. The dyad is indicated (purple). Histones H3, H4, H2A, and H2B are shown in cartoon representation and colored blue, green, yellow, and red, respectively. Nucleosomal DNA is shown as sticks (light blue). (b) Enlarged view showing one H3/H4 heterodimer bound to DNA containing three TA steps (other histones are not shown for clarity purposes). Backbone phosphates bound to the H3/H4 histone folds are shown in space-filling representation as indicated. Secondary structure elements of dimer are shown.
Why is the TA base step so flexible? The fewer hydrogen bonds between the bases do allow for greater flexibility compared to base steps containing GC base pairs. However, this cannot be sufficient since TT, AA, and AT base steps are not as flexible despite having the same number of Watson–Crick hydrogen bonds. A simple structural explanation is that the stacking of bases is minimal in the TA base step, allowing greater flexibility for roll, propeller twisting and other distortions than the TT, AA, or AT base steps (Figure 8). The methyl group on the thymine base also plays an important role because the relatively bulky methyl group must be accommodated when a T-A base pair is distorted. In the TA base step, the minimal base stacking and the position of the methyl group allow for large roll or propeller twisting without the thymine methyl clashing with other atoms. It is for a similar reason that the eukaryotic transcriptional initiation TATA box is distorted so dramatically upon binding of the TBP TATA binding protein. (69-71) In contrast, the TT, AA, and AT base steps each offer steric challenges to distortions including roll and propeller twisting. It is worth emphasizing that the geometry of the TA base steps is distinct from the AT base step despite the common alternating A/T sequence. The other base step with fewer constraints to distortions is CA = TG, and this base step has been found to be among the most flexible in protein–DNA structures. (72)
Figure 8
Figure 8. Minimal base stacking in TA and CA compared to other base pair steps. TA, CA, AA, and AT base pair steps colored as follows: T = yellow, A = blue, G = green, C = red. The thymine methyl groups are shown highlighted in space-filling representation (dark yellow), all other non-hydrogen atoms shown in sticks representation. The minimal base stacking and the absence of atoms close to the thymine methyl group permit greater flexibility of the TA and CA base pair steps.
The concept that the flexible TA base steps located between critical pressure points in nucleosomal DNA provides an explanation for surprising results of experiments studying the ability of RNA polymerases to progress through a nucleosome. Studitsky and colleagues found that transcriptional elongation by yeast and human RNA polymerase II were blocked by a nucleosome reconstituted with the 601 positioning sequence in one but not the opposite orientation (73) (Figure 9). This blockage was mediated by the H3/H4 histone tetramer since the same effect was observed in the absence of the H2A/H2B dimer. Similar results were obtained for the Widom 603 and 605 nucleosome positioning sequences. These were puzzling findings because the symmetrical nature of the nucleosome structure made it difficult to imagine why a sequence block would function in one orientation but not in the opposite orientation. However, inspection of the DNA sequences with a focus on TA base steps shows a strong correlation between TA base steps at SHL +0.5, +1.5, and +2.5 and the ability to block RNA polymerase II progression. For each of the Widom 601, 603, and 605 nucleosome positioning sequences, RNA polymerase II transcriptional elongation was blocked when the TA base steps are positioned at the pressure points facing the H3/H4 tetramer downstream of the dyad (Figure 9). At first glance, the fact that the block to transcriptional elongation occurs when tight binding to the nucleosome is downstream of the dyad seems counterintuitive. If we imagine RNA polymerase II as an engine peeling off DNA from a one-dimensional histone track, we might expect that TA base steps positioned upstream of the dyad to be more efficient at blocking. The problem, of course, is that this Flatland (74) analysis ignores the three-dimensionality of molecules instead of focusing on how RNA polymerase interacts with the architecture of the three-dimensional nucleosome. The finding that tight binding of nucleosomal DNA to the histone tetramer downstream of the dyad blocks RNA polymerase II indicates the ability of RNA polymerase II to unwrap DNA from the tetramer on the downstream side is a critical aspect of the mechanism of passing through a nucleosome. This insight was exploited by Studitsky and colleagues in a subsequent study where they propose a structural mechanism for this very process. (62)
Figure 9
Figure 9. RNA polymerase II blocking by nucleosome positioning sequences. Sequences of NCP601, NCP603, and NCP605 sequences and their reversed counterparts together with ability to block RNA polymerase II. (73) Multiple TA steps bound to the H3/H4 tetramer downstream (red) of the dyad (purple) blocks RNA polymerase II passage as compared with upstream (blue) of the dyad. TA steps bound to the H2A/H2B dimers are shown in orange. The sequence shown for the 601 sequence is the reverse complement of what is shown in Figure 6 to be consistent with ref 73.
3.3 DNA Stretching in the Nucleosome
The original 2.8 Å crystal structure of the nucleosome core particle contained 146 bp of symmetrized human alpha satellite DNA. Since the nucleosomal dyad lies on a base pair, there was necessarily 73 bp on one side of the dyad base pair and 72 bp on the other side. Thus, the structure was asymmetrical despite the fact that the nucleosomal DNA was symmetrical. To date, every single nucleosome core particle crystallized by itself has packed in the crystal essentially the same way: in space group P212121 with end-to-end DNA contacts between individual nucleosomes (with the possible exception of the CENP-A centromeric nucleosome where the DNA ends are not visible in the crystal structure (28)) (Figure 10). In order for the DNA ends to pack against each other in the crystal, the DNA on the 72 bp side had to stretch by one bp localized around SHL −2 (Figure 11a). Subsequent nucleosome core particle crystal structures containing human alpha satellite DNA of variant length or sequence show stretching of one bp around SHL ± 2 and SHL ± 5 but not at other locations. For example, the NCP146b nucleosome, which incorporates a symmetrical version of a different human α-satellite half-repeat, is stretched around SHL −5, (61) while nucleosomes prepared from the original human α-satellite 146 bp DNA and recombinant human histones display stretching at SHL −2 and ±5. (25) In contrast, a 147 bp pseudosymmetric human α-satellite sequence (pseudosymmetric because the symmetry of the two 73 bp halves was broken at the dyad) displayed no DNA stretching, (17) and it is generally accepted that the human alpha satellite sequence forms a 147 bp nucleosome core particle in solution.
Figure 10
Figure 10. DNA end-to-end packing in nucleosome core particle crystals. Three nucleosome core particles from one plane of the high resolution NCP crystal structure (PDB ID 1KX5) colored yellow, red and blue. (a) Full and (b) enlarged views of the alignment of the DNA ends from adjacent NCP in the structure. The DNA end-to-end packing exists in all crystals of the nucleosome core particle on its own.
Figure 11
Figure 11. DNA stretching in nucleosome core particle structures. Cartoon representation of structure of approximately half of the nucleosomal DNA for (a) 146 bp human alpha-satellite (HAS146) (PDB ID 1AOI, blue) and (b) 145 bp 601 (PDB ID 3LZO, red) nucleosome positioning sequences relative to the HAS147 sequence (PDB ID 1KX5, yellow) (top). Stretching of 1 bp is observed at superhelical location (SHL) −2 with the HAS146 sequence and 1 bp each at SHL ± 5 with the 145 bp 601 sequence. SHLs and the dyad = SHL 0 are indicated. The length of DNA wrapped on each side of the NCP for each of the sequences is also shown (bottom).
When we crystallized the chromatin factor RCC1 in complex with the 601 nucleosome, we anticipated that the Widom 601 sequence would also likewise form a 147 bp nucleosome core particle. We therefore employed a 147 bp 601 DNA sequence in our crystallization studies. To our surprise, structure determination showed that the 601 nucleosome in the RCC1/nucleosome structure forms a 145 bp nucleosome core particle due to stretching of DNA by one bp at SHL ± 5 (52) (Figure 11b). Two lines of evidence indicate that the DNA stretching in the 601 nucleosome did not result from crystal contacts. First, unlike crystals of nucleosome core particles on their own, the RCC1/nucleosome complex does not make DNA end to DNA end crystal contacts. A symmetry related RCC1 does make important crystal contacts with one DNA end, but the DNA end on other end of the nucleosome makes no crystal contacts. (75) Second, the 601 nucleosome core particle on its own (i.e., in the absence of RCC1) crystallized in a different space group (P212121 vs P21 for RCC1/nucleosome) and via different crystal packing interactions than in the complex with RCC1. (52, 53) Despite the different crystal packing arrangement, the 601 nucleosome particle on its own also exhibits stretching at SHL ± 5. This stretching can explain why the 601 nucleosome core particle had evaded crystallization for such a long time: the 147 bp 601 nucleosome core particles with its extra bp extending beyond the nucleosome core particle would prevent the canonical DNA end to DNA end crystal packing found in all nucleosome only crystals. We were fortunate that our use of the 147 bp 601 nucleosome not only did not prevent the RCC1/nucleosome from crystallizing but was in fact important for the RCC1-nucleosomal DNA end crystal contact to occur. (75)
4 Recognition of the Nucleosome Core by Chromatin Factors
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The recruitment of macromolecular chromatin factors to genomic loci is controlled at many levels. Factors can be actively sequestered in or excluded from the nucleus. The accessibility of large territories of the genome can be regulated by altering the degree of chromatin compaction. At a more local level, the binding of chromatin factors can be tuned by the positioning of nucleosomes. Some chromatin factors including many transcription factors bind to specific DNA sequences only in nucleosome free regions. On the other hand, many chromatin factors require nucleosomes for binding to chromatin. Obvious examples are histone-modifying and chromatin remodeling enzymes that by definition modify the chemical composition or architecture/location of nucleosomes, respectively. However, many other chromatin factors also bind to nucleosomal regions including high-mobility group proteins, (76) heterochromatin scaffolding proteins, (77, 78) and even viral proteins. (79) As described above, the nucleosome provides a diverse platform for binding of macromolecular chromatin factors. This platform is further diversified by replacement of canonical histones with histone variants and the chemical modification of both histone and DNA components of the nucleosome.
Chromatin factors can bind the nucleosome using one or more of the three following nucleosome surfaces: (1) the histone N- and C-terminal tails; (2) the disk faces of the histone octamer; and/or (3) the nucleosomal DNA. [Of particular note, the nucleosomal DNA affords the possibility of novel protein–DNA interactions, given its unique curvature as well as the alignment of the nucleosomal DNA gyres.] Much of the research regarding binding to histone tails is centered around histone PTMs. The molecular recognition of histone tails by numerous catalytic domains that establish and remove histone PTMs and diverse protein domains that bind tails modified at specific residues are thoroughly reviewed elsewhere. (3, 80-82) Due to technical challenges in the structural characterization of the nucleosome core bound to chromatin factors, much less is understood regarding the molecular recognition of the disk surfaces of the histone octamer and nucleosomal DNA. However, recent advances in the cocrystallization of macromolecular chromatin factors bound to the nucleosome core particle have permitted new atomic scale depictions of recognition of the nucleosomal disk. All crystal structures of macromolecules bound to the nucleosome solved to date share one common interaction motif, an arginine bound to the H2A/H2B acidic patch. Details of each of these structures, a discussion of this shared acidic patch arginine-anchor, and insights from further modes of nucleosomal recognition from NMR and cryo-EM studies are discussed below.
4.1 H4 N-Terminal Tail
The first instance of a protein segment binding to the nucleosomal H2A/H2B acidic patch was observed in a crystal contact of the 1997 nucleosome core particle structure. (9) In these crystals, residues 16 to 25 of one H4 tail contact the acidic patch on an adjacent NCP forming a charged interaction surface (Notably, the other tail is not resolved N-terminal to residue 20 even though NMR experiments suggest the H4 tail is structured starting with residue 16 (83)). The H4 K16 side chain projects into an acidic cavity generated by H2A acidic patch residues E61, D90, and E92 of the neighboring NCP surface. Other positively charged amino acids in the H4 tail also interact with negative side chains in the H2A/H2B acidic patch, including H4 R19-H2A E64, H4 K20–H2B E110 and H4 R23 which contacts both H2B E110 and H2A E56. Similar interactions are observed for H4 K20 and R23 in the crystal lattice of the 1.9 Å NCP structure though the other basic H4 side chains point toward intranucleosomal DNA. (17) A functional role for the H4 N-terminal tail in chromatin structure has been confirmed using nucleosome arrays in solution. Truncation of the N-terminal H4 tail prior to residue 20 (84) or the charge neutralizing acetylation of H4 K16 (5, 85, 86) leads to incomplete cation-mediated compaction of the 30 nm fiber in vitro. The case for a role in chromatin structure is furthered by disulfide formation between spatially adjacent mutant nucleosomes containing H4 V21C and H2A E64C both within a chromatin array (87) and between arrays. (88) It is important to note that the observed structure of the H4 N-terminal tail seen in the crystal lattice may not reflect a native conformation in chromatin fibers. Recent modeling of the H4 N-terminal tail suggests that H4 residues 15 to 20 exhibit a propensity for α helix formation. This allows H4 K16, R17, R19, and K20 to occupy a single helical face, which can be accommodated within the acidic patch groove. (89) Though the evidence for the H4 tail-acidic patch in higher order chromatin structure is compelling, higher resolution structural characterization is required to accurately define the molecular details of this interaction.
4.2 Viral LANA Peptide
In 2006, Barbera et al. demonstrated that the nucleosome docking region of the Kaposi’s sarcoma-associated herpes virus (KSHV) latency-associated nuclear antigen (LANA) also interacts with the H2A/H2B acidic patch. (79) The KSHV genome is contained within an episome, which is tethered to mitotic chromosomes using the basic N-terminal region of LANA by anchoring to the nucleosomal acidic patch. The 2.9 Å crystal structure of the LANA nucleosome recognition sequence bound to the nucleosome core particle was solved by soaking the peptide corresponding to LANA residues 1–23 into NCP crystals. LANA peptide forms a hairpin that fits in the acidic patch groove between the αC and α1 helices of H2B and makes multiple charged and hydrophobic interactions (Figure 12). The LANA R9 side chain inserts into the acidic patch to form ionic interactions with H2A residues E61, D90, and D92. This acidic patch “arginine-anchor” (our terminology) is shared with all crystal structures of chromatin factors bound to the NCP reported to date and overlaps the H4 K16 binding site observed in the original NCP structure crystal lattice. (9) LANA R9 is critical in nucleosome binding as mutation of this residue eliminates LANA’s chromatin association. (79, 90) An additional LANA arginine (R7) forms an ionic interaction with H2B E110 and LANA S10 hydrogen bonds to H2A E64. LANA hydrophobic residues M6 and L8 bind a hydrophobic surface adjacent to the acidic patch including H2A residues Y50, V54, and Y57. Overall the regions of LANA observed to interact with the NCP correlate with those required for the virus to tether its episomal DNA to chromosomes.
Figure 12
Figure 12. Nucleosome recognition using the acidic patch arginine-anchor. From top to bottom, structures of RCC1 (PDB ID 3MVD), (52) Sir3 (PDB ID 3TU4), (78) PRC1 (PDB ID 4R8P), (111) LANA peptide (PDB ID 1ZLA), (79) and CENP-C peptide (PDB ID 4INM) (107) bound to the nucleosome core particle. Overview of structures as viewed from opposite the dyad (right) and zoomed view of acidic patch (left) with arginine-anchor in space-filling representation and key H2A residues shown as sticks. Locations of RCC1 switchback loop (1), DNA binding loop (2), and N-terminus (N) and Sir3 loop 3 (3) and N-terminus (N) are indicated. Histones H3, H4, H2A, and H2B are shown in cartoon representation and colored cornflower blue, light green, wheat, and pink, respectively. DNA (light pink) is shown as sticks.
4.3 Ran Guanine Exchange Factor RCC1
In 2010, we solved the structure of the Drosophila melanogaster β-propeller protein RCC1 (Regulator of chromatin condensation) bound to the nucleosome core particle. (52) In contrast to the LANA-NCP structure, the RCC1-NCP structure was solved by cocrystallization of RCC1 on the NCP. RCC1 is a guanine exchange factor for the Ran GTPase (or RanGEF) that establishes a gradient of the GTP bound form of Ran around chromatin. This gradient plays roles in nuclear-cytoplasmic transport, mitotic spindle formation, and formation of the nuclear envelope following mitosis. (91-93) RCC1 binds to the nucleosome resulting in an increase in its ability to catalyze Ran guanine exchange. (94) Our 2.9 Å structure of the RCC1-NCP complex shows that RCC1 interacts with both the acidic patch and nucleosomal DNA using two β-propeller loops and its N-terminal tail (52) (Figure 12). One loop, termed the switchback loop, binds to the H2A/H2B acidic patch using an intricate network of ionic and hydrogen bonds and van der Waals contacts. The switchback loop contains the RCC1 arginine-anchor residue 223 that binds H2A E61, D90, and E92 nearly identically to LANA R9. Much like LANA, RCC1 uses a second arginine 216 for additional interactions with H2A E61 and E64. Both RCC1 R223 and R216 are important for nucleosome binding in solution. (95) RCC1 S217 forms hydrogen bonds with H2A V45 and E64. An additional hydrogen bond is observed between RCC1 S214 and H2A E64. These ionic and hydrogen bonding interactions are complemented by van der Waals contacts, especially with residues in the H2B αC helix that form a ridge at the edge of the acidic patch.
In addition to the acidic patch, RCC1 also binds to nucleosomal DNA using a distinct β-propeller loop and its N-terminus. The RCC1 DNA binding loop interacts with the phosphodiester backbone across a major groove near SHL ± 6 forming hydrogen bonds or charged interactions with the side chains of K241 and R239. An additional hydrogen bond is formed by the side chain of RCC1 259. The N-terminal tail of RCC1 is also implicated in nucleosome binding. (94) While the N-terminal residues 2–27 are not visible in our RCC1-NCP structure, residues 28 and 29 are positioned to allow the N-terminal tail to enter the major groove of nucleosomal DNA adjacent to the DNA binding loop. Alignment of RCC1 from the RCC1-NCP and RCC1-Ran structures suggest that Ran approaches but does not contact the nucleosome surface. Therefore, either RCC1 or Ran must undergo conformational changes to allow for Ran-NCP interactions to enhance RCC1’s RanGEF activity. Further experiments are required to resolve this issue.
4.4 Silencing Protein Sir3
In 2011, Armache et al. solved the crystal structure of the BAH (bromo-associated homology) domain of the yeast silent information regulator protein Sir3 bound to the nucleosome core particle. (78)Saccharomyces cerevisiae uses SIR (silent information regulator) proteins Sir1, Sir2, Sir3, and Sir4 to establish a transcriptionally repressive chromatin state at telomeres, ribosomal DNA loci and silent mating-type loci. (96) Silencing is thought to be accomplished in part though direct chromatin compaction by Sir3 as demonstrated in vitro. (97-99) This 3.0 Å structure illustrates one of the most extensive interaction surfaces of the published high resolution chromatin factor-NCP structures, including 28 Sir3 BAH domain residues and greater than 30 histone residues (and potentially nucleosomal DNA at the BAH domain N-terminus) (78) (Figure 12). The structure also suggests that weak self-interactions of the BAH domain observed in the crystal lattice and in solution may contribute to its ability to compact chromatin fibers. Analogous to LANA and RCC1, the Sir3 BAH domain binds to the H2A/H2B acidic patch, but also interacts with the nucleosome in three additional regions: the H4 N-terminal tail, surfaces of H3/H4 in the loss of rDNA silencing (LRS) domain, and the H2B C-terminal helices. Sixteen Sir3 BAH domain residues from loops 2 and 4, strand B5 and the A1 helix interact with H4 tail residues 13–23. This region of the H4 tail is often unstructured in NCP crystals in the absence of crystal lattice contacts. The Sir3 BAH domain-H4 tail interface is predominantly electrostatic in nature owing to the positively charged H4 tail and negatively charged complementary Sir3 surface. Importantly, the H4 K16 side chain forms several hydrogen bonds and ionic interactions with acidic Sir3 side chains, offering a molecular mechanism for the observed loss of Sir3 binding upon H4 K16 acetylation. (98) The nucleosomal recognition surface of the Sir3 BAH domain also includes the α1 helix and L1 loop of H3, the α2 helix and L2 loop of H4 as well as the α3 and αC helices of H2B. These interactions are mediated by the Sir3 BAH loop 3 which becomes structured upon NCP binding and strands B6 and B8. This interaction surface includes the LRS domain residues 76–80 of H3 that are required for silencing in yeast. (100) Much like H4 K16 acetylation, H3 K79 methylation disrupts Sir3 binding. (101-103) The interactions in this region offer insight into the preference for H3 K79 in the unmodified state due to loss of potential hydrogen bonds with Sir3 BAH side chains. This is further exemplified by two recent structures of the N-α-acetylated Sir3 BAH domain bound to the NCP. (104, 105) The native N-terminally acetylated residue specifically interacts with regions of the Sir3 BAH domain further structuring its loop 3, enhancing contacts with the nucleosome surface in the LRS region and leading to a 30-fold increase in affinity for the NCP. (105) Sir3 BAH also contacts the H2A/H2B acidic patch using the loop 1 region that is unstructured in the absence of the NCP. (106) While electron density is weaker in this region of the Sir3 BAH-NCP structure, it appears that multiple arginines (R28, R29, R30, R32, and R34) line the acidic patch groove to make charged interactions with the NCP surface. Notably, Sir3 R32 occupies an identical binding site to that observed for LANA R9 and RCC1 R223 in a cavity surrounded by H2A E61, D90, and D92. Overall, the multifaceted interaction of the Sir3 BAH with the NCP explains many silencing defects observed upon mutation of both histones and Sir3 in yeast.
4.5 Centromeric Protein CENP-C
The crystal structure of the central region of rat centromere protein CENP-C bound to the nucleosome core particle illustrates the ability of a chromatin factor to recognize specific features of a variant histone in the context of the nucleosome. (107) Proper segregation of chromosomes in mitosis requires the mitotic spindle to attach to the kinetochore at the centromere of each chromosome. (108) Centromeric chromatin contains H3 variant CENP-A and a complex of 16 centromeric proteins including CENP-C. (109, 110)The crystal structure of the CENP-C nucleosome binding peptide in complex with a chimeric NCP in which the C-terminal region of H3 was replaced with the LEEGLG solvent exposed sequence of CENP-A was solved by Kato et al. in 2013. (107) In the structure, CENP-C forms an elongated conformation, which contacts the acidic patch and the CENP-A specific regions of the chimeric NCP (Figure 12). CENP-C binds to the acidic patch using two arginines, R717 and R719. The CENP-C R717 arginine-anchor binds in the now characteristic H2A E61, D90, E92 pocket; R719 makes additional ionic interactions with H2A E61 and E64. In the CENP-A specific region of the chimeric nucleosome, CENP-C residue Y725 binds in a hydrophobic pocket created by CENP-A residues I133 and L137. While CENP-A proteins are not highly conserved, they all contain at least one large hydrophobic residue in the CENP-C binding region that is not found in canonical H3. (107) This 3.5 Å structure was validated by extensive NMR experiments using chemical shift perturbations and site-specific incorporation of paramagnetic spin labels. (107)
4.6 Polycomb Repressive Complex 1 (PRC1) Ubiquitylation Module
Recently, we solved the crystal structure of the PRC1 ubiquitylation module, containing the Ring1B-Bmi1 ubiquitin E3 ligase RING heterodimer together with the E2 enzyme UbcH5c, bound to its nucleosome core particle substrate at 3.3 Å resolution (Figure 12). (111) PRC1 is a member of the Polycomb group family of complexes and plays a role in transcriptional repression of developmentally regulated genes at least in part through H2A K119 ubiquitylation and intrinsic chromatin compaction. (112-114) PRC1 contains a RING-type ubiquitin E3 ligase composed of RING domains from Ring1B and Bmi1 that can pair with one of several ubiquitin E2 conjugating enzymes, including UbcH5c. (115) Our structure reveals that all three proteins in the PRC1 ubiquitylation module bind to the nucleosome surface, together contacting all components of the nucleosome core particle. The Ring1B-Bmi1 RING heterodimer forms a saddle over the proximal end of the H2B αC helix, anchored on each side by histone interactions. The RING domain of Ring1B binds the H2A/H2B acidic patch using multiple positively charged side chains including an arginine anchor residue R98 (Figure 12). The substantial Ring1B-acidic patch interface contrasts a more modest Bmi1-nucleosome interface. The RING domain of Bmi1 interacts with a smaller acidic surface on the H3/H4 tetramer and forms a cap on the distal end of the H3 α1 helix. The structure is consistent with mutagenesis experiments implicating both the H2A/H2B acidic patch, the arginine anchor, and several other basic side chains. (116, 117) In addition to these E3 ligase-histone interactions, the E2 UbcH5c binds to nucleosomal DNA in two positions. Near the DNA end, the UbcH5c antiparallel β-sheet aligns several charged and polar side chains for interaction with the adjacent nucleosomal DNA backbone. UbcH5c binds nucleosomal DNA again at the dyad using basic α3 side chains. Mutagenesis at these novel E2-substrate interfaces diminishes nucleosome binding and activity by the PRC1 ubiquitylation module. (111) Importantly, unlike other histone modifying enzymes structurally characterized to date, the PRC1 ubiquitylation module does not appear to directly recognize its targeted primary sequence. Rather the E3 and E2 components bind to topologically unique nucleosome surfaces distant from the site of catalysis to position the E2 active site directly over the H2A C-terminal tail near the target lysine.
4.7 NMR-Based Model of HMGN2
As a complementary approach to X-ray crystallography, Bai and colleagues have introduced NMR-based techniques to characterize chromatin factor-nucleosome interactions. (107, 118, 119) The combination of methyl-TROSY and paramagnetic spin label NMR experiments allowed Kato et al. to map the nucleosomal surface bound by high mobility group nucleosomal protein HMGN2. (103) A model for the HMGN2-NCP complex was created based on comprehensive NMR-based restraints. HMGN proteins are chromatin architectural proteins with roles in DNA damage repair, chromatin remodeling, and histone PTMs. (76, 120-122) They can also directly decompact chromatin and compete for chromatin binding with the linker histone H1. (123) HMGNs share a common N-terminal nucleosome binding domain with the conserved basic octapeptide sequence, RRSARLSA. (123) Kato et al. observed chemical shift perturbations of labeled side chain methyl groups of H2A L65 and H2B V45 and L103 upon HMGN binding. (118) These residues are in proximity to the H2A/H2B acidic patch. Based on many NMR experimental restraints, a model of the HMGN2-NCP complex was proposed, suggesting arginines in the conserved HMGN nucleosome binding domain (R22, R23, and R26) interact with the H2A/H2B acidic patch. HMGN2 lysines 39, 41, and 42 were also proposed to interact with DNA near the entry/exit from the NCP. The two NCP-binding regions of HMGN2 are separated by a rigid proline-rich linker. Of note, two HMGN2 proteins bind the pseudosymmetry-related faces of the NCP with positive cooperativity (Kd of 1.5 and 0.17 μM for the first and second binding events, respectively, giving a Hill coefficient of 1.4). The authors suggest that HMGN-NCP interactions staple the DNA end to the histone octamer blocking the activity of chromatin remodelers. Moreover, the model explains the release of HMGNs from chromatin during mitosis following phosphorylation of S24 and S28. (124) The negative charge carried on the phosphorylated serines would create repulsive interactions with the neighboring acidic patch.
4.8 Acidic Patch Arginine-Anchor As a Common Motif for Nucleosome Recognition
All crystal structures of chromatin factors bound to the nucleosome core particle share a common structural motif: an arginine-anchor that binds to a specific cavity generated by H2A E61, D90, and E92 side chains in the H2A/H2B acidic patch. And these are not the only examples of chromatin enzymes and factors relying on the acidic patch for nucleosome binding and/or activity. Ubiquitin E3 ligases RNF168 and BRCA1 exhibit defective ubiquitylation when the acidic patch is mutated (111, 125) and IL-33 binds to the acidic patch, likely in a manner similar to the LANA nucleosome targeting peptide. (126) So why would such a common motif for recognition of a complex as large as the nucleosome exist? The simple answer is that the acidic patch is the most unique region of the nucleosome surface. It is topologically poised for chromatin factor interaction as a deep groove with a complex surface. The width of the groove allows the binding of multiple types of structures including loops (RCC1, Sir3), hairpins (LANA), extended conformations (HMGN2, CENP-C) but can also accommodate helical and β-strand secondary structure elements. (89, 127) In addition, the acidic patch carries the greatest net charge of the solvent exposed region of the histone octamer disk surface. Furthermore, the guanidinium group of the arginine-anchor is optimal for ionic interaction with all three H2A acidic side chains in the shared acidic-patch binding pocket.
Why would other distinct surfaces not be targeted to minimize interference? Luger and colleagues propose that the overlap of binding sites may serve a regulatory role in the determination of chromatin structure. (127) That is, competition for the acidic patch between factors that condense chromatin (H4 tail and Sir3) with other macromolecules (HMGN2, RCC1, etc.) may tune the higher-order state of chromatin. Many questions still remain regarding this emerging paradigm for nucleosome binding. How common is it? Are we seeing it so frequently in biochemical experiments because we know to look for it? Are chromatin factors that bind to the acidic patch just easier to crystallize owing to binding affinities or resultant crystal packing opportunities? How is the binding of multiple chromatin factors to the same nucleosomal surface regulated? Are other acidic patch binders post-translationally modified to tune their binding affinities similar to HMGNs? Currently, the sample size is too small to address most of these questions. However, as described above, recent strides have been made in the structural characterization of chromatin factor-nucleosome complexes. This will provide a foundation for future work to address these unanswered questions and undoubtedly uncover new paradigms for nucleosomal recognition.
4.9 Cryo-EM Models of Chromatin Factor-Nucleosome Complexes
In addition to X-ray crystallography and NMR structures, several cryo-EM structures have enhanced our understanding of nucleosome recognition by chromatin enzymes and factors. The chromatin factors studied by cryo-EM to date fall into three functional categories: chromatin remodeling enzymes, histone modification enzyme complexes, and chromatin architectural proteins. They include large and dynamic macromolecules that present difficulties to crystallographers and NMR spectroscopists. Of note, all reported cryo-EM structures of chromatin factor-nucleosome complexes were solved at resolutions greater than ∼20 Å. This allows overall architecture to be revealed but precludes molecular description of NCP interactions. Studies using multimodality approaches, pairing cryo-EM with crystallographic characterization of subcomplexes, cross-linking mass spectrometry and/or comprehensive biochemical analysis permit nearly residue-specific understanding of interactions and thus heighten mechanistic insight as compared to cryo-EM reconstructions alone.
ATP-dependent chromatin remodeling complexes can alter the position and composition of nucleosomes by sliding them along DNA, unwrapping nucleosomal DNA, or ejecting/exchanging histone dimers or octamers. (128) There are four principal families of chromatin remodelers: SWI/SNF, ISWI, Mi-2/CHD, and SWR/INO80. Nucleosome binding of representatives of all families except the Mi-2/CHD family have been characterized by cryo-EM. These three families interact with the nucleosome in distinct ways. In 2008, Chaban et al. used negative stain reconstructions of the SWI/SNF family remodeling complex RSC to show that RSC nearly engulfs the NCP, (129) consistent with DNaseI protection experiments (130, 131) and the 2007 Leschziner et al. model docking the NCP into the central cavity of a reconstruction of RSC alone. (132) Interestingly, some density was missing for both nucleosomal DNA and one H2A/H2B dimer, suggesting RSC-mediated remodeling even in the absence of ATP.
Cryo-EM reconstructions of two ISWI family remodeling complexes bound to nucleosomes show less extensive interactions. In a 2009 study, Racki et al. demonstrated that the ACF catalytic subunit, Snf2, binds with 2:1 stoichiometry to the nucleosome. (133) While the ATPase domains and linker DNA are not visible in their reconstructions, biochemical data suggests that the ATPase domain binds the nucleosome at SHL ± 2 and the interaction also involves the H4 N-terminal tail. The authors propose a competition mechanism through which nucleosome spacing is accomplished by competitive sliding by two Snf2 subunits bound to opposite sides of the nucleosome. A different mechanism for nucleosome spacing was suggested for ISWI family member ISW1a by Yamada et al. based on combined cryo-EM and crystallographic data. (134) Cryo-EM reconstructions of ISW1a in the absence of its ATPase domain bound to nucleosomes containing one or two DNA extensions revealed two modes of interactions with linker DNA. Together with a crystal structure of the ISW1a construct bound to free DNA, these cryo-EM reconstructions led to a model in which ISW1a acts as a ruler, using its size and shape to space adjacent nucleosomes.
Two cryo-EM studies also offered insight into the nucleosome binding of SWR/INO80 family remodelers. In 2012, Saravanan et al. generated a model for the 2:1 Arp8:NCP complex using crystal structures of the components and a 21 Å cryo-EM reconstruction. (135) In this model, Arp8 interacts with the H3/H4 surface though the molecular details are unclear. More recently, Tosi et al. used cryo-EM and cross-linking mass spectrometry (XL-MS) to extensively characterize the interaction of the holo-INO80 complex with the NCP. (136) The architecture of the INO80 complex alone shows four domains: the Rvb1/2 dodecamer head, the Ino80 ATPase-Ies2-Arp5-Ies6 neck, the Ino80 N-terminus-Nhp10-Ies1-Ies3-Ies5 body, and the Ino80 HAS-Act1-Arp4-Arp8-Ies4-Taf14 foot. While heterogeneity in the INO80-NCP cryo-EM images prevented proper 3D reconstruction, extensive XL-MS was observed between all four domains of INO80 and the nucleosome disk and tails. Notably, some cross-linking was observed to surfaces of the H2A/H2B dimer that are buried in the NCP structure. These contacts may facilitate opening of the NCP structure required for INO80 mediated H2A/H2B exchange. The authors created a model for the INO80-NCP complex based on the XL-MS data in which the NCP rests on a cradle surrounded by all four domains of the INO80 complex.
NCP bound cryo-EM reconstitutions have also been reported for the Saccharomyces cerevisiae Piccolo NuA4 histone acetyltransferase (HAT) complex (137) and the HP1-like heterochromatin protein Swi6 from Schizosaccharomyces pombe. (77) Piccolo NuA4 is an H4- and H2A-specific HAT complex that functions alone and as part of the larger NuA4 complex. (138) Piccolo’s Esa1 catalytic subunit is unable to bind and acetylate nucleosomes without its accessory subunits Epl1 and Yng2. (138) The 2011 Chittuluru et al. cryo-EM reconstruction shows that Piccolo binds to the NCP with 1:1 stoichiometry using two prongs that contact the NCP opposite to the dyad and over the H4 histone-fold with flexibility observed between Piccolo and the NCP. (137) Subsequent cross-linking experiments place the Esa1 Tudor domain in proximity to nucleosomal DNA and the Epl1 EPcA domain near the N-terminal tail of H2A. (139)
Swi6 is an HP1 ortholog that binds to trimethylated H3 K9 to enable the spreading of heterochromatin. The 2013 25 Å cryo-EM reconstitution of two Swi6 dimers bound to the NCP in open/disinhibited forms reported by Canzio et al. suggests that one Swi6 chromodomain and one chromoshadow domain from each dimer contact the nucleosome near the exit of the H3 tail and at the nucleosomal DNA at SHL ± 5, respectively. (77) The other chromodomain in each dimer is poised for binding H3 K9me3 in a neighboring nucleosome to facilitate spreading of Swi6 across chromatin. While the authors could thoroughly investigate the autoinhibitory function of Swi6, the low resolution of the cryo-EM structure prevented a molecular understanding of the Swi6-NCP interactions.
These cryo-EM studies offer unique views of nucleosome recognition by large and complex chromatin enzymes and factors. At this time, cryo-EM allows for the general architecture of chromatin factor-NCP complexes to be ascertained. The molecular workhorse remains other modalities, including crystallography of subcomplexes, XL-MS and comprehensive biochemistry. Yet, with technological advances in sample preparation together with higher resolution and faster detectors and more powerful image alignment algorithms, cryo-EM holds promise to complement if not rival or surpass crystallographic and NMR methods for atomic-resolution determination of chromatin factor-nucleosome complex structures.
5 Structural Studies of the Chromatosome and 30 nm Fiber
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The structure of the chromatosome and the structure, and even relevance, of the 30 nm fiber are two of the most highly studied, yet controversial, topics in chromatin biology. It is clear linker histone H1 (or H5) binds to the nucleosome core particle and linker DNA and promotes compaction of chromatin arrays into 30 nm fibers. (140) Linker histones contain a central globular domain and unstructured N- and C-terminal extensions. The globular domain gH1 and the C-terminal domain are primarily involved in chromatin binding and compaction. Many years of biochemical, biophysical and computational experiments have led to two distinct classes of gH1-NCP interactions: (1) symmetric in which gH1 binds at the dyad and interacts with linker DNA extending from both sides of the core particle; (141-144) and (2) asymmetric in which gH1 binds near the dyad and interacts with 10–20 bp of linker DNA extending from one side of the nucleosome core. (119, 143, 145-149) Two recent structural studies offer unique insights into H1 binding in the chromatosome and the 30 nm fiber.
5.1 NMR and Cryo-EM Models of the Chromatosome
In 2013, Zhou et al. used extensive NMR measurements to generate a unique residue-specific model for the gH1/NCP complex. (119) The authors first observed chemical shift perturbation of isotopically labeled gH1 residues 37–211 and nucleosome core particles to define the regions of each involved in gH1/NCP complex formation. Then they incorporated paramagnetic spin labels to define distances between regions of gH1 and the NCP to orient the complex. Finally, they performed computational docking to generate models of the gH1/NCP complex. The favored model correlates with asymmetric binding to the nucleosome consistent with strong effects observed with spin labels attached to H3 R37 and H2A T119, as these observations are incompatible with symmetric binding models. Similar results were seen with the H1 tails that are unstructured in the chromatosome. gH1 only interacts with the 10 bp extending from the NCP given that no differences were seen with longer segments of nucleosomal DNA. In the favored computational model, gH1 uses two positively charged surfaces defined by NMR experiments (residues 119–125 and 164–174) to bridge the nucleosome core surface and 10 bp of linker DNA on one side of the NCP. However, the authors could not rule out weaker binding to the other linker DNA. No evidence was seen of gH1 binding histones within the nucleosomal disk. However, gH1 binding imparts structural organization of the H2A C-terminal tail consistent with a direct interaction. This explains the decreased binding of gH1 to H2A.Z containing nucleosomes that have been attributed to divergent C-terminal sequences. (150, 151) In 2014, Song et al. reported an 11 Å reconstruction of the 30 nm fiber reconstituted with histone H1. The density for the gH1 domain showed a 1:1 H1:nucleosome binding with H1 binding asymmetrically near, but off-center from, the dyad and interacting with both entry and exit linker DNAs. These studies provide exciting views of the position of H1 within the chromatosome and bolster growing evidence for the asymmetric binding model. Precise molecular details await higher-resolution structural solutions.
5.2 11 Å Cryo-EM Structure of a Two-Start 30 nm Fiber
Traditional dogma holds that the 11 nm unfolded chromatin strand (often referred to as beads on a string) compacts into a 30 nm fiber with side-to-side packing of nucleosomes perpendicular to the fiber axis. (152, 153) The folding of the 30 nm fiber is encouraged by the interiorly positioned linker histones. (154) The 30 nm fiber further condenses into progressively higher-order chromatin states. Most models for the 30 nm fiber fall into two categories: (1) one-start models are solenoidal with sequential nucleosomes connected by bent linker DNA segments arranged along a helical path; (12, 152-154) (2) two-start models separate sequential nucleosomes by straight linker DNA in a zigzag patter either longitudinally along the fiber (helical ribbon) or radially across the fiber (crossed-linker). (13, 154-157)
Studies by two groups in the mid 2000s used reconstituted arrays with defined nucleosome positions to build opposing two- and one-start molecular models. Richmond and colleagues proposed a two-start model based on digestion patterns of short, cross-linked nucleosomal arrays. (87) The two-start model was further supported by a 9 Å tetranucleosome crystal structure showing two stacks of two nucleosomes separated by a zig-zagging pattern of straight linker DNA. (13) This structure was used to generate an idealized model for the two-start crossed-linker-type 30 nm fiber (Figure 13). Of note, the modeled fiber has a smaller diameter owing to the 167 nucleosome repeat length used for the tetranucleosome structure and the dependence of crossed-linker-type fiber diameters on linker length. Meanwhile, Rhodes and colleagues characterized longer H1-containing 30 nm fibers by cryo-EM. (12) They observed similar fiber diameter over widely varying linker lengths characteristic of a one-start solenoid structure (Figure 13). Later modeling also suggested potential two-start solutions to the cryo-EM data in addition to the original one-start model. (158)
Figure 13
Figure 13. Models of the 30 nm fiber. Orthogonal views perpendicular to the 30 nm fiber axis (top) and down the axis (bottom) of the Richmond two-start model (left), Rhodes one-start model (center) and Li-Zhu tetranucleosome-unit repeat two-start model (right). The sequence of nucleosomes in each model is indicated. In the Richmond model, each sequential pair of nucleosomes across the fiber is colored similarly. For the Rhodes model, all nucleosomes in the same turn of the solenoid are colored similarly. In the Li-Zhu model, each tetranucleosome repeating unit is colored similarly. Unlabeled nucleosomes in the two-start models are not shown in the bottom views for figure clarity. Linker DNA is not present in the Rhodes model but, given the nature of the solenoidal structure, must be bent. The B-form DNA double helix is shown for comparison (far right). All models shown in space-filling representation and scaled as indicated.
Despite this long-standing hierarchical paradigm for chromatin folding, recent cryo-EM and SAXS measurements with mitotic chromosomes show no evidence of the 30 nm fiber. (159-162) Rather mitotic chromatin may assume a fractal-like state. Similar experiments with chicken erythrocytes did show evidence of the 30 nm fiber. (162) Altogether, this implies that the 30 nm fiber does exist in certain cell-types and/or cell-cycle stages, but may not be as pervasive as once thought. (163)
In 2014, Song et al. solved 11 Å cryo-EM structures of 30 nm fibers reconstituted with 12 × 177 and 12 × 187 bp of the Widom 601 nucleosome positioning sequence. (11) The structures clearly show a left-handed parallel double helix similar to that proposed by Richmond and colleagues (Figure 13). This structure bears some resemblance to the DNA double helix, although the DNA double helix contains right-handed antiparallel strands. The diameter of the fiber is dependent on small changes in DNA linker length, suggesting a two-start model in which straight linker DNA crossing the central channel determines the fiber diameter. This two-start crossed-linker type model is confirmed by clear density for straight segments of linker DNA crossing the center channel of the fiber. The repeating unit of the fiber is a tetranucleosome with two stacked nucleosomes on opposing sides of the superhelix. The arrangement of nucleosomes places the thinner half of the nucleosome near the dyad close to the interior of the fiber where the fiber diameter is smaller. Save the different linker length, the tetranucleosome repeating unit is very similar to the 9 Å tetranucleosome crystal structure. (13) Unexpectedly, gH1 from neighboring nucleosomes in the 30 nm fiber align with alternating head-to-head arrangements within the tetranucleosomal unit and tail-to-tail arrangements between tetranucleosomal units. The tail-to-tail aligned gH1s interact with one another, imparting an additional twist to the fiber. This results in different internucleosomal contacts between adjacent stacked nucleosomes in the tetranucleosome unit and between tetranucleosome units. Notably, the H4 tail-H2A/H2B acidic patch interaction is plausible between tetranucleosome units, as in the idealized model from Richmond and colleagues. This interaction is not possible within the tetranucleosome unit due to juxtaposition of the H2B αC helix of one nucleosome with the acidic patch H2A α2 helix of the neighboring nucleosome. This phenomenon was also seen in the tetranucleosome crystal structure. While controversy remains regarding the prevalence of the 30 nm fiber in cell-cycle specific structures of chromatin in different cell types in vivo, this model provides a higher resolution view of the 30 nm fiber, which gives new insights into the orientation of H1 within each nucleosome and across the chromatin fiber.
6 Perspective
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Over the past 5 years, major strides have been made in the understanding of nucleosome structure and function. Molecular details of the sequence-dependence of nucleosomal DNA structure have been elucidated. Co-crystallization and comprehensive NMR analysis of chromatin factors and the nucleosome core particle have established the acidic patch arginine-anchor as a paradigm for nucleosome recognition. Advancements in cryo-EM technology have aided in the visualization of the two-start 30 nm fiber and characterization of nucleosome binding by large and conformationally flexible chromatin enzymes. Despite this extraordinary progress, it is clear that we have only scratched the surface of the tremendously complex role of the nucleosome in coordinating chromatin-templated processes. Additional work will undoubtedly establish new paradigms for nucleosome recognition and bring about a heightened understanding of the role of the 30 nm fiber and other chromatin structures in the functional organization of the eukaryotic genome.
Biographies
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Robert K. McGinty
Robert K. McGinty received his Ph.D. from The Rockefeller University in 2010 and M.D. from Weill Cornell Medical College as a member of the Tri-Institutional M.D.–Ph.D. program. During his graduate studies under the supervision of Prof. Tom Muir, McGinty used protein chemistry to study crosstalk between histone modifications. In 2011, he joined the laboratory of Prof. Song Tan in the Center for Eukaryotic Gene Regulation at Penn State, where he is using X-ray crystallography to study the molecular recognition of the nucleosome by histone modifying enzymes. He is currently a Damon Runyon postdoctoral fellow.
Song Tan
Song Tan studied physics as an undergraduate at Cornell University (1985) before pursuing his Ph.D. at the MRC Laboratory of Molecular Biology as a Marshall Scholar (1989). He continued his training as a postdoctoral fellow and project leader under Tim Richmond at the ETH-Zürich (Swiss Federal Institute of Technology) where he determined crystal structures of several transcription factor/DNA complexes. Dr. Tan joined the Center for Eukaryotic Gene Regulation and the faculty of the Department of Biochemistry and Molecular Biology in 1998. He was named a Pew Scholar in the Biomedical Sciences in 2001. Dr. Tan’s laboratory investigates how chromatin enzymes and factors interact with their nucleosome substrates through biochemical and structural approaches. His laboratory determined the first chromatin factor-nucleosome crystal structure (RCC1-nucleosome) in 2010 and, with Dr. McGinty, the first chromatin enzyme-nucleosome crystal structure (PRC1-nucleosome) in 2014.
Acknowledgment
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This work was supported by NIGMS Grants GM060489-09S1, GM088236, and GM111651 to S.T. R.K.M. is supported by a Damon Runyon Postdoctoral fellowship (DRG 2107-12).We would like to thank Tim Richmond for providing the coordinates for his two-start model of the 30 nm fiber; Phil Robinson and Daniela Rhodes for providing coordinates for their one-start model of the 30 nm fiber; and Guohong Li and Ping Zhu for providing coordinates for their two-start 30 nm fiber model.
| NCP | nucleosome core particle |
| LANA | latency-associated nuclear antigen |
| RCC1 | regulator of chromosome condensation 1 |
| Sir3 | silent information regulator 3 |
| CENP-C | centromeric protein C |
| PRC1 | Polycomb repressive complex 1 |
| cryo-EM | cryogenic electron microscopy |
| SHL | superhelical location |
| KSHV | Kaposi’s sarcoma-associated herpesvirus |
| BAH | bromo-adjacent homology |
| CENP-A | centromeric protein A |
| HMGN2 | high-mobility group nucleosome binding 2 |
| NMR | nuclear magnetic resonance |
| methyl-TROSY | methyl transverse relaxation optimized spectroscopy |
References
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This article references 164 other publications.
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- 9Luger, K.; Mäder, A. W.; Richmond, R. K.; Sargent, D. F.; Richmond, T. J. Nature 1997, 389, 251Google Scholar9Crystal structure of the nucleosome core particle at 2.8 Å resolutionLuger, Karolin; Mader, Armin W.; Richmond, Robin K.; Sargent, David F.; Richmond, Timothy J.Nature (London) (1997), 389 (6648), 251-260CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)The x-ray crystal structure of the nucleosome core particle of chromatin shows in at. detail how the histone protein octamer is assembled and how 146 base pairs of DNA are organized into a superhelix around it. Both histone/histone and histone/DNA interactions depend on the histone fold domains and addnl., well ordered structure elements extending from this motif. Histone amino-terminal tails pass over and between the gyres of the DNA superhelix to contact neighboring particles. The lack of uniformity between multiple histone/DNA-binding sites causes the DNA to deviate from ideal superhelix geometry.
- 10Lowary, P. T.; Widom, J. J. Mol. Biol. 1998, 276, 19Google Scholar10New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioningLowary, P. T.; Widom, J.Journal of Molecular Biology (1998), 276 (1), 19-42CODEN: JMOBAK; ISSN:0022-2836. (Academic Press Ltd.)DNA sequences that position nucleosomes are of increasing interest because of their relationship to gene regulation in vivo and because of their utility in studies of nucleosome structure and function in vitro. However, at present our understanding of the rules for DNA sequence-directed nucleosome positioning is fragmentary, and existing positioning sequences have many limitations. We carried out a SELEX expt. starting with a large pool of chem. synthetic random DNA mols. to identify those individuals having the highest affinity for histone octamer. A set of highest-affinity mols. were selected, cloned, and sequenced, their affinities (free energies) for histone octamer in nucleosome reconstitution measured, and their ability to position nucleosomes in vitro assessed by native gel electrophoresis. The selected sequences have higher affinity than previously known natural or non-natural sequences, and have a correspondingly strong nucleosome positioning ability. A variety of analyses including Fourier transform, real-space correlation, and direct counting computations were carried out to assess non-random features in the selected sequences. The results reveal sequence rules that were already identified in earlier studies of natural nucleosomal DNA, together with a large set of new rules having even stronger statistical significance. Possible phys. origins of the selected mols.' high affinities are discussed. The sequences isolated in this study should prove valuable for studies of chromatin structure and function in vitro and, potentially, for studies in vivo.
- 11Song, F.; Chen, P.; Sun, D.; Wang, M.; Dong, L.; Liang, D.; Xu, R.-M.; Zhu, P.; Li, G. Science 2014, 344, 376Google Scholar11Cryo-EM Study of the Chromatin Fiber Reveals a Double Helix Twisted by Tetranucleosomal UnitsSong, Feng; Chen, Ping; Sun, Dapeng; Wang, Mingzhu; Dong, Liping; Liang, Dan; Xu, Rui-Ming; Zhu, Ping; Li, GuohongScience (Washington, DC, United States) (2014), 344 (6182), 376-380CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The hierarchical packaging of eukaryotic chromatin plays a central role in transcriptional regulation and other DNA-related biol. processes. Here, we report the 11-angstrom-resoln. cryogenic electron microscopy (cryo-EM) structures of 30-nm chromatin fibers reconstituted in the presence of linker histone H1 and with different nucleosome repeat lengths. The structures show a histone H1-dependent left-handed twist of the repeating tetranucleosomal structural units, within which the four nucleosomes zigzag back and forth with a straight linker DNA. The asym. binding and the location of histone H1 in chromatin play a role in the formation of the 30-nm fiber. Our results provide mechanistic insights into how nucleosomes compact into higher-order chromatin fibers.
- 12Robinson, P. J. J.; Fairall, L.; Huynh, V. A. T.; Rhodes, D. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 6506Google Scholar12EM measurements define the dimensions of the "30-nm" chromatin fiber: evidence for a compact, interdigitated structureRobinson, Philip J. J.; Fairall, Louise; Huynh, Van A. T.; Rhodes, DanielaProceedings of the National Academy of Sciences of the United States of America (2006), 103 (17), 6506-6511CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Chromatin structure plays a fundamental role in the regulation of nuclear processes such as DNA transcription, replication, recombination, and repair. Despite considerable efforts during three decades, the structure of the 30-nm chromatin fiber remains controversial. To define fiber dimensions accurately, we have produced very long and regularly folded 30-nm fibers from in vitro reconstituted nucleosome arrays contg. the linker histone and with increasing nucleosome repeat lengths (10 to 70 bp of linker DNA). EM measurements show that the dimensions of these fully folded fibers do not increase linearly with increasing linker length, a finding that is inconsistent with two-start helix models. Instead, we find that there are two distinct classes of fiber structure, both with unexpectedly high nucleosome d.: arrays with 10 to 40 bp of linker DNA all produce fibers with a diam. of 33 nm and 11 nucleosomes per 11 nm, whereas arrays with 50 to 70 bp of linker DNA all produce 44-nm-wide fibers with 15 nucleosomes per 11 nm. Using the phys. constraints imposed by these measurements, we have built a model in which tight nucleosome packing is achieved through the interdigitation of nucleosomes from adjacent helical gyres. Importantly, the model closely matches raw image projections of folded chromatin arrays recorded in the soln. state by using electron cryo-microscopy.
- 13Schalch, T.; Duda, S.; Sargent, D. F.; Richmond, T. J. Nature 2005, 436, 138Google Scholar13X-ray structure of a tetranucleosome and its implications for the chromatin fiberSchalch, Thomas; Duda, Sylwia; Sargent, David F.; Richmond, Timothy J.Nature (London, United Kingdom) (2005), 436 (7047), 138-141CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)DNA in eukaryotic chromosomes is organized in arrays of nucleosomes compacted into chromatin fibers. This higher-order structure of nucleosomes is the substrate for DNA replication, recombination, transcription and repair. Although the structure of the nucleosome core is known at near-at. resoln., even the most fundamental information about the organization of nucleosomes in the fiber is controversial. Here we report the crystal structure of an oligonucleosome (a compact tetranucleosome) at 9 Å resoln., solved by mol. replacement using the nucleosome core structure. The structure shows that linker DNA zigzags back and forth between two stacks of nucleosome cores, which form a truncated two-start helix, and does not follow a path compatible with a one-start solenoidal helix. The length of linker DNA is most probably buffered by stretching of the DNA contained in the nucleosome cores. We have built continuous fiber models by successively stacking tetranucleosomes one on another. The resulting models are nearly fully compacted and most closely resemble the previously described crossed-linker model. They suggest that the interfaces between nucleosomes along a single helix start are polymorphic.
- 14Flaus, A.; Luger, K.; Tan, S.; Richmond, T. J. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 1370Google Scholar14Mapping nucleosome position at single base-pair resolution by using site-directed hydroxyl radicalsFlaus A; Luger K; Tan S; Richmond T JProceedings of the National Academy of Sciences of the United States of America (1996), 93 (4), 1370-5 ISSN:0027-8424.A base-pair resolution method for determining nucleosome position in vitro has been developed to com- plement existing, less accurate methods. Cysteaminyl EDTA was tethered to a recombinant histone octamer via a mutant histone H4 with serine 47 replaced by cysteine. When assembled into nucleosome core particles, the DNA could be cut site specifically by hydroxyl radical-catalyzed chain scission by using the Fenton reaction. Strand cleavage occurs mainly at a single nucleotide close to the dyad axis of the core particle, and assignment of this location via the symmetry of the nucleosome allows base-pair resolution mapping of the histone octamer position on the DNA. The positions of the histone octamer and H3H4 tetramer were mapped on a 146-bp Lytechinus variegatus 5S rRNA sequence and a twofold-symmetric derivative. The weakness of translational determinants of nucleosome positioning relative to the overall affinity of the histone proteins for this DNA is clearly demonstrated. The predominant location of both histone octamer and H3H4 tetramer assembled on the 5S rDNA is off center. Shifting the nucleosome core particle position along DNA within a conserved rotational phase could be induced under physiologically relevant conditions. Since nucleosome shifting has important consequences for chromatin structure and gene regulation, an approach to the thermodynamic characterization of this movement is proposed. This mapping method is potentially adaptable for determining nucleosome position in chromatin in vivo.
- 15Arents, G.; Burlingame, R. W.; Wang, B. C.; Love, W. E.; Moudrianakis, E. N. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 10148Google Scholar15The nucleosomal core histone octamer at 3.1 Å resolution: a tripartite protein assembly and a left-handed superhelixArents, Gina; Burlingame, Rufus W.; Wang, Bi Cheng; Love, Warner E.; Moudrianakis, Evangelos N.Proceedings of the National Academy of Sciences of the United States of America (1991), 88 (22), 10148-52CODEN: PNASA6; ISSN:0027-8424.The structure of the octameric histone core of the nucleosome has been detd. by x-ray crystallog. to a resoln. of 3.1 Å. The histone octamer is a tripartite assembly in which a centrally located (H3-H4)2 tetramer is flanked by 2 H2A-H2B dimers. It has a complex outer surface; depending on the perspective, the structure appears as a wedge or as a flat disk. The disk represents the planar projection of a left-handed proteinaceous superhelix with ≈28 Å pitch. The diam. of the particle is 65 Å and the length is 60 Å at its max. and ≈10 Å at its min. extension; these dimensions are in agreement with those reported earlier by A. Klug et al. (1980). The folded histone chains are elongated rather than globular and are assembled in a characteristic handshake motif. The individual polypeptides share a common central structural element of the helix-loop-helix type, which is named the histone fold.
- 16Arents, G.; Moudrianakis, E. N. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 11170Google Scholar16The histone fold: a ubiquitous architectural motif utilized in DNA compaction and protein dimerizationArents, Gina; Moudrianakis, Evangelos N.Proceedings of the National Academy of Sciences of the United States of America (1995), 92 (24), 11170-4CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The histones of all eukaryotes show only a low degree of primary structure homol., but our earlier crystallog. results defined a three-dimensional structural motif, the histone fold, common to all core histones. We now examine the specific architectural patterns within the fold and analyze the nature of the amino acid residues within its functional segments. The histone fold emerges as a fundamental protein dimerization motif while the differentiations of the tips of the histone dimers appear to provide the rules of core octamer assembly and the basis for nucleosome regulation. We present evidence for the occurrence of the fold from archaebacteria to mammals and propose the use of this structural motif to define a distinct family of proteins, the histone fold superfamily. It appears that evolution has conserved the conformation of the fold even through variations in primary structure and among proteins with various functional roles.
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- 19Preez, du L. L.; Patterton, H.-G. Subcell. Biochem. 2013, 61, 37Google ScholarThere is no corresponding record for this reference.
- 20Zheng, C.; Hayes, J. J. J. Biol. Chem. 2003, 278, 24217Google Scholar20Intra- and Inter-nucleosomal Protein-DNA Interactions of the Core Histone Tail Domains in a Model SystemZheng, Chunyang; Hayes, Jeffrey J.Journal of Biological Chemistry (2003), 278 (26), 24217-24224CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)The core histone tail domains are key regulators of eukaryotic chromatin structure and function, and alterations in the tail-directed folding of chromatin fibers and higher order structures are the probable outcome of much of the post-translational modifications occurring in these domains. The functions of the tail domains are likely to involve complex intra- and inter-nucleosomal histone-DNA interactions, yet little is known about either the structures or interactions of these domains. Here the authors introduce a method for examg. inter-nucleosome interactions of the tail domains in a model dinucleosome and det. the propensity of each of the four N-terminal tail domains to mediate such interactions in this system. Using a strong nucleosome "positioning" sequence, the authors reconstituted a nucleosome contg. a single histone site specifically modified with a photoinducible crosslinker within the histone tail domain, and a second nucleosome contg. a radiolabeled DNA template. These two nucleosomes were then ligated together and crosslinking induced by brief UV irradn. under various soln. conditions. After crosslinking, the two templates were again sepd. so that crosslinking representing inter-nucleosomal histone-DNA interactions could be unambiguously distinguished from intra-nucleosomal crosslinks. The results show that the N-terminal tails of H2A and H2B, but not of H3 and H4, make internucleosomal histone-DNA interactions within the dinucleosome. The relative extent of intra- to inter-nucleosome interactions was not strongly dependent on ionic strength. Addnl., the authors find that binding of a linker histone to the dinucleosome increased the assocn. of the H3 and H4 tails with the linker DNA region.
- 21Kan, P. Y.; Lu, X.; Hansen, J. C.; Hayes, J. J. Mol. Cell. Biol. 2007, 27, 2084Google Scholar21The H3 tail domain participates in multiple interactions during folding and self-association of nucleosome arraysKan, Pu-Yeh; Lu, Xu; Hansen, Jeffrey C.; Hayes, Jeffrey J.Molecular and Cellular Biology (2007), 27 (6), 2084-2091CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)The core histone tail domains play a central role in chromatin structure and epigenetic processes controlling gene expression. Although little is known regarding the mol. details of tail interactions, it is likely that they participate in both short-range and long-range interactions between nucleosomes. Previously, we demonstrated that the H3 tail domain participates in internucleosome interactions during MgCl2-dependent condensation of model nucleosome arrays. However, these studies did not distinguish whether these internucleosome interactions represented short-range intra-array or longer-range interarray interactions. To better understand the complex interactions of the H3 tail domain during chromatin condensation, we have developed a new site-directed crosslinking method to identify and quantify interarray interactions mediated by histone tail domains. Interarray crosslinking was undetectable under salt conditions that induced only local folding, but was detected concomitant with salt-dependent interarray oligomerization at higher MgCl2 concns. Interestingly, lysine-to-glutamine mutations in the H3 tail domain to mimic acetylation resulted in little or no redn. in interarray crosslinking. In contrast, binding of a linker histone caused a much greater enhancement of interarray interactions for unmodified H3 tails compared to "acetylated" H3 tails. Collectively, these results indicate that H3 tail domain performs multiple functions during chromatin condensation via distinct mol. interactions that can be differentially regulated by acetylation or binding of linker histones.
- 22Kan, P. Y.; Caterino, T. L.; Hayes, J. J. Mol. Cell. Biol. 2009, 29, 538Google Scholar22The H4 tail domain participates in intra- and internucleosome interactions with protein and DNA during folding and oligomerization of nucleosome arraysKan, Pu-Yeh; Caterino, Tamara L.; Hayes, Jeffrey J.Molecular and Cellular Biology (2009), 29 (2), 538-546CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)The condensation of nucleosome arrays into higher-order secondary and tertiary chromatin structures likely involves long-range internucleosomal interactions mediated by the core histone tail domains. We have characterized interarray interactions mediated by the H4 tail domain, known to play a predominant role in the formation of such structures. We find that the N-terminal end of the H4 tail mediates interarray contacts with DNA during self-assocn. of oligonucleosome arrays similar to that found previously for the H3 tail domain. However, a site near the histone fold domain of H4 participates in a distinct set of interactions, contacting both DNA and H2A in condensed structures. Moreover, we also find that H4-H2A interactions occur via an intra- as well as an internucleosomal fashion, supporting an addnl. intranucleosomal function for the tail. Interestingly, acetylation of the H4 tail has little effect on interarray interactions by itself but overrides the strong stimulation of interarray interactions induced by linker histones. Our results indicate that the H4 tail facilitates secondary and tertiary chromatin structure formation via a complex array of potentially exclusive interactions that are distinct from those of the H3 tail domain.
- 23White, C. L.; Suto, R. K.; Luger, K. EMBO J. 2001, 20, 5207Google Scholar23Structure of the yeast nucleosome core particle reveals fundamental changes in internucleosome interactionsWhite, Cindy L.; Suto, Robert K.; Luger, KarolinEMBO Journal (2001), 20 (18), 5207-5218CODEN: EMJODG; ISSN:0261-4189. (Oxford University Press)Chromatin is composed of nucleosomes, the universally repeating protein-DNA complex in eukaryotic cells. The crystal structure of the nucleosome core particle from Saccharomyces cerevisiae reveals that the structure and function of this fundamental complex is conserved between single-cell organisms and metazoans. Our results show that yeast nucleosomes are likely to be subtly destabilized as compared with nucleosomes from higher eukaryotes, consistent with the idea that much of the yeast genome remains constitutively open during much of its life cycle. Importantly, minor sequence variations lead to dramatic changes in the way in which nucleosomes pack against each other within the crystal lattice. This has important implications for our understanding of the formation of higher-order chromatin structure and its modulation by post-translational modifications. Finally, the yeast nucleosome core particle provides a structural context by which to interpret genetic data obtained from yeast. Coordinates have been deposited with the Protein Data Bank under accession no. 1ID3.
- 24Clapier, C. R.; Chakravarthy, S.; Petosa, C.; Fernández-Tornero, C.; Luger, K.; Müller, C. W. Proteins 2008, 71, 1Google Scholar24Structure of the Drosophila nucleosome core particle highlights evolutionary constraints on the H2A-H2B histone dimerClapier, Cedric R.; Chakravarthy, Srinivas; Petosa, Carlo; Fernandez-Tornero, Carlos; Luger, Karolin; Muller, Christoph W.Proteins: Structure, Function, and Bioinformatics (2008), 71 (1), 1-7CODEN: PSFBAF ISSN:. (Wiley-Liss, Inc.)We detd. the 2.45 Å crystal structure of the nucleosome core particle from Drosophila melanogaster and compared it to that of Xenopus laevis bound to the identical 147 base-pair DNA fragment derived from human α-satellite DNA. Differences between the two structures primarily reflect 16 amino acid substitutions between species, 15 of which are in histones H2A and H2B. Four of these involve histone tail residues, resulting in subtly altered protein-DNA interactions that exemplify the structural plasticity of these tails. Of the 12 substitutions occurring within the histone core regions, five involve small, solvent-exposed residues not involved in intraparticle interactions. The remaining seven involve buried hydrophobic residues, and appear to have coevolved so as to preserve the vol. of side chains within the H2A hydrophobic core and H2A-H2B dimer interface. Thus, apart from variations in the histone tails, amino acid substitutions that differentiate Drosophila from Xenopus histones occur in mutually compensatory combinations. This highlights the tight evolutionary constraints exerted on histones since the vertebrate and invertebrate lineages diverged.
- 25Tsunaka, Y.; Kajimura, N.; Tate, S.-I.; Morikawa, K. Nucleic Acids Res. 2005, 33, 3424Google Scholar25Alteration of the nucleosomal DNA path in the crystal structure of a human nucleosome core particleTsunaka, Yasuo; Kajimura, Naoko; Tate, Shin-ichi; Morikawa, KosukeNucleic Acids Research (2005), 33 (10), 3424-3434CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Gene expression in eukaryotes depends upon positioning, mobility and packaging of nucleosomes; thus, we need the detailed information of the human nucleosome core particle (NCP) structure, which could clarify chromatin properties. Here, we report the 2.5 Å crystal structure of a human NCP. The overall structure is similar to those of other NCPs reported previously. However, the DNA path of human NCP is remarkably different from that taken within other NCPs with an identical DNA sequence. A comparison of the structural parameters between human and Xenopus laevis DNA reveals that the DNA path of human NCP consecutively shifts by 1 bp in the regions of superhelix axis location -5.0 to -2.0 and 5.0 to 7.0. This alteration of the human DNA path is caused predominantly by tight DNA-DNA contacts within the crystal. It is also likely that the conformational change in the human H2B tail induces the local alteration of the DNA path. In human NCP, the region with the altered DNA path lacks Mn2+ ions and the B-factors of the DNA phosphate groups are substantially high. Therefore, in contrast to the histone octamer, the nucleosomal DNA is sufficiently flexible and mobile and can undergo drastic conformational changes, depending upon the environment.
- 26Sugiyama, M.; Arimura, Y.; Shirayama, K.; Fujita, R.; Oba, Y.; Sato, N.; Inoue, R.; Oda, T.; Sato, M.; Heenan, R. K.; Kurumizaka, H. Biophys. J. 2014, 106, 2206Google Scholar26Distinct features of the histone core structure in nucleosomes containing the histone H2A.B variantSugiyama, Masaaki; Arimura, Yasuhiro; Shirayama, Kazuyoshi; Fujita, Risa; Oba, Yojiro; Sato, Nobuhiro; Inoue, Rintaro; Oda, Takashi; Sato, Mamoru; Heenan, Richard K.; Kurumizaka, HitoshiBiophysical Journal (2014), 106 (10), 2206-2213CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)Nucleosomes contg. a human histone variant, H2A.B, were analyzed in aq. soln. by SANS utilizing a contrast variation technique. Comparisons with the canonical H2A nucleosome structure revealed that the DNA termini of the H2A.B nucleosome were detached from the histone core surface, and flexibly expanded toward the solvent. In contrast, the histone tails were compacted in H2A.B nucleosomes compared to those in canonical H2A nucleosomes, suggesting that they bind to the surface of the histone core and/or DNA. Thus, the histone tail dynamics may function to regulate the flexibility of the DNA termini in the nucleosomes.
- 27Urahama, T.; Horikoshi, N.; Osakabe, A.; Tachiwana, H.; Kurumizaka, H. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2014, 70, 444Google Scholar27Structure of human nucleosome containing the testis-specific histone variant TSH2BUrahama, Takashi; Horikoshi, Naoki; Osakabe, Akihisa; Tachiwana, Hiroaki; Kurumizaka, HitoshiActa Crystallographica, Section F: Structural Biology Communications (2014), 70 (4), 444-449CODEN: ACSFEN; ISSN:2053-230X. (International Union of Crystallography)The human histone H2B variant TSH2B is highly expressed in testis and may function in the chromatin transition during spermatogenesis. In the present study, the crystal structure of the human testis-specific nucleosome contg. TSH2B was detd. at 2.8 Å resoln. A local structural difference between TSH2B and canonical H2B in nucleosomes was detected around the TSH2B-specific amino-acid residue Ser85. The TSH2B Ser85 residue does not interact with H4 in the nucleosome, but in the canonical nucleosome the H2B Asn84 residue (corresponding to the TSH2B Ser85 residue) forms water-mediated hydrogen bonds with the H4 Arg78 residue. In contrast, the other TSH2B-specific amino-acid residues did not induce any significant local structural changes in the TSH2B nucleosome. These findings may provide important information for understanding how testis-specific histone variants form nucleosomes during spermatogenesis.
- 28Tachiwana, H.; Kagawa, W.; Shiga, T.; Osakabe, A.; Miya, Y.; Saito, K.; Hayashi-Takanaka, Y.; Oda, T.; Sato, M.; Park, S.-Y.; Kimura, H.; Kurumizaka, H. Nature 2011, 476, 232Google Scholar28Crystal structure of the human centromeric nucleosome containing CENP-ATachiwana, Hiroaki; Kagawa, Wataru; Shiga, Tatsuya; Osakabe, Akihisa; Miya, Yuta; Saito, Kengo; Hayashi-Takanaka, Yoko; Oda, Takashi; Sato, Mamoru; Park, Sam-Yong; Kimura, Hiroshi; Kurumizaka, HitoshiNature (London, United Kingdom) (2011), 476 (7359), 232-235CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)In eukaryotes, accurate chromosome segregation during mitosis and meiosis is coordinated by kinetochores, which are unique chromosomal sites for microtubule attachment. Centromeres specify the kinetochore formation sites on individual chromosomes, and are epigenetically marked by the assembly of nucleosomes contg. the centromere-specific histone H3 variant, CENP-A. Although the underlying mechanism is unclear, centromere inheritance is probably dictated by the architecture of the centromeric nucleosome. Here we report the crystal structure of the human centromeric nucleosome contg. CENP-A and its cognate α-satellite DNA deriv. (147 base pairs). In the human CENP-A nucleosome, the DNA is wrapped around the histone octamer, consisting of two each of histones H2A, H2B, H4 and CENP-A, in a left-handed orientation. However, unlike the canonical H3 nucleosome, only the central 121 base pairs of the DNA are visible. The thirteen base pairs from both ends of the DNA are invisible in the crystal structure, and the αN helix of CENP-A is shorter than that of H3, which is known to be important for the orientation of the DNA ends in the canonical H3 nucleosome. A structural comparison of the CENP-A and H3 nucleosomes revealed that CENP-A contains two extra amino acid residues (Arg 80 and Gly 81) in the loop 1 region, which is completely exposed to the solvent. Mutations of the CENP-A loop 1 residues reduced CENP-A retention at the centromeres in human cells. Therefore, the CENP-A loop 1 may function in stabilizing the centromeric chromatin contg. CENP-A, possibly by providing a binding site for trans-acting factors. The structure provides the first at.-resoln. picture of the centromere-specific nucleosome.
- 29Tachiwana, H.; Osakabe, A.; Shiga, T.; Miya, Y.; Kimura, H.; Kagawa, W.; Kurumizaka, H. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 578Google Scholar29Structures of human nucleosomes containing major histone H3 variantsTachiwana, Hiroaki; Osakabe, Akihisa; Shiga, Tatsuya; Miya, Yuta; Kimura, Hiroshi; Kagawa, Wataru; Kurumizaka, HitoshiActa Crystallographica, Section D: Biological Crystallography (2011), 67 (6), 578-583CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)The nucleosome is the fundamental repeating unit of chromatin, via which genomic DNA is packaged into the nucleus in eukaryotes. In the nucleosome, 2 copies of each core histone (H2A, H2B, H3, and H4) form a histone octamer which wraps 146 base pairs of DNA around itself. All of the core histones except for histone H4 have nonallelic isoforms called histone variants. In humans, 8 histone H3 variants (H3.1, H3.2, H3.3, H3T, H3.5, H3.X, H3.Y, and CENP-A) have been reported to date. Previous studies have suggested that histone H3 variants possess distinct functions in the formation of specific chromosome regions and/or in the regulation of transcription and replication. Histones H3.1, H3.2, and H3.3 are the most abundant H3 variants. Here, crystal structures of human HeLa cell nucleosomes contg. either H3.2 or H3.3 were solved. The structures were essentially the same as that of the H3.1 nucleosome. Since the amino acid residues specific for H3.2 and H3.3 are located on the accessible surface of the H3/H4 tetramer, they may be potential interaction sites for H3.2- and H3.3-specific chaperones.
- 30Tachiwana, H.; Kagawa, W.; Osakabe, A.; Kawaguchi, K.; Shiga, T.; Hayashi-Takanaka, Y.; Kimura, H.; Kurumizaka, H. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 10454Google Scholar30Structural basis of instability of the nucleosome containing a testis-specific histone variant, human H3TTachiwana, Hiroaki; Kagawa, Wataru; Osakabe, Akihisa; Kawaguchi, Koichiro; Shiga, Tatsuya; Hayashi-Takanaka, Yoko; Kimura, Hiroshi; Kurumizaka, HitoshiProceedings of the National Academy of Sciences of the United States of America (2010), 107 (23), 10454-10459, S10454/1-S10454/5CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A histone H3 variant, H3T, is highly expressed in the testis, suggesting that it may play an important role in the chromatin reorganization required for. meiosis and/or spermatogenesis. In the present study, the authors found that the nucleosome contg. human H3T is significantly unstable both in vitro and in vivo, as compared to the conventional nucleosome contg. H3.1. The crystal structure of the H3T nucleosome revealed structural differences in the H3T regions on both ends of the central αa2 helix, as compared to those of H3.1. The H3T-specific residues (Met71 and Val111) are the source of the structural differences obsd. between H3T and H3.1. A mutational anal. revealed that these residues are responsible for the reduced stability of the H3T-contg. nucleosome. These phys. and structural properties of the H3T-contg. nucleosome may provide the basis of chromatin reorganization during spermatogenesis.
- 31Arimura, Y.; Kimura, H.; Oda, T.; Sato, K.; Osakabe, A.; Tachiwana, H.; Sato, Y.; Kinugasa, Y.; Ikura, T.; Sugiyama, M.; Sato, M.; Kurumizaka, H. Sci. Rep. 2013, 3, 3510Google Scholar31Structural basis of a nucleosome containing histone H2A.B/H2A.Bbd that transiently associates with reorganized chromatinArimura Yasuhiro; Sato Koichi; Osakabe Akihisa; Tachiwana Hiroaki; Kurumizaka Hitoshi; Kimura Hiroshi; Sato Yuko; Oda Takashi; Kinugasa Yasuha; Ikura Tsuyoshi; Sugiyama Masaaki; Sato MamoruScientific reports (2013), 3 (), 3510 ISSN:.Human histone H2A.B (formerly H2A.Bbd), a non-allelic H2A variant, exchanges rapidly as compared to canonical H2A, and preferentially associates with actively transcribed genes. We found that H2A.B transiently accumulated at DNA replication and repair foci in living cells. To explore the biochemical function of H2A.B, we performed nucleosome reconstitution analyses using various lengths of DNA. Two types of H2A.B nucleosomes, octasome and hexasome, were formed with 116, 124, or 130 base pairs (bp) of DNA, and only the octasome was formed with 136 or 146 bp DNA. In contrast, only hexasome formation was observed by canonical H2A with 116 or 124 bp DNA. A small-angle X-ray scattering analysis revealed that the H2A.B octasome is more extended, due to the flexible detachment of the DNA regions at the entry/exit sites from the histone surface. These results suggested that H2A.B rapidly and transiently forms nucleosomes with short DNA segments during chromatin reorganization.
- 32Chakravarthy, S.; Bao, Y.; Roberts, V. A.; Tremethick, D.; Luger, K. Cold Spring Harbor Symp. Quant. Biol. 2004, 69, 227Google Scholar32Structural characterization of histone H2A variantsChakravarthy, S.; Bao, Y.; Roberts, V. A.; Tremethick, D.; Luger, K.Cold Spring Harbor Symposia on Quantitative Biology (2004), 69 (), 227-234CODEN: CSHSAZ; ISSN:0091-7451. (Cold Spring Harbor Laboratory Press)A review on available structural information on nucleosomes and chromatin contg. histone H2A variants and how structure relates to their varied function. A hypothesis why "true" histone variants have been identified for only histone H2A and H3 is presented, and data in support of this hypothesis that particular regions in the H2A amino acid sequence appeared to have been targets during the evolution of H2A histone variants is shown.
- 33Chakravarthy, S.; Gundimella, S. K. Y.; Caron, C.; Perche, P.-Y.; Pehrson, J. R.; Khochbin, S.; Luger, K. Mol. Cell. Biol. 2005, 25, 7616Google Scholar33Structural characterization of the histone variant macroH2AChakravarthy, Srinivas; Gundimella, Sampath Kumar Y.; Caron, Cecile; Perche, Pierre-Yves; Pehrson, John R.; Khochbin, Saadi; Luger, KarolinMolecular and Cellular Biology (2005), 25 (17), 7616-7624CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)MacroH2A is an H2A variant with a highly unusual structural organization. It has a C-terminal domain connected to the N-terminal histone domain by a linker. Crystallog. and biochem. studies show that changes in the L1 loop in the histone fold region of macroH2A impact the structure and potentially the function of nucleosomes. The 1.6-A x-ray structure of the nonhistone region reveals an α/β fold which has previously been found in a functionally diverse group of proteins. This region assocs. with histone deacetylases and affects the acetylation status of nucleosomes contg. macroH2A. Thus, the unusual domain structure of macroH2A integrates independent functions that are instrumental in establishing a structurally and functionally unique chromatin domain.
- 34Chakravarthy, S.; Luger, K. J. Biol. Chem. 2006, 281, 25522Google Scholar34The hstone variant macro-H2A preferentially forms "hybrid nucleosomes"Chakravarthy, Srinivas; Luger, KarolinJournal of Biological Chemistry (2006), 281 (35), 25522-25531CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)The histone domain of macro-H2A, which constitutes the N-terminal one-third of this histone variant, is only 64% identical to major H2A. We have shown previously that the main structural differences in a nucleosome in which both H2A moieties have been replaced by macro-H2A reside in the only point of contact between the two histone dimers, the L1-L1 interface of macro-H2A. Here we show that the L1 loop of macro-H2A is responsible for the increased salt-dependent stability of the histone octamer, with implications for the nucleosome assembly pathway. It is unknown whether only one or both of the H2A-H2B dimers within a nucleosome are replaced with H2A variant contg. nucleosomes in vivo. We demonstrate that macro-H2A preferentially forms hybrid nucleosomes contg. one chain each of major H2A and macro-HA in vitro. The 2.9-Å crystal structure of such a hybrid nucleosome shows significant structural differences in the L1-L1 interface when comparing with homotypic major H2A- and macro-H2A-contg. nucleosomes. Both homotypic and hybrid macro-nucleosome core particles (NCPs) are resistant to chaperone-assisted H2A-H2B dimer exchange. Together, our findings suggest that the histone domain of macro-H2A modifies the dynamic properties of the nucleosome. We propose that the possibility of forming hybrid macro-NCP adds yet another level of complexity to variant nucleosome structure and function.
- 35Abbott, D. W.; Laszczak, M.; Lewis, J. D.; Su, H.; Moore, S. C.; Hills, M.; Dimitrov, S.; Ausio, J. Biochemistry 2004, 43, 1352Google Scholar35Structural Characterization of MacroH2A Containing ChromatinAbbott, D. Wade; Laszczak, Mario; Lewis, John D.; Su, Harvey; Moore, Susan C.; Hills, Melissa; Dimitrov, Stefan; Ausio, JuanBiochemistry (2004), 43 (5), 1352-1359CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)MacroH2A (mH2A) is one of the most recently identified members of the heteromorphous histone variant family. It is unique among the members of this group because it contains an unusually large non-histone C-terminal end, from where its name derives, and appears to be restricted to subphylum vertebrata. Although a concerted effort has been carried out to characterize the physiol. relevance of mH2A, little is known in comparison about the structural importance of the mol. Elucidating the biophys. and conformational properties of mH2A in chromatin may provide clues into the links between this histone variant and its unique function(s). In this paper, the authors look first at the heterogeneous tissue-specific distribution of this protein in different vertebrate classes. This is followed by a structural comparison between mH2A and H2A protein and by the characterization of the nucleosome core particles with which these histone subtypes are assocd. The authors find that the highly α-helical C-terminus of mH2A confers an asym. conformation to nucleosomes and that this variant is tightly bound to chromatin fragments in a way that does not depend on the overall extent of acetylation of the other core histones.
- 36Eirín-López, J. M.; Ishibashi, T.; Ausio, J. FASEB J. 2008, 22, 316Google Scholar36H2A.Bbd: a quickly evolving hypervariable mammalian histone that destabilizes nucleosomes in an acetylation-independent wayEirin-Lopez, Jose Maria; Ishibashi, Toyotaka; Ausio, JuanFASEB Journal (2008), 22 (1), 316-326, 10.1096/fj.07-9255comCODEN: FAJOEC; ISSN:0892-6638. (Federation of American Societies for Experimental Biology)Mol. evolutionary analyses revealed that histone H2A.Bbd is a highly variable quickly evolving mammalian replacement histone variant, in striking contrast to all other histones. At the nucleotide level, this variability appears to be the result of a larger amt. of nonsynonymous variation, which affects to a lesser extent, the structural domain of the protein comprising the histone fold. The resulting amino acid sequence diversity can be predicted to affect the internucleosomal and intranucleosomal histone interactions. Our phylogenetic anal. has allowed us to identify several of the residues involved. The biophys. characterization of nucleosomes reconstituted with recombinant mouse H2A.Bbd and their comparison to similar data obtained with human H2A.Bbd clearly support this notion. Despite the high interspecific amino acid sequence variability, all of the H2A.Bbd variants exert similar structural effects at the nucleosome level, which result in an unfolded highly unstable nucleoprotein complex. Such structure resembles that previously described for the highly dynamically acetylated nucleosomes assocd. with transcriptionally active regions of the genome. Nevertheless, the structure of nucleosome core particles reconstituted from H2A.Bbd is not affected by the presence of a hyperacetylated histone complement. This suggests that replacement by H2A.Bbd provides an alternative mechanism to unfold chromatin structure, possibly in euchromatic regions, in a way that is not dependent on acetylation.
- 37Horikoshi, N.; Sato, K.; Shimada, K.; Arimura, Y.; Osakabe, A.; Tachiwana, H.; Hayashi-Takanaka, Y.; Iwasaki, W.; Kagawa, W.; Harata, M.; Kimura, H.; Kurumizaka, H. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2013, 69, 2431Google Scholar37Structural polymorphism in the L1 loop regions of human H2A.Z.1 and H2A.Z.2Horikoshi, Naoki; Sato, Koichi; Shimada, Keisuke; Arimura, Yasuhiro; Osakabe, Akihisa; Tachiwana, Hiroaki; Hayashi-Takanaka, Yoko; Iwasaki, Wakana; Kagawa, Wataru; Harata, Masahiko; Kimura, Hiroshi; Kurumizaka, HitoshiActa Crystallographica, Section D: Biological Crystallography (2013), 69 (12), 2431-2439CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)The histone H2A.Z variant is widely conserved among eukaryotes. Two isoforms, H2A.Z.1 and H2A.Z.2, have been identified in vertebrates and may have distinct functions in cell growth and gene expression. However, no structural differences between H2A.Z.1 and H2A.Z.2 have been reported. In the present study, the crystal structures of nucleosomes contg. human H2A.Z.1 and H2A.Z.2 were detd. The structures of the L1 loop regions were found to clearly differ between H2A.Z.1 and H2A.Z.2, although their amino-acid sequences in this region are identical. This structural polymorphism may have been induced by a substitution that evolutionarily occurred at the position of amino acid 38 and by the flexible nature of the L1 loops of H2A.Z.1 and H2A.Z.2. It was also found that in living cells nucleosomal H2A.Z.1 exchanges more rapidly than H2A.Z.2. A mutational anal. revealed that the amino-acid difference at position 38 is at least partially responsible for the distinctive dynamics of H2A.Z.1 and H2A.Z.2. These findings provide important new information for understanding the differences in the regulation and functions of H2A.Z.1 and H2A.Z.2 in cells.
- 38Suto, R. K.; Clarkson, M. J.; Tremethick, D. J.; Luger, K. Nat. Struct. Biol. 2000, 7, 1121Google Scholar38Crystal structure of a nucleosome core particle containing the variant histone H2A.ZSuto, Robert K.; Clarkson, Michael J.; Tremethick, David J.; Luger, KarolinNature Structural Biology (2000), 7 (12), 1121-1124CODEN: NSBIEW; ISSN:1072-8368. (Nature America Inc.)Activation of transcription within chromatin has been correlated with the incorporation of the essential histone variant H2A.Z into nucleosomes. H2A.Z and other histone variants may establish structurally distinct chromosomal domains; however, the mol. mechanism by which they function is largely unknown. Here we report the 2.6 Å crystal structure of a nucleosome core particle contg. the histone variant H2A.Z. The overall structure is similar to that of the previously reported 2.8 Å nucleosome structure contg. major histone proteins. However, distinct localized changes result in the subtle destabilization of the interaction between the (H2A.Z-H2B) dimer and the (H3-H4)2 tetramer. Moreover, H2A.Z nucleosomes have an altered surface that includes a metal ion. This altered surface may lead to changes in higher order structure, and/or could result in the assocn. of specific nuclear proteins with H2A.Z. Finally, incorporation of H2A.Z and H2A within the same nucleosome is unlikely, due to significant changes in the interface between the two H2A.Z-H2B dimers.
- 39Dechassa, M. L.; Wyns, K.; Li, M.; Hall, M. A.; Wang, M. D.; Luger, K. Nat. Commun. 2011, 2, 313Google Scholar39Structure and Scm3-mediated assembly of budding yeast centromeric nucleosomesDechassa Mekonnen Lemma; Wyns Katharina; Li Ming; Hall Michael A; Wang Michelle D; Luger KarolinNature communications (2011), 2 (), 313 ISSN:.Much controversy exists regarding the structural organization of the yeast centromeric nucleosome and the role of the nonhistone protein, Scm3, in its assembly and architecture. Here we show that the substitution of H3 with its centromeric variant Cse4 results in octameric nucleosomes that organize DNA in a left-handed superhelix. We demonstrate by single-molecule approaches, micrococcal nuclease digestion and small-angle X-ray scattering that Cse4-nucleosomes exhibit an open conformation with weakly bound terminal DNA segments. The Cse4-octamer does not preferentially form nucleosomes on its cognate centromeric DNA. We show that Scm3 functions as a Cse4-specific nucleosome assembly factor, and that the resulting octameric nucleosomes do not contain Scm3 as a stably bound component. Taken together, our data provide insights into the assembly and structural features of the budding yeast centromeric nucleosome.
- 40Conde e Silva, N.; Black, B. E.; Sivolob, A.; Filipski, J.; Cleveland, D. W.; Prunell, A. J. Mol. Biol. 2007, 370, 555Google Scholar40CENP-A-containing Nucleosomes: Easier Disassembly versus Exclusive Centromeric LocalizationConde e Silva, Natalia; Black, Ben E.; Sivolob, Andrei; Filipski, Jan; Cleveland, Don W.; Prunell, ArielJournal of Molecular Biology (2007), 370 (3), 555-573CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)CENP-A is a histone variant that replaces conventional H3 in nucleosomes of functional centromeres. We report here, from reconstitutions of CENP-A- and H3-contg. nucleosomes on linear DNA fragments and the comparison of their electrophoretic mobility, that CENP-A induces some positioning of its own and some unwrapping at the entry-exit relative to canonical nucleosomes on both 5 S DNA and the α-satellite sequence on which it is normally loaded. This steady-state unwrapping was quantified to 7(±2) bp by nucleosome reconstitutions on a series of DNA minicircles, followed by their relaxation with topoisomerase I. The unwrapping was found to ease nucleosome invasion by exonuclease III, to hinder the binding of a linker histone, and to promote the release of an H2A-H2B dimer by nucleosome assembly protein 1 (NAP-1). The (CENP-A-H4)2 tetramer was also more readily destabilized with heparin than the (H3-H4)2 tetramer, suggesting that CENP-A has evolved to confer its nucleosome a specific ability to disassemble. This dual relative instability is proposed to facilitate the progressive clearance of CENP-A nucleosomes that assemble promiscuously in euchromatin, esp. as is seen following CENP-A transient over-expression.
- 41Sekulic, N.; Bassett, E. A.; Rogers, D. J.; Black, B. E. Nature 2010, 467, 347Google Scholar41The structure of (CENP-AH4)2 reveals physical features that mark centromeresSekulic, Nikolina; Bassett, Emily A.; Rogers, Danielle J.; Black, Ben E.Nature (London, United Kingdom) (2010), 467 (7313), 347-351CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Centromeres are specified epigenetically, and the histone H3 variant CENP-A is assembled into the chromatin of all active centromeres. Divergence from H3 raises the possibility that CENP-A generates unique chromatin features to phys. mark centromere location. Here we report the crystal structure of a subnucleosomal heterotetramer, human (CENP-AH4)2, that reveals three distinguishing properties encoded by the residues that comprise the CENP-A targeting domain (CATD): (1) a CENP-ACENP-A interface that is substantially rotated relative to the H3H3 interface; (2) a protruding loop L1 of the opposite charge compared to that on H3; and (3) strong hydrophobic contacts that rigidify the CENP-AH4 interface. Residues involved in the CENP-ACENP-A rotation are required for efficient incorporation into centromeric chromatin, indicating specificity for an unconventional nucleosome shape. DNA topol. anal. indicates that CENP-A-contg. nucleosomes are octameric with conventional left-handed DNA wrapping, in contrast to other recent proposals. Our results indicate that CENP-A marks centromere location by restructuring the nucleosome from within its folded histone core.
- 42Kingston, I. J.; Yung, J. S. Y.; Singleton, M. R. J. Biol. Chem. 2011, 286, 4021Google Scholar42Biophysical Characterization of the Centromere-specific Nucleosome from Budding YeastKingston, Isabel J.; Yung, Jasmine S. Y.; Singleton, Martin R.Journal of Biological Chemistry (2011), 286 (5), 4021-4026CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)The centromeric DNA of all eukaryotes is assembled upon a specialized nucleosome contg. a histone H3 variant known as CenH3. Despite the importance and conserved nature of this protein, the characteristics of the centromeric nucleosome are still poorly understood. In particular, the stoichiometry and DNA-binding properties of the CenH3 nucleosome have been the subject of some debate. We have characterized the budding yeast centromeric nucleosome by biochem. and biophys. methods and show that it forms a stable octamer contg. two copies of the Cse4 protein and wraps DNA in a left-handed supercoil, similar to the canonical H3 nucleosome. The DNA-binding properties of the recombinant nucleosome are identical to those obsd. in vivo, demonstrating that the octameric structure is physiol. relevant.
- 43Panchenko, T.; Sorensen, T. C.; Woodcock, C. L.; Kan, Z.-Y.; Wood, S.; Resch, M. G.; Luger, K.; Englander, S. W.; Hansen, J. C.; Black, B. E. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 16588Google Scholar43Replacement of histone H3 with CENP-A directs global nucleosome array condensation and loosening of nucleosome superhelical terminiPanchenko, Tanya; Sorensen, Troy C.; Woodcock, Christopher L.; Kan, Zhong-yuan; Wood, Stacey; Resch, Michael G.; Luger, Karolin; Englander, S. Walter; Hansen, Jeffrey C.; Black, Ben E.Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (40), 16588-16593, S16588/1-S16588/7CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Centromere protein A (CENP-A) is a histone H3 variant that marks centromere location on the chromosome. To study the subunit structure and folding of human CENP-A-contg. chromatin, we generated a set of nucleosomal arrays with canonical core histones and another set with CENP-A substituted for H3. At the level of quaternary structure and assembly, we find that CENP-A arrays are composed of octameric nucleosomes that assemble in a stepwise mechanism, recapitulating conventional array assembly with canonical histones. At intermediate structural resoln., we find that CENP-A-contg. arrays are globally condensed relative to arrays with the canonical histones. At high structural resoln., using hydrogen-deuterium exchange coupled to mass spectrometry (H/DX-MS), we find that the DNA superhelical termini within each nucleosome are loosely connected to CENP-A, and we identify the key amino acid substitution that is largely responsible for this behavior. Also the C terminus of histone H2A undergoes rapid hydrogen exchange-relative to canonical arrays and does so in a manner that is independent of nucleosomal array folding. These findings have implications for understanding CENP-A-contg. nucleosome structure and higher-order chromatin folding at the centromere.
- 44Doyen, C.-M.; Montel, F.; Gautier, T.; Menoni, H.; Claudet, C.; Delacour-Larose, M.; Angelov, D.; Hamiche, A.; Bednar, J.; Faivre-Moskalenko, C.; Bouvet, P.; Dimitrov, S. EMBO J. 2006, 25, 4234Google Scholar44Dissection of the unusual structural and functional properties of the variant H2A.Bbd nucleosomeDoyen, Cecile-Marie; Montel, Fabien; Gautier, Thierry; Menoni, Herve; Claudet, Cyril; Delacour-Larose, Marlene; Angelov, Dimitri; Hamiche, Ali; Bednar, Jan; Faivre-Moskalenko, Cendrine; Bouvet, Philippe; Dimitrov, StefanEMBO Journal (2006), 25 (18), 4234-4244CODEN: EMJODG; ISSN:0261-4189. (Nature Publishing Group)The histone variant H2A.Bbd appeared to be assocd. with active chromatin, but how it functions is unknown. We have dissected the properties of nucleosome contg. H2A.Bbd. At. force microscopy (AFM) and electron cryo-microscopy (cryo-EM) showed that the H2A.Bbd histone octamer organizes only ∼130 bp of DNA, suggesting that 10 bp of each end of nucleosomal DNA are released from the octamer. In agreement with this, the entry/exit angle of the nucleosomal DNA ends formed an angle close to 180° and the physico-chem. anal. pointed to a lower stability of the variant particle. Reconstitution of nucleosomes with swapped-tail mutants demonstrated that the N-terminus of H2A.Bbd has no impact on the nucleosome properties. AFM, cryo-EM and chromatin remodeling expts. showed that the overall structure and stability of the particle, but not its property to interfere with the SWI/SNF induced remodeling, were detd. to a considerable extent by the H2A.Bbd docking domain. These data show that the whole H2A.Bbd histone fold domain is responsible for the unusual properties of the H2A.Bbd nucleosome.
- 45Cosgrove, M. S.; Wolberger, C. Biochem. Cell Biol. 2005, 83, 468Google Scholar45How does the histone code work?Cosgrove, Michael S.; Wolberger, CynthiaBiochemistry and Cell Biology (2005), 83 (4), 468-476CODEN: BCBIEQ; ISSN:0829-8211. (National Research Council of Canada)A review. Patterns of histone post-translational modifications correlate with distinct chromosomal states that regulate access to DNA, leading to the histone-code hypothesis. However, it is not clear how modification of flexible histone tails leads to changes in nucleosome dynamics and, thus, chromatin structure. The recent discovery that, like the flexible histone tails, the structured globular domain of the nucleosome core particle is also extensively modified adds a new and exciting dimension to the histone-code hypothesis, and calls for the re-examn. of current models for the epigenetic regulation of chromatin structure. Here, the authors review these findings and other recent studies that suggest the structured globular domain of the nucleosome core particle plays a key role regulating chromatin dynamics.
- 46Cosgrove, M. S.; Boeke, J. D.; Wolberger, C. Nat. Struct. Mol. Biol. 2004, 11, 1037Google Scholar46Regulated nucleosome mobility and the histone codeCosgrove, Michael S.; Boeke, Jef D.; Wolberger, CynthiaNature Structural & Molecular Biology (2004), 11 (11), 1037-1043CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)A review. Post-translational modifications of the histone tails are correlated with distinct chromatin states that regulate access to DNA. Recent proteomic analyses have revealed several new modifications in the globular nucleosome core, many of which lie at the histone-DNA interface. We interpret these modifications in light of previously published data and propose a new and testable model for how cells implement the histone code by modulating nucleosome dynamics.
- 47Mersfelder, E. L.; Parthun, M. R. Nucleic Acids Res. 2006, 34, 2653Google Scholar47The tale beyond the tail: histone core domain modifications and the regulation of chromatin structureMersfelder, Erica L.; Parthun, Mark R.Nucleic Acids Research (2006), 34 (9), 2653-2662CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)A review. Histone post-translational modifications occur, not only in the N-terminal tail domains, but also in the core domains. While modifications in the N-terminal tail function largely through the regulation of the binding of non-histone proteins to chromatin, based on their location in the nucleosome, core domain modifications may also function through distinct mechanisms involving structural alterations to the nucleosome. Here, the authors review recent developments with regard to these novel histone modifications and discuss their important role in the regulation of chromatin structure.
- 48North, J. A.; Šimon, M.; Ferdinand, M. B.; Shoffner, M. A.; Picking, J. W.; Howard, C. J.; Mooney, A. M.; van Noort, J.; Poirier, M. G.; Ottesen, J. J. Nucleic Acids Res. 2014, 42, 4922Google Scholar48Histone H3 phosphorylation near the nucleosome dyad alters chromatin structureNorth, Justin A.; Simon, Marek; Ferdinand, Michelle B.; Shoffner, Matthew A.; Picking, Jonathan W.; Howard, Cecil J.; Mooney, Alex M.; van Noort, John; Poirier, Michael G.; Ottesen, Jennifer J.Nucleic Acids Research (2014), 42 (8), 4922-4933CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Nucleosomes contain ∼146 bp of DNA wrapped around a histone protein octamer that controls DNA accessibility to transcription and repair complexes. Post-translational modification (PTM) of histone proteins regulates nucleosome function. To date, only modest changes in nucleosome structure have been directly attributed to histone PTMs. Histone residue H3(T118) is located near the nucleosome dyad and can be phosphorylated. This PTM destabilizes nucleosomes and is implicated in the regulation of transcription and repair. Here, we report gel electrophoretic mobility, sucrose gradient sedimentation, thermal disassembly, micrococcal nuclease digestion and at. force microscopy measurements of two DNA-histone complexes that are structurally distinct from nucleosomes. We find that H3(T118ph) facilitates the formation of a nucleosome duplex with two DNA mols. wrapped around two histone octamers, and an altosome complex that contains one DNA mol. wrapped around two histone octamers. The nucleosome duplex complex forms within short ∼150 bp DNA mols., whereas altosomes require at least ∼250 bp of DNA and form repeatedly along 3000 bp DNA mols. These results are the first report of a histone PTM significantly altering the nucleosome structure.
- 49North, J. A.; Javaid, S.; Ferdinand, M. B.; Chatterjee, N.; Picking, J. W.; Shoffner, M.; Nakkula, R. J.; Bartholomew, B.; Ottesen, J. J.; Fishel, R.; Poirier, M. G. Nucleic Acids Res. 2011, 39, 6465Google Scholar49Phosphorylation of histone H3(T118) alters nucleosome dynamics and remodelingNorth, Justin A.; Javaid, Sarah; Ferdinand, Michelle B.; Chatterjee, Nilanjana; Picking, Jonathan W.; Shoffner, Matthew; Nakkula, Robin J.; Bartholomew, Blaine; Ottesen, Jennifer J.; Fishel, Richard; Poirier, Michael G.Nucleic Acids Research (2011), 39 (15), 6465-6474CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Nucleosomes, the fundamental units of chromatin structure, are regulators and barriers to transcription, replication, and repair. Post-translational modifications (PTMs) of the histone proteins within nucleosomes regulate these DNA processes. Histone H3(T118) is a site of phosphorylation [H3(T118ph)] and is implicated in regulation of transcription and DNA repair. Here, the authors prepd. H3(T118ph) by expressed protein ligation and detd. its influence on nucleosome dynamics. It was found that H3(T118ph) reduced DNA-histone binding by 2 kcal/mol, increased nucleosome mobility by 28-fold, and increased DNA accessibility near the dyad region by 6-fold. Moreover, H3(T118ph) increased the rate of hMSH2-hMSH6 nucleosome disassembly and enabled nucleosome disassembly by the SWI/SNF chromatin remodeler. These studies suggest that H3(T118ph) directly enhances and may reprogram chromatin remodeling reactions.
- 50Manohar, M.; Mooney, A. M.; North, J. A.; Nakkula, R. J.; Picking, J. W.; Edon, A.; Fishel, R.; Poirier, M. G.; Ottesen, J. J. J. Biol. Chem. 2009, 284, 23312Google Scholar50Acetylation of Histone H3 at the Nucleosome Dyad Alters DNA-Histone BindingManohar, Mridula; Mooney, Alex M.; North, Justin A.; Nakkula, Robin J.; Picking, Jonathan W.; Edon, Annick; Fishel, Richard; Poirier, Michael G.; Ottesen, Jennifer J.Journal of Biological Chemistry (2009), 284 (35), 23312-23321CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Histone post-translational modifications are essential for regulating and facilitating biol. processes such as RNA transcription and DNA repair. Fifteen modifications are located in the DNA-histone dyad interface and include the acetylation of H3-K115 (H3-K115Ac) and H3-K122 (H3-K122Ac), but the functional consequences of these modifications are unknown. We have prepd. semisynthetic histone H3 acetylated at Lys-115 and/or Lys-122 by expressed protein ligation and incorporated them into single nucleosomes. Competitive reconstitution anal. demonstrated that the acetylation of H3-K115 and H3-K122 reduces the free energy of histone octamer binding. Restriction enzyme kinetic anal. suggests that these histone modifications do not alter DNA accessibility near the sites of modification. However, acetylation of H3-K122 increases the rate of thermal repositioning. Remarkably, Lys→Gln substitution mutations, which are used to mimic Lys acetylation, do not fully duplicate the effects of the H3-K115Ac or H3-K122Ac modifications. Our results are consistent with the conclusion that acetylation in the dyad interface reduces DNA-histone interaction(s), which may facilitate nucleosome repositioning and/or assembly/disassembly.
- 51Šimon, M.; North, J. A.; Shimko, J. C.; Forties, R. A.; Ferdinand, M. B.; Manohar, M.; Zhang, M.; Fishel, R.; Ottesen, J. J.; Poirier, M. G. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 12711Google Scholar51Histone fold modifications control nucleosome unwrapping and disassemblySimon, Marek; North, Justin A.; Shimko, John C.; Forties, Robert A.; Ferdinand, Michelle B.; Manohar, Mridula; Zhang, Meng; Fishel, Richard; Ottesen, Jennifer J.; Poirier, Michael G.Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (31), 12711-12716, S12711/1-S12711/9CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Nucleosomes are stable DNA-histone protein complexes that must be unwrapped and disassembled for genome expression, replication, and repair. Histone posttranslational modifications (PTMs) are major regulatory factors of these nucleosome structural changes, but the mol. mechanisms assocd. with PTM function remains poorly understood. Here we demonstrate that histone PTMs within distinct structured regions of the nucleosome directly regulate the inherent dynamic properties of the nucleosome. Precise PTMs were introduced into nucleosomes by chem. ligation. Single mol. magnetic tweezers measurements detd. that only PTMs near the nucleosome dyad increase the rate of histone release in unwrapped nucleosomes. In contrast, FRET and restriction enzyme anal. reveal that only PTMs throughout the DNA entry-exit region increase unwrapping and enhance transcription factor binding to nucleosomal DNA. These results demonstrate that PTMs in sep. structural regions of the nucleosome control distinct dynamic events, where the dyad regulates disassembly while the DNA entry-exit region regulates unwrapping. These studies are consistent with the conclusion that histone PTMs may independently influence nucleosome dynamics and assocd. chromatin functions.
- 52Makde, R. D.; England, J. R.; Yennawar, H. P.; Tan, S. Nature 2010, 467, 562Google Scholar52Structure of RCC1 chromatin factor bound to the nucleosome core particleMakde, Ravindra D.; England, Joseph R.; Yennawar, Hemant P.; Tan, SongNature (London, United Kingdom) (2010), 467 (7315), 562-566CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)The small GTPase Ran enzyme regulates crit. eukaryotic cellular functions including nuclear transport and mitosis through the creation of a RanGTP gradient around the chromosomes. This concn. gradient is created by the chromatin-bound RCC1 (regulator of chromosome condensation) protein, which recruits Ran to nucleosomes and activates Ran's nucleotide exchange activity. Although RCC1 has been shown to bind directly with the nucleosome, the mol. details of this interaction were not known. Here we det. the crystal structure of a complex of Drosophila RCC1 and the nucleosome core particle at 2.9 resoln., providing an at. view of how a chromatin protein interacts with the histone and DNA components of the nucleosome. Our structure also suggests that the Widom601 DNA positioning sequence present in the nucleosomes forms a 145-base-pair nucleosome core particle, not the expected canonical 147-base-pair particle.
- 53Vasudevan, D.; Chua, E. Y. D.; Davey, C. A. J. Mol. Biol. 2010, 403, 1Google Scholar53Crystal Structures of Nucleosome Core Particles Containing the 601' Strong Positioning SequenceVasudevan, Dileep; Chua, Eugene Y. D.; Davey, Curt A.Journal of Molecular Biology (2010), 403 (1), 1-10CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)Nucleosome positioning plays a key role in genomic regulation by defining histone-DNA context and by modulating access to specific sites. Moreover, the histone-DNA register influences the double-helix structure, which in turn can affect the assocn. of small mols. and protein factors. Anal. of genomic and synthetic DNA has revealed sequence motifs that direct nucleosome positioning in vitro; thus, establishing the basis for the DNA sequence dependence of positioning would shed light on the mechanics of the double helix and its contribution to chromatin structure in vivo. However, acquisition of well-diffracting nucleosome core particle (NCP) crystals is extremely dependent on the DNA fragment used for assembly, and all previous NCP crystal structures have been based on human α-satellite sequences. Here, we describe the crystal structures of Xenopus NCPs contg. one of the strongest known histone octamer binding and positioning sequences, the so-called 601' DNA. Two distinct 145-bp 601 crystal forms display the same histone-DNA register, which coincides with the occurrence of DNA stretching-overtwisting in both halves of the particle around five double-helical turns from the nucleosome center, giving the DNA aneffective length' of 147 bp. As we have found previously with stretching around two turns from the nucleosome center for a centromere-based sequence, the terminal stretching obsd. in the 601 constructs is assocd. with extreme kinking into the minor groove at purine-purine (pyrimidine-pyrimidine) dinucleotide steps. In other contexts, these step types display an overall nonflexible behavior, which raises the possibility that DNA stretching in the nucleosome or extreme distortions in general have unique sequence dependency characteristics. Our findings indicate that DNA stretching is an intrinsically predisposed site-specific property of the nucleosome and suggest how NCP crystal structures with diverse DNA sequences can be obtained.
- 54Finch, J. T.; Brown, R. S.; Richmond, T.; Rushton, B.; Lutter, L. C.; Klug, A. J. Mol. Biol. 1981, 145, 757Google Scholar54X-ray diffraction study of a new crystal form of the nucleosome core showing higher resolutionFinch J T; Brown R S; Richmond T; Rushton B; Lutter L C; Klug AJournal of molecular biology (1981), 145 (4), 757-69 ISSN:0022-2836.There is no expanded citation for this reference.
- 55Richmond, T. J.; Finch, J. T.; Rushton, B.; Rhodes, D.; Klug, A. Nature 1984, 311, 532Google Scholar55Structure of the nucleosome core particle at 7 Å resolutionRichmond, T. J.; Finch, J. T.; Rushton, B.; Rhodes, D.; Klug, A.Nature (London, United Kingdom) (1984), 311 (5986), 532-7CODEN: NATUAS; ISSN:0028-0836.The crystal structure of the nucleosome core particle was solved to 7 Å resoln. The right-handed B-DNA superhelix on the outside contains several sharp bends and numerous interactions with the histone octamer within. The central turn of the superhelix and the H3-H4 tetramer have dyad symmetry, but the H2A-H2B dimers show departures due to interparticle assocns.
- 56Richmond, T. J.; Finch, J. T.; Klug, A. Cold Spring Harbor Symp. Quant. Biol. 1983, 47, 493Google Scholar56Studies of nucleosome structureRichmond, T. J.; Finch, J. T.; Klug, AaronCold Spring Harbor Symposia on Quantitative Biology (1983), 47 (1), 493-501CODEN: CSHSAZ; ISSN:0091-7451.A review and discussion with 43 refs., of the structure of the nucleosome and its assembly from its constituents. The soln. of the the crystal structure of the nucleosome core particle is emphasized.
- 57Richmond, T. J.; Searles, M. A.; Simpson, R. T. J. Mol. Biol. 1988, 199, 161Google Scholar57Crystals of a nucleosome core particle containing defined sequence DNARichmond, Timothy J.; Searles, M. Alexandra; Simpson, Robert T.Journal of Molecular Biology (1988), 199 (1), 161-70CODEN: JMOBAK; ISSN:0022-2836.Nucleosome core particles were reconstituted from a DNA restriction fragment and histone octamers, crystd., and the crystals examd. by x-ray diffraction. A DNA fragment was engineered by site-directed mutagenesis to obtain a 146 base-pair sequence that takes up a sym. arrangement in the core particle. The resulting DNA sequence was cloned in multiple copies into pUC9 and excised as monomer via EcoRV to produce it in milligram quantities. Nucleosome core particles incorporating the DNA were reconstituted by salt gradient dialysis and purified by anion-exchange HPLC. DNase I digestion was used to demonstrate that the termini of the restriction fragment are located 73 base pairs from the mol. dyad axis of the particle. The diffraction limits of crystals of defined sequence core particles extend along the principal direction to a = ∼4, b = ∼5, and c = ∼3 Å, giving about a 2-fold increase in the no. of measurable x-ray reflections over previous crystals contg. mixed sequence DNA. The methods developed here should be useful in the study of other large protein-DNA complexes.
- 58Simpson, R. T.; Stafford, D. W. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 51Google Scholar58Structural features of a phased nucleosome core particleSimpson, Robert T.; Stafford, Darrell W.Proceedings of the National Academy of Sciences of the United States of America (1983), 80 (1), 51-5CODEN: PNASA6; ISSN:0027-8424.Chicken erythrocyte inner histones assoc. with a cloned 260-base-pair (bp) segment of Lytechinus variegatus DNA in a unique location. The fragment contains a 120-bp segment encoding 5 S rRNA, a 90-bp flanking sequence to the 5'-side of the transcribed segment, and a 50-bp downstream flanking sequence. Assocn. of DNA, uniquely labeled at one end and at either the 3'- or the 5'-terminus of a given strand, with histones at 0.1M strength leads to the formation of a compact complex which sediments at ∼13 S. Anal. of the complex by DNase I cleavage shows that protection from the nuclease is confined to a region beginning 20 bp from the left end of the segment and extending to ∼165 bp from the left end. Within the protected region, the 2 DNA strands differ in susceptibility to nuclease, the precise location of nuclease cleavage sites, and the spacing between these sites, and the relative susceptibility of specific cleavage locations. Information present in DNA and the histone octomer is apparently sufficient to create a precisely phased nucleosome in which interactions of the 2 DNA strands with histone differ. The structure of this unique nucleosome is not predicted by the intellectual model based on studies of mixed population of nucleosome core particles.
- 59Harp, J. M.; Palmer, E. L.; York, M. H.; Gewiess, A.; Davis, M.; Bunick, G. J. Electrophoresis 1995, 16, 1861Google Scholar59Preparative separation of nucleosome core particles containing defined-sequence DNA in multiple translational phasesHarp, Joel M.; Palmer, Elise L.; York, Melissa H.; Gewiess, Andreas; Davis, Matthew; Bunick, Gerard J.Electrophoresis (1995), 16 (10), 1861-4CODEN: ELCTDN; ISSN:0173-0835. (VCH)The nucleosome core particle is composed of an octamer of core histone proteins and about 146 bp of DNA. When reconstituted from purified histone octamer and defined-sequence, nucleosome positioning DNA fragments, the DNA will bind to the histone core in a no. of translational phases with respect to the dyad symmetry axis of the histone octamer. Only one of these phases contains sym. bound DNA, and it is this species which is required for crystn. and X-ray diffraction studies. We have developed a technique for sepg. nucleosome core particles, contg. defined-sequence 146 bp DNA, which differ only in translational phasing of the DNA with respect to the histone octamer core.
- 60Satchwell, S. C.; Drew, H. R.; Travers, A. A. J. Mol. Biol. 1986, 191, 659Google Scholar60Sequence periodicities in chicken nucleosome core DNASatchwell, Sandra C.; Drew, Horace R.; Travers, Andrew A.Journal of Molecular Biology (1986), 191 (4), 659-75CODEN: JMOBAK; ISSN:0022-2836.The rotational positioning of DNA about the histone octamer appears to be detd. by certain sequence-dependent modulations of DNA structure. To establish the detailed nature of these interactions, the sequences of 177 different DNA mols. from chicken erythrocyte core particles were analyzed. All variations in the sequence content of these mols., which may be attributed to sequence-dependent preferences for DNA bending, correlate well with the detailed path of the DNA as it wraps around the histone octamer in the crystal structure of the nucleosome core. The sequence-dependent preferences that correlate most closely with the rotational orientation of the DNA, relative to the surface of the protein, are of two kinds: ApApA/TpTpT and ApApT/ApTpT, the minor grooves of which face predominantly in towards the protein; and also GpGpC/GpCpC and ApGpC/GpCpT, whose minor grooves face outward. Fourier anal. has been used to obtain fractional variations in occurrence for all ten dinucleotide and all 32 trinucleotide arrangements. These sequence preferences should apply generally to many other cases of protein-DNA recognition, where the DNA wraps around a protein. In addn., it is obsd. that long runs of homopolymer (dA)·(dT) prefer to occupy the ends of core DNA, five to six turns away from the dyad. These same sequences are apparently excluded from the near-center of core DNA, two to three turns from the dyad. Hence, the translational positioning of any single histone octamer along a DNA mol. of defined sequence may be strongly influenced by the placement of (dA)·(dT) sequences. It may also be influenced by any aversion of the protein for sequences in the linker region, the sequence content of which remains to be detd.
- 61Chua, E. Y. D.; Vasudevan, D.; Davey, G. E.; Wu, B.; Davey, C. A. Nucleic Acids Res. 2012, 40, 6338Google Scholar61The mechanics behind DNA sequence-dependent properties of the nucleosomeChua, Eugene Y. D.; Vasudevan, Dileep; Davey, Gabriela E.; Wu, Bin; Davey, Curt A.Nucleic Acids Research (2012), 40 (13), 6338-6352CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Chromatin organization and compn. impart sophisticated regulatory features crit. to eukaryotic genomic function. Although DNA sequence-dependent histone octamer binding is important for nucleosome activity, many aspects of this phenomenon have remained elusive. We studied nucleosome structure and stability with diverse DNA sequences, including Widom 601 derivs. with the highest known octamer affinities, to establish a simple model behind the mechanics of sequence dependency. This uncovers the unique but unexpected role of TA dinucleotides and a propensity for GC-rich sequence elements to conform energetically favorably at most locations around the histone octamer, which rationalizes GC% as the most predictive factor for nucleosome occupancy in vivo. In addn., our findings reveal dominant constraints on double helix conformation by H3-H4 relative to H2A-H2B binding and DNA sequence context-dependency underlying nucleosome structure, positioning and stability. This provides a basis for improved prediction of nucleosomal properties and the design of tailored DNA constructs for chromatin investigations.
- 62Kulaeva, O. I.; Gaykalova, D. A.; Pestov, N. A.; Golovastov, V. V.; Vassylyev, D. G.; Artsimovitch, I.; Studitsky, V. M. Nat. Struct. Mol. Biol. 2009, 16, 1272Google Scholar62Mechanism of chromatin remodeling and recovery during passage of RNA polymerase IIKulaeva, Olga I.; Gaykalova, Daria A.; Pestov, Nikolai A.; Golovastov, Viktor V.; Vassylyev, Dmitry G.; Artsimovitch, Irina; Studitsky, Vasily M.Nature Structural & Molecular Biology (2009), 16 (12), 1272-1278CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)Transcription of eukaryotic genes by RNA polymerase II (Pol II) is typically accompanied by nucleosome survival and minimal exchange of histones H3 and H4. The mechanism of nucleosome survival and recovery of chromatin structure remains obscure. Here we show how transcription through chromatin by Pol II is uniquely coupled with nucleosome survival. Structural modeling and functional anal. of the intermediates of transcription through a nucleosome indicated that when Pol II approaches an area of strong DNA-histone interactions, a small intranucleosomal DNA loop (zero-size or O-loop) contg. transcribing enzyme is formed. During formation of the O-loop, the recovery of DNA-histone interactions behind Pol II is tightly coupled with their disruption ahead of the enzyme. This coupling is a distinct feature of the Pol II-type mechanism that allows further transcription through the nucleosome, prevents nucleosome translocation and minimizes displacement of H3 and H4 histones from DNA during enzyme passage.
- 63Cui, F.; Zhurkin, V. B. J. Biomol. Struct. Dyn. 2010, 27, 821Google Scholar63Structure-based analysis of DNA sequence patterns guiding nucleosome positioning in vitroCui Feng; Zhurkin Victor BJournal of biomolecular structure & dynamics (2010), 27 (6), 821-41 ISSN:.Recent studies of genome-wide nucleosomal organization suggest that the DNA sequence is one of the major determinants of nucleosome positioning. Although the search for underlying patterns encoded in nucleosomal DNA has been going on for about 30 years, our knowledge of these patterns still remains limited. Based on our evaluations of DNA deformation energy, we developed new scoring functions to predict nucleosome positioning. There are three principal differences between our approach and earlier studies: (i) we assume that the length of nucleosomal DNA varies from 146 to 147 bp; (ii) we consider the anisotropic flexibility of pyrimidine-purine (YR) dimeric steps in the context of their neighbors (e.g., YYRR versus RYRY); (iii) we postulate that alternating AT-rich and GC-rich motifs reflect sequence-dependent interactions between histone arginines and DNA in the minor groove. Using these functions, we analyzed 20 nucleosome positions mapped in vitro at single nucleotide resolution (including clones 601, 603, 605, the pGUB plasmid, chicken beta-globin and three 5S rDNA genes). We predicted 15 of the 20 positions with 1-bp precision, and two positions with 2-bp precision. The predicted position of the '601' nucleosome (i.e., the optimum of the computed score) deviates from the experimentally determined unique position by no more than 1 bp - an accuracy exceeding that of earlier predictions. Our analysis reveals a clear heterogeneity of the nucleosomal sequences which can be divided into two groups based on the positioning 'rules' they follow. The sequences of one group are enriched by highly deformable YR/YYRR motifs at the minor-groove bending sites SHL+/- 3.5 and +/- 5.5, which is similar to the alpha-satellite sequence used in most crystallized nucleosomes. Apparently, the positioning of these nucleosomes is determined by the interactions between histones H2A/H2B and the terminal parts of nucleosomal DNA. In the other group (that includes the '601' clone) the same YR/YYRR motifs occur predominantly at the sites SHL +/- 1.5. The interaction between the H3/H4 tetramer and the central part of the nucleosomal DNA is likely to be responsible for the positioning of nucleosomes of this group, and the DNA trajectory in these nucleosomes may differ in detail from the published structures. Thus, from the stereochemical perspective, the in vitro nucleosomes studied here follow either an X-ray-like pattern (with strong deformations in the terminal parts of nucleosomal DNA), or an alternative pattern (with the deformations occurring predominantly in the central part of the nucleosomal DNA). The results presented here may be useful for genome-wide classification of nucleosomes, linking together structural and thermodynamic characteristics of nucleosomes with the underlying DNA sequence patterns guiding their positions.
- 64Tolstorukov, M. Y.; Colasanti, A. V.; McCandlish, D. M.; Olson, W. K.; Zhurkin, V. B. J. Mol. Biol. 2007, 371, 725Google Scholar64A Novel Roll-and-Slide Mechanism of DNA Folding in Chromatin: Implications for Nucleosome PositioningTolstorukov, Michael Y.; Colasanti, Andrew V.; McCandlish, David M.; Olson, Wilma K.; Zhurkin, Victor B.Journal of Molecular Biology (2007), 371 (3), 725-738CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)How eukaryotic genomes encode the folding of DNA into nucleosomes and how this intrinsic organization of chromatin guides biol. function are questions of wide interest. The phys. basis of nucleosome positioning lies in the sequence-dependent propensity of DNA to adopt the tightly bent configuration imposed by the binding of the histone proteins. Traditionally, only DNA bending and twisting deformations are considered, while the effects of the lateral displacements of adjacent base pairs are neglected. We demonstrate, however, that these displacements have a much more important structural role than ever imagined. Specifically, the lateral Slide deformations obsd. at sites of local anisotropic bending of DNA define its superhelical trajectory in chromatin. Furthermore, the computed cost of deforming DNA on the nucleosome is sequence-specific: in optimally positioned sequences the most easily deformed base-pair steps (CA:TG and TA) occur at sites of large pos. Slide and neg. Roll (where the DNA bends into the minor groove). These conclusions rest upon a treatment of DNA that goes beyond the conventional ribbon model, incorporating all essential degrees of freedom of "real" duplexes in the estn. of DNA deformation energies. Indeed, only after lateral Slide displacements are considered are we able to account for the sequence-specific folding of DNA found in nucleosome structures. The close correspondence between the predicted and obsd. nucleosome locations demonstrates the potential advantage of our "structural" approach in the computer mapping of nucleosome positioning.
- 65Travers, A. A. Philos. Trans. R. Soc., A 2004, 362, 1423Google Scholar65The structural basis of DNA flexibilityTravers, A. A.Philosophical Transactions of the Royal Society of London, Series A: Mathematical, Physical and Engineering Sciences (2004), 362 (1820), 1423-1438CODEN: PTRMAD; ISSN:1364-503X. (Royal Society)Although the av. physico-chem. properties of a long DNA mol. may approx. to those of a thin isotropic homogeneous rod, DNA behaves more locally as an anisotropic heterogeneous rod. This bending anisotropy is sequence dependent and to a first approxn. reflects both the geometry and stability of individual base steps. The biol. manipulation and packaging of the mol. often depend crucially on local variations in both bending and torsional flexibility. However, whereas the probability of DNA untwisting can be strongly correlated with a high bending flexibility, DNA bending, esp. when the mol. is tightly wrapped on a protein surface, may be energetically favored by a less flexible sequence whose preferred configuration conforms more closely to that of the complementary protein surface. In the latter situation the lower bending flexibility may be more than compensated for on binding by a reduced required deformation energy relative to a fully isotropic DNA mol.
- 66Travers, A. A.; Muskhelishvili, G.; Thompson, J. M. T. Philos. Trans. R. Soc., A 2012, 370, 2960Google Scholar66DNA information: from digital code to analogue structureTravers, A. A.; Muskhelishvili, G.; Thompson, J. M. T.Philosophical Transactions of the Royal Society, A: Mathematical, Physical & Engineering Sciences (2012), 370 (1969), 2960-2986CODEN: PTRMAD; ISSN:1364-503X. (Royal Society)A review. The digital linear coding carried by the base pairs in the DNA double helix is now known to have an important component that acts by altering, along its length, the natural shape and stiffness of the mol. In this way, one region of DNA is structurally distinguished from another, constituting an addnl. form of encoded information manifest in three-dimensional space. These shape and stiffness variations help in guiding and facilitating the DNA during its three-dimensional spatial interactions. Such interactions with itself allow communication between genes and enhanced wrapping and histone-octamer binding within the nucleosome core particle. Meanwhile, interactions with proteins can have a reduced entropic binding penalty owing to advantageous sequence-dependent bending anisotropy. Sequence periodicity within the DNA, giving a corresponding structural periodicity of shape and stiffness, also influences the supercoiling of the mol., which, in turn, plays an important facilitating role. In effect, the super-helical d. acts as an analog regulatory mode in contrast to the more commonly acknowledged purely digital mode. Many of these ideas are still poorly understood, and represent a fundamental and outstanding biol. question. This review gives an overview of very recent developments, and hopefully identifies promising future lines of enquiry.
- 67Olson, W. K.; Zhurkin, V. B. Curr. Opin. Struct. Biol. 2011, 21, 348Google Scholar67Working the kinks out of nucleosomal DNAOlson, Wilma K.; Zhurkin, Victor B.Current Opinion in Structural Biology (2011), 21 (3), 348-357CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)A review. Condensation of DNA in the nucleosome takes advantage of its double-helical architecture. The DNA deforms at sites where the base pairs face the histone octamer. The largest so-called kink-and-slide deformations occur in the vicinity of Arg residues that penetrate the minor groove. Nucleosome structures formed from the 601 positioning sequence differ subtly from those incorporating an AT-rich human α-satellite DNA. Restraints imposed by the histone Arg residues on the displacement of base pairs can modulate the sequence-dependent deformability of DNA and potentially contribute to the unique features of the different nucleosomes. Steric barriers mimicking constraints found in the nucleosome induce the simulated large-scale rearrangement of canonical B DNA to kink-and-slide states. The pathway to these states shows nonharmonic behavior consistent with bending profiles inferred from AFM measurements.
- 68Richmond, T. J.; Davey, C. A. Nature 2003, 423, 145Google Scholar68The structure of DNA in the nucleosome coreRichmond, Timothy J.; Davey, Curt A.Nature (London, United Kingdom) (2003), 423 (6936), 145-150CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)The 1.9-Å-resoln. crystal structure of the nucleosome core particle contg. 147 DNA base pairs reveals the conformation of nucleosomal DNA with unprecedented accuracy. The DNA structure is remarkably different from that in oligonucleotides and non-histone protein-DNA complexes. The DNA base-pair-step geometry has, overall, twice the curvature necessary to accommodate the DNA superhelical path in the nucleosome. DNA segments bent into the minor groove are either kinked or alternately shifted. The unusual DNA conformational parameters induced by the binding of histone protein have implications for sequence-dependent protein recognition and nucleosome positioning and mobility. Comparison of the 147-base-pair structure with two 146-base-pair structures reveals alterations in DNA twist that are evidently common in bulk chromatin, and which are of probable importance for chromatin fiber formation and chromatin remodelling.
- 69Juo, Z. S.; Chiu, T. K.; Leiberman, P. M.; Baikalov, I.; Berk, A. J.; Dickerson, R. E. J. Mol. Biol. 1996, 261, 239Google Scholar69How proteins recognize the TATA boxJuo, Zong Sean; Chiu, Thang Kien; Lieberman, Paul M.; Baikalov, Igor; Berk, Arnold J.; Dickerson, Richard E.Journal of Molecular Biology (1996), 261 (2), 239-254CODEN: JMOBAK; ISSN:0022-2836. (Academic)The crystal structure of a complex of human TATA-binding protein with TATA-sequence DNA has been solved, complementing earlier TBP/DNA analyses from Saccharomyces cerevisiae and Arabidopsis thaliana. Special insight into TATA box specificity is provided by considering the TBP/DNA complex, not as a protein mol. with bound DNA, but as a DNA duplex with a particularly large minor groove ligand. This point of view provides explanations for: (1) why T·A base-pairs are required rather than C·G; (2) why an alternation of T and A bases is needed; (3) how TBP recognizes the upstream and downstream ends of the TATA box to bind properly; and (4) why the second half of the TATA box can be more variable than the first.
- 70Kim, Y.; Geiger, J. H.; Hahn, S.; Sigler, P. B. Nature 1993, 365, 512Google Scholar70Crystal structure of a yeast TBP/TATA-box complexKim, Youngchang; Geiger, James H.; Hahn, Steven; Sigler, Paul B.Nature (London, United Kingdom) (1993), 365 (6446), 512-20CODEN: NATUAS; ISSN:0028-0836.The 2.5 Å crystal structure of a TATA-box complex with yeast TBP shows that the eight base pairs of the TATA box bind to the concave surface of TBP by bending towards the major groove with unprecedented severity. This produces a wide open, underwound, shallow minor groove which forms a primarily hydrophobic interface with the entire under-surface of the TBP saddle. The severe bend and a pos. writhe radically alter the trajectory of the flanking B-form DNA.
- 71Kim, J. L.; Nikolov, D. B.; Burley, S. K. Nature 1993, 365, 520Google Scholar71Co-crystal structure of TBP recognizing the minor groove of a TATA elementKim, Joseph L.; Nikolov, Dimitar B.; Burley, Stephen K.Nature (London, United Kingdom) (1993), 365 (6446), 520-7CODEN: NATUAS; ISSN:0028-0836.The three-dimensional structure of a TATA-box binding polypeptide (TBP or TFIIDτ) complexed with the TATA element of the adenovirus major late promoter has been detd. by x-ray crystallog. at 2.25 Å resoln. Binding of the saddle-shaped protein induces a conformational change in the DNA, inducing sharp kinks at either end of the sequence TATAAAAG. Between the kinks, the right-handed double helix is smoothly curved and partially unwound, presenting a widened minor groove to TBP's concave, antiparallel β-sheet. Side-chain/base interactions are restricted to the minor groove, and include hydrogen bonds, van der Waals contacts and phenylalanine base stacking interactions.
- 72Olson, W. K.; Gorin, A. A.; Lu, X.-J.; Hock, L. M.; Zhurkin, V. B. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 11163Google Scholar72DNA sequence-dependent deformability deduced from protein-DNA crystal complexesOlson, Wilma K.; Gorin, Andrey A.; Lu, Xiang-Jun; Hock, Lynette M.; Zhurkin, Victor B.Proceedings of the National Academy of Sciences of the United States of America (1998), 95 (19), 11163-11168CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The deformability of double helical DNA is crit. for its packaging in the cell, recognition by other mols., and transient opening during biochem. important processes. Here, a complete set of sequence-dependent empirical energy functions suitable for describing such behavior is extd. from the fluctuations and correlations of structural parameters in DNA-protein crystal complexes. These elastic functions provide useful stereochem. measures of the local base step movements operative in sequence-specific recognition and protein-induced deformations. In particular, the pyrimidine-purine dimers stand out as the most variable steps in the DNA-protein complexes, apparently acting as flexible "hinges" fitting the duplex to the protein surface. In addn. to the angular parameters widely used to describe DNA deformations (i.e., the bend and twist angles), the translational parameters describing the displacements of base pairs along and across the helical axis are analyzed. The obsd. correlations of base pair bending and shearing motions are important for nonplanar folding of DNA in nucleosomes and other nucleoprotein complexes. The knowledge-based energies also offer realistic three-dimensional models for the study of long DNA polymers at the global level, incorporating structural features beyond the scope of conventional elastic rod treatments and adding a new dimension to literal analyses of genomic sequences.
- 73Bondarenko, V. A.; Steele, L. M.; Ujvári, A.; Gaykalova, D. A.; Kulaeva, O. I.; Polikanov, Y. S.; Luse, D. S.; Studitsky, V. M. Mol. Cell 2006, 24, 469Google Scholar73Nucleosomes can form a polar barrier to transcript elongation by RNA polymerase IIBondarenko, Vladimir A.; Steele, Louise M.; Ujvari, Andrea; Gaykalova, Daria A.; Kulaeva, Olga I.; Polikanov, Yury S.; Luse, Donal S.; Studitsky, Vasily M.Molecular Cell (2006), 24 (3), 469-479CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)Nucleosomes uniquely positioned on high-affinity DNA sequences present a polar barrier to transcription by human and yeast RNA polymerase II (Pol II). In one transcriptional orientation, these nucleosomes provide a strong, factor- and salt-insensitive barrier at the entry into the H3/H4 tetramer that can be recapitulated without H2A/H2B dimers. The same nucleosomes transcribed in the opposite orientation form a weaker, more diffuse barrier that is largely relieved by higher salt, TFIIS, or FACT. Barrier properties are therefore dictated by both the local nucleosome structure (influenced by the strength of the histone-DNA interactions) and the location of the high-affinity DNA region within the nucleosome. Pol II transcribes DNA sequences at the entry into the tetramer much less efficiently than the same sequences located distal to the nucleosome dyad. Thus, entry into the tetramer by Pol II facilitates further transcription, perhaps due to partial unfolding of the tetramer from DNA.
- 74Abbott, E. A. Flatland: A romance of many dimensions; Seely & Co.: UK, 1884.Google ScholarThere is no corresponding record for this reference.
- 75Makde, R. D.; Tan, S. Anal. Biochem. 2013, 442, 138Google Scholar75Strategies for crystallizing a chromatin protein in complex with the nucleosome core particleMakde, Ravindra D.; Tan, SongAnalytical Biochemistry (2013), 442 (2), 138-145CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)The mol. details of how chromatin factors and enzymes interact with the nucleosome are crit. to understanding fundamental genetic processes including cell division and gene regulation. A structural understanding of such processes has been hindered by the difficulty in producing diffraction-quality crystals of chromatin proteins in complex with the nucleosome. We describe here the steps used to grow crystals of the 300-kDa RCC1 chromatin factor/nucleosome core particle complex that diffract to 2.9-Å resoln. These steps include both pre- and postcrystn. strategies potentially useful to other complexes. We screened multiple variant RCC1/nucleosome core particle complexes assembled using different RCC1 homologs and deletion variants, and nucleosomes contg. nucleosomal DNA with different sequences and lengths, as well as histone deletion variants. We found that using RCC1 from different species produced different crystal forms of the RCC1/nucleosome complex consistent with key crystal packing interactions mediated by RCC1. Optimization of postcrystn. soaks to dehydrate the crystals dramatically improved the diffraction quality of the RCC1/nucleosome crystal from 5.0- to 2.9-Å resoln.
- 76Hock, R.; Furusawa, T.; Ueda, T.; Bustin, M. Trends Cell Biol. 2007, 17, 72Google Scholar76HMG chromosomal proteins in development and diseaseHock, Robert; Furusawa, Takashi; Ueda, Tetsuya; Bustin, MichaelTrends in Cell Biology (2007), 17 (2), 72-79CODEN: TCBIEK; ISSN:0962-8924. (Elsevier B.V.)A review. The high mobility group (HMG) proteins are a superfamily of abundant and ubiquitous nuclear proteins that bind to DNA and nucleosomes and induce structural changes in the chromatin fiber. They are important in chromatin dynamics and influence DNA processing in the context of chromatin. Results emerging from studies of human disease, genetically modified mice and cells with altered HMG expression indicate that the expression of the HMG proteins is developmentally regulated and that changes in HMG protein levels alter the cellular phenotype and can lead to developmental abnormalities and disease. Here, we focus on the biol. function of HMG proteins and highlight their possible roles in cellular differentiation and in the etiol. of various diseases.
- 77Canzio, D.; Liao, M.; Naber, N.; Pate, E.; Larson, A.; Wu, S.; Marina, D. B.; Garcia, J. F.; Madhani, H. D.; Cooke, R.; Schuck, P.; Cheng, Y.; Narlikar, G. J. Nature 2013, 496, 377Google Scholar77A conformational switch in HP1 releases auto-inhibition to drive heterochromatin assemblyCanzio, Daniele; Liao, Maofu; Naber, Nariman; Pate, Edward; Larson, Adam; Wu, Shenping; Marina, Diana B.; Garcia, Jennifer F.; Madhani, Hiten D.; Cooke, Roger; Schuck, Peter; Cheng, Yifan; Narlikar, Geeta J.Nature (London, United Kingdom) (2013), 496 (7445), 377-381CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)A hallmark of histone H3 lysine 9 (H3K9)-methylated heterochromatin, conserved from the fission yeast Schizosaccharomyces pombe to humans, is its ability to spread to adjacent genomic regions. Central to heterochromatin spread is heterochromatin protein 1 (HP1), which recognizes H3K9-methylated chromatin, oligomerizes and forms a versatile platform that participates in diverse nuclear functions, ranging from gene silencing to chromosome segregation. How HP1 proteins assemble on methylated nucleosomal templates and how the HP1-nucleosome complex achieves functional versatility remain poorly understood. Here we show that binding of the key S. pombe HP1 protein, Swi6, to methylated nucleosomes drives a switch from an auto-inhibited state to a spreading-competent state. In the auto-inhibited state, a histone-mimic sequence in one Swi6 monomer blocks methyl-mark recognition by the chromodomain of another monomer. Auto-inhibition is relieved by recognition of two template features, the H3K9 Me mark and nucleosomal DNA. Cryo-electron-microscopy-based reconstruction of the Swi6-nucleosome complex provides the overall architecture of the spreading-competent state in which two unbound chromodomain sticky ends appear exposed. Disruption of the switch between the auto-inhibited and spreading-competent states disrupts heterochromatin assembly and gene silencing in vivo. These findings are reminiscent of other conditionally activated polymn. processes, such as actin nucleation, and open up a new class of regulatory mechanisms that operate on chromatin in vivo.
- 78Armache, K. J.; Garlick, J. D.; Canzio, D.; Narlikar, G. J.; Kingston, R. E. Science 2011, 334, 977Google Scholar78Structural Basis of Silencing: Sir3 BAH Domain in Complex with a Nucleosome at 3.0 Å ResolutionArmache, Karim-Jean; Garlick, Joseph D.; Canzio, Daniele; Narlikar, Geeta J.; Kingston, Robert E.Science (Washington, DC, United States) (2011), 334 (6058), 977-982CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Gene silencing is essential for regulating cell fate in eukaryotes. Altered chromatin architectures contribute to maintaining the silenced state in a variety of species. The silent information regulator (Sir) proteins regulate mating type in Saccharomyces cerevisiae. One of these proteins, Sir3, interacts directly with the nucleosome to help generate silenced domains. We detd. the crystal structure of a complex of the yeast Sir3 BAH (bromo-assocd. homol.) domain and the nucleosome core particle at 3.0 angstrom resoln. We see multiple mol. interactions between the protein surfaces of the nucleosome and the BAH domain that explain numerous genetic mutations. These interactions are accompanied by structural rearrangements in both the nucleosome and the BAH domain. The structure explains how covalent modifications on H4K16 and H3K79 regulate formation of a silencing complex that contains the nucleosome as a central component.
- 79Barbera, A. J.; Chodaparambil, J. V.; Kelley-Clarke, B.; Joukov, V.; Walter, J. C.; Luger, K.; Kaye, K. M. Science 2006, 311, 856Google Scholar79The nucleosomal surface as a docking station for Kaposi's sarcoma herpesvirus LANABarbera, Andrew J.; Chodaparambil, Jayanth V.; Kelley-Clarke, Brenna; Joukov, Vladimir; Walter, Johannes C.; Luger, Karolin; Kaye, Kenneth M.Science (Washington, DC, United States) (2006), 311 (5762), 856-861CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Kaposi's sarcoma-assocd. herpesvirus (KSHV) latency-assocd. nuclear antigen (LANA) mediates viral genome attachment to mitotic chromosomes. We find that N-terminal LANA docks onto chromosomes by binding nucleosomes through the folded region of histones H2A-H2B. The same LANA residues were required for both H2A-H2B binding and chromosome assocn. Further, LANA did not bind Xenopus sperm chromatin, which is deficient in H2A-H2B; chromatin binding was rescued after assembly of nucleosomes contg. H2A-H2B. We also describe the 2.9-angstrom crystal structure of a nucleosome complexed with the first 23 LANA amino acids. The LANA peptide forms a hairpin that interacts exclusively with an acidic H2A-H2B region that is implicated in the formation of higher order chromatin structure. Our findings present a paradigm for how nucleosomes may serve as binding platforms for viral and cellular proteins and reveal a previously unknown mechanism for KSHV latency.
- 80Marmorstein, R.; Trievel, R. C. Biochim. Biophys. Acta, Gene Regul. Mech. 2009, 1789, 58Google Scholar80Histone modifying enzymes: Structures, mechanisms, and specificitiesMarmorstein, Ronen; Trievel, Raymond C.Biochimica et Biophysica Acta, Gene Regulatory Mechanisms (2009), 1789 (1), 58-68CODEN: BBAGC6; ISSN:1874-9399. (Elsevier B.V.)A review. Histone modifying enzymes catalyze the addn. or removal of an array of covalent modifications in histone and non-histone proteins. Within the context of chromatin, these modifications regulate gene expression as well as other genomic functions and have been implicated in establishing and maintaining a heritable epigenetic code that contributes to defining cell identity and fate. Biochem. and structural characterization of histone modifying enzymes has yielded important insights into their resp. catalytic mechanisms, substrate specificities, and regulation. In this review, we summarize recent advances in understanding these enzymes, highlighting studies of the histone acetyltransferases (HATs) p300 (also now known as KAT3B) and Rtt109 (KAT11) and the histone lysine demethylases (HDMs) LSD1 (KDM1) and JMJD2A (KDM4A), present overriding themes that derive from these studies, and pose remaining questions concerning their regulatory roles in mediating DNA transactions.
- 81Musselman, C. A.; Lalonde, M.-E.; Côté, J.; Kutateladze, T. G. Nat. Struct. Mol. Biol. 2012, 19, 1218Google Scholar81Perceiving the epigenetic landscape through histone readersMusselman, Catherine A.; Lalonde, Marie-Eve; Cote, Jacques; Kutateladze, Tatiana G.Nature Structural & Molecular Biology (2012), 19 (12), 1218-1227CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)A review. Post-translational modifications (PTMs) of histones provide a fine-tuned mechanism for regulating chromatin structure and dynamics. PTMs can alter direct interactions between histones and DNA and serve as docking sites for protein effectors, or readers, of these PTMs. Binding of the readers recruits or stabilizes various components of the nuclear signaling machinery at specific genomic sites, mediating fundamental DNA-templated processes, including gene transcription and DNA recombination, replication and repair. In this review, we highlight the latest advances in characterizing histone-binding mechanisms and identifying new epigenetic readers and summarize the functional significance of PTM recognition.
- 82Ruthenburg, A. J.; Li, H.; Patel, D. J.; Allis, C. D. Nat. Rev. Mol. Cell Biol. 2007, 8, 983Google Scholar82Multivalent engagement of chromatin modifications by linked binding modulesRuthenburg, Alexander J.; Li, Haitao; Patel, Dinshaw J.; Allis, C. DavidNature Reviews Molecular Cell Biology (2007), 8 (12), 983-994CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. Histone post-translational modifications (PTMs) play crucial roles in genome management, in part by recruiting specific factors that alter the structural properties of chromatin. These so-called effector complexes often comprise multiple histone-binding modules that may act in concert to regulate chromatin structure and DNA-related activities. Various chem. modifications on histones and regions of assocd. DNA play crucial roles in genome management by binding specific factors that, in turn, serve to alter the structural properties of chromatin. These so-called effector proteins have typically been studied with the biochemist's paring knife; the capacity to recognize specific chromatin PTMs has been mapped to an increasing no. of domains that frequently appear in the nuclear subset of the proteome, often present in large, multisubunit complexes that bristle with modification-dependent binding potential. The authors propose that multivalent interactions on a single histone tail and beyond may have a significant, if not dominant, role in chromatin transactions.
- 83Zhou, B.-R.; Feng, H.; Ghirlando, R.; Kato, H.; Gruschus, J.; Bai, Y. J. Mol. Biol. 2012, 421, 30Google Scholar83Histone H4 K16Q Mutation, an Acetylation Mimic, Causes Structural Disorder of Its N-Terminal Basic Patch in the NucleosomeZhou, Bing-Rui; Feng, Hanqiao; Ghirlando, Rodolfo; Kato, Hidenori; Gruschus, James; Bai, YawenJournal of Molecular Biology (2012), 421 (1), 30-37CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)Histone tails and their posttranslational modifications play important roles in regulating the structure and dynamics of chromatin. For histone H4, the basic patch K16R17H18R19 in the N-terminal tail modulates chromatin compaction and nucleosome sliding catalyzed by ATP-dependent ISWI chromatin remodeling enzymes while acetylation of H4 K16 affects both functions. The structural basis for the effects of this acetylation is unknown. Here, we investigated the conformation of histone tails in the nucleosome by soln. NMR. We found that backbone amides of the N-terminal tails of histones H2A, H2B, and H3 are largely observable due to their conformational disorder. However, only residues 1-15 in H4 can be detected, indicating that residues 16-22 in the tails of both H4 histones fold onto the nucleosome core. Surprisingly, we found that K16Q mutation in H4, a mimic of K16 acetylation, leads to a structural disorder of the basic patch. Thus, our study suggests that the folded structure of the H4 basic patch in the nucleosome is important for chromatin compaction and nucleosome remodeling by ISWI enzymes while K16 acetylation affects both functions by causing structural disorder of the basic patch K16R17H18R19.
- 84Dorigo, B.; Schalch, T.; Bystricky, K.; Richmond, T. J. J. Mol. Biol. 2003, 327, 85Google Scholar84Chromatin Fiber Folding: Requirement for the Histone H4 N-terminal TailDorigo, Benedetta; Schalch, Thomas; Bystricky, Kerstin; Richmond, Timothy J.Journal of Molecular Biology (2003), 327 (1), 85-96CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Science Ltd.)We have developed a self-assembly system for nucleosome arrays in which recombinant, post-translationally unmodified histone proteins are combined with DNA of defined-sequence to form chromatin higher-order structure. The nucleosome arrays obtained are highly homogeneous and sediment at 53 S when maximally folded in 1 mM or 100 mM MgCl2. The folding properties are comparable to established systems. Anal. ultracentrifugation is used to det. the consequence of individual histone tail domain deletions on array folding. Fully compacted chromatin fibers are obtained with any one of the histone tails deleted with the exception of the H4 N terminus. The region of the H4 tail, which mediates compaction, resides in the stretch of amino acids 14-19.
- 85Robinson, P. J. J.; An, W.; Routh, A.; Martino, F.; Chapman, L.; Roeder, R. G.; Rhodes, D. J. Mol. Biol. 2008, 381, 816Google Scholar8530 nm Chromatin Fibre Decompaction Requires both H4-K16 Acetylation and Linker Histone EvictionRobinson, Philip J. J.; An, Woojin; Routh, Andrew; Martino, Fabrizio; Chapman, Lynda; Roeder, Robert G.; Rhodes, DanielaJournal of Molecular Biology (2008), 381 (4), 816-825CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)The mechanism by which chromatin is decondensed to permit access to DNA is largely unknown. Here, using a model nucleosome array reconstituted from recombinant histone octamers, we have defined the relative contribution of the individual histone octamer N-terminal tails as well as the effect of a targeted histone tail acetylation on the compaction state of the 30 nm chromatin fiber. This study goes beyond previous studies as it is based on a nucleosome array that is very long (61 nucleosomes) and contains a stoichiometric concn. of bound linker histone, which is essential for the formation of the 30 nm chromatin fiber. We find that compaction is regulated in two steps: Introduction of H4 acetylated to 30% on K16 inhibits compaction to a greater degree than deletion of the H4 N-terminal tail. Further decompaction is achieved by removal of the linker histone.
- 86Allahverdi, A.; Yang, R.; Korolev, N.; Fan, Y.; Davey, C. A.; Liu, C. F.; Nordenskiold, L. Nucleic Acids Res. 2011, 39, 1680Google Scholar86The effects of histone H4 tail acetylations on cation-induced chromatin folding and self-associationAllahverdi, Abdollah; Yang, Renliang; Korolev, Nikolay; Fan, Yanping; Davey, Curt A.; Liu, Chuan-Fa; Nordenskioeld, LarsNucleic Acids Research (2011), 39 (5), 1680-1691CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Understanding the mol. mechanisms behind regulation of chromatin folding through covalent modifications of the histone N-terminal tails is hampered by a lack of accessible chromatin contg. precisely modified histones. We study the internal folding and intermol. self-assocn. of a chromatin system consisting of satd. 12-mer nucleosome arrays contg. various combinations of completely acetylated lysines at positions 5, 8, 12 and 16 of histone H4, induced by the cations Na+, K+, Mg2+, Ca2+, cobalt-hexammine3+, spermidine3+ and spermine4+. Histones were prepd. using a novel semi-synthetic approach with native chem. ligation. Acetylation of H4-K16, but not its glutamine mutation, drastically reduces cation-induced folding of the array. Neither acetylations nor mutations of all the sites K5, K8 and K12 can induce a similar degree of array unfolding. The ubiquitous K+, (as well as Rb+ and Cs+) showed an unfolding effect on unmodified arrays almost similar to that of H4-K16 acetylation. We propose that K+ (and Rb+/Cs+) binding to a site on the H2B histone (R96-L99) disrupts H4K16 ε-amino group binding to this specific site, thereby deranging H4 tail-mediated nucleosome-nucleosome stacking and that a similar mechanism operates in the case of H4-K16 acetylation. Inter-array self-assocn. follows electrostatic behavior and is largely insensitive to the position or nature of the H4 tail charge modification.
- 87Dorigo, B.; Schalch, T.; Kulangara, A.; Duda, S.; Schroeder, R. R.; Richmond, T. J. Science 2004, 306, 1571Google Scholar87Nucleosome arrays reveal the two-start organization of the chromatin fiberDorigo, Benedetta; Schalch, Thomas; Kulangaral, Alexandra; Duda, Sylwia; Schroeder, Rasmus R.; Richmond, Timothy J.Science (Washington, DC, United States) (2004), 306 (5701), 1571-1573CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Chromatin folding dets. the accessibility of DNA constituting eukaryotic genomes and consequently is profoundly important in the mechanisms of nuclear processes such as gene regulation. Nucleosome arrays compact to form a 30-nm chromatin fiber of hitherto disputed structure. Two competing classes of models have been proposed in which nucleosomes are either arranged linearly in a one-start higher order helix or zigzag back and forth in a two-start helix. We analyzed compacted nucleosome arrays stabilized by introduction of disulfide cross-links and show that the chromatin fiber comprises two stacks of nucleosomes in accord with the two-start model.
- 88Sinha, D.; Shogren-Knaak, M. A. J. Biol. Chem. 2010, 285, 16572Google Scholar88Role of Direct Interactions between the Histone H4 Tail and the H2A Core in Long Range Nucleosome ContactsSinha, Divya; Shogren-Knaak, Michael A.Journal of Biological Chemistry (2010), 285 (22), 16572-16581CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)In eukaryotic nuclei the majority of genomic DNA is believed to exist in higher order chromatin structures. Nonetheless, the nature of direct, long range nucleosome interactions that contribute to these structures is poorly understood. To det. whether these interactions are directly mediated by contacts between the histone H4 amino-terminal tail and the acidic patch of the H2A/H2B interface, as previously demonstrated for short range nucleosomal interactions, we have characterized the extent and effect of disulfide crosslinking between residues in histones contained in different strands of nucleosomal arrays. We show that in 208-12 5 S rDNA and 601-177-12 nucleosomal array systems, direct interactions between histones H4-V21C and H2A-E64C can be captured. This interaction depends on the extent of initial cross-strand assocn. but does not require these specific residues, because interactions with residues flanking H4-V21C can also be captured. Addnl., we find that trapping H2A-H4 intra-array interactions antagonizes the ability of these arrays to undergo intermol. self-assocn.
- 89Yang, D.; Arya, G. Phys. Chem. Chem. Phys. 2011, 13, 2911Google Scholar89Structure and binding of the H4 histone tail and the effects of lysine 16 acetylationYang, Darren; Arya, GauravPhysical Chemistry Chemical Physics (2011), 13 (7), 2911-2921CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)The H4 histone tail plays a crit. role in chromatin folding and regulation - it mediates strong interactions with the acidic patch of proximal nucleosomes and its acetylation at lysine 16 (K16) leads to partial unfolding of chromatin. The mol. mechanism assocd. with the H4 tail/acidic patch interactions and its modulation via K16 acetylation remains unknown. Here we employ a combination of mol. dynamics simulations, mol. docking calcns., and free energy computations to investigate the structure of the H4 tail in soln., the binding of the H4 tail with the acidic patch, and the effects of K16 acetylation. The H4 tail exhibits a disordered configuration except in the region Ala15-Lys20, where it exhibits a strong propensity for an α-helical structure. This α-helical region is found to dock very favorably into the acidic patch groove of a nucleosome with a binding free energy of approx. -7 kcal mol-1. We have identified the specific interactions that stabilize this binding as well as the assocd. energetics. The acetylation of K16 is found to reduce the α-helix-forming propensity of the H4 tail and K16's accessibility for mediating external interactions. More importantly, K16 acetylation destabilizes the binding of the H4 tail at the acidic patch by mitigating specific salt bridges and longer-range electrostatic interactions mediated by K16. Our study thus provides new microscopic insights into the compaction of chromatin and its regulation via post-translational modifications of histone tails, which could be of interest to chromatin biol., cancer, epigenetics, and drug design.
- 90Barbera, A. J.; Ballestas, M. E.; Kaye, K. M. J. Virol. 2004, 78, 294Google Scholar90The Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 1 N terminus is essential for chromosome association, DNA replication, and episome persistenceBarbera, Andrew J.; Ballestas, Mary E.; Kaye, Kenneth M.Journal of Virology (2004), 78 (1), 294-301CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)To persist in latently infected, proliferating cells, Kaposi's sarcoma-assocd. herpesvirus (KSHV) episomes must replicate and efficiently segregate to progeny nuclei. Episome persistence in uninfected cells requires latency-assocd. nuclear antigen 1 (LANA1) in trans and cis-acting KSHV terminal repeat (TR) DNA. The LANA1 C terminus binds TR DNA, and LANA1 mediates TR-assocd. DNA replication in transient assays. LANA1 also concs. at sites of KSHV TR DNA episomes along mitotic chromosomes, consistent with a tethering role to efficiently segregate episomes to progeny nuclei. LANA1 amino acids 5-22 constitute a chromosome assocn. region. We now investigate LANA1 residues 5-22 with scanning alanine substitutions. Mutations targeting LANA1 5GMR7, 8LRS10, and 11GRS13 eliminated chromosome assocn., DNA replication, and episome persistence. LANA1 mutated at 14TG15 retained the ability to assoc. with chromosomes but was partially deficient in DNA replication and episome persistence. These results provide genetic support for a key role of the LANA1 N terminus in chromosome assocn., LANA1-mediated DNA replication, and episome persistence.
- 91Clarke, P. R.; Zhang, C. Nat. Rev. Mol. Cell Biol. 2008, 9, 464Google Scholar91Spatial and temporal coordination of mitosis by Ran GTPaseClarke, Paul R.; Zhang, ChuanmaoNature Reviews Molecular Cell Biology (2008), 9 (6), 464-477CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. Small nuclear GTPase Ran controls the directionality of macromol. transport between the nucleus and the cytoplasm. Ran also plays important roles during mitosis, when the nucleus is dramatically reorganized to allow chromosome segregation. Ran directs the assembly of the mitotic spindle, nuclear envelope dynamics, and the timing of cell-cycle transitions. The mechanisms that underlie these functions provide insights into the spatial and temporal coordination of the changes that occur in intracellular organization during the cell-division cycle.
- 92Carazo-Salas, R. E.; Guarguaglini, G.; Gruss, O. J.; Segref, A.; Karsenti, E.; Mattaj, I. W. Nature 1999, 400, 178Google Scholar92Generation of GTP-bound Ran by RCC1 is required for chromatin-induced mitotic spindle formationCarazo-Salas, Rafael E.; Guarguaglini, Giulia; Gruss, Oliver J.; Segref, Alexandra; Karsenti, Eric; Mattaj, Iain W.Nature (London) (1999), 400 (6740), 178-181CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)Chromosomes are segregated by two antiparallel arrays of microtubules arranged to form the spindle app. During cell division, the nucleation of cytosolic microtubules is prevented and spindle microtubules nucleate from centrosomes (in mitotic animal cells) or around chromosomes (in plants and some meiotic cells). The mol. mechanism by which chromosomes induce local microtubule nucleation in the absence of centrosomes is unknown, but it can be studied by adding chromatin beads to Xenopus egg exts. The beads nucleate microtubules that eventually reorganize into a bipolar spindle. RCC1, the guanine-nucleotide-exchange factor for the GTPase protein Ran, is a component of chromatin. Using the chromatin bead assay, the authors show here that the activity of chromosome-assocd. RCC1 protein is required for spindle formation. Ran itself, when in the GTP-bound state (Ran-GTP), induces microtubule nucleation and spindle-like structures in M-phase ext. The authors propose that RCC1 generates a high local concn. of Ran-GTP around chromatin which in turn induces the local nucleation of microtubules.
- 93Kalab, P.; Heald, R. J. Cell Sci. 2008, 121, 1577Google Scholar93The RanGTP gradient - a GPS for the mitotic spindleKalab, Petr; Heald, RebeccaJournal of Cell Science (2008), 121 (10), 1577-1586CODEN: JNCSAI; ISSN:0021-9533. (Company of Biologists Ltd.)A review. GTPase Ran plays a key role in nuclear import and export, mitotic spindle assembly, and nuclear envelope formation. The cycling of Ran between its GTP- and GDP-bound forms is catalyzed by the chromatin-bound guanine nucleotide exchange factor RCC1 and the cytoplasmic Ran GTPase-activating protein, RanGAP. The result is an intracellular concn. gradient of RanGTP that equips eukaryotic cells with a 'genome-positioning system' (GPS). The binding of RanGTP to nuclear transport receptors (NTRs) of the importin-β superfamily mediates the effects of the gradient and generates further downstream gradients, which have been elucidated by FRET imaging and computational modeling. The Ran-dependent GPS spatially directs many functions required for genome segregation by the mitotic spindle during mitosis. Through exportin 1, RanGTP recruits essential centrosome and kinetochore components, whereas the RanGTP-induced release of spindle assembly factors (SAFs) from importins activates SAFs to nucleate, bind, and organize nascent spindle microtubules. Although a considerable fraction of cytoplasmic SAFs is active and RanGTP induces only partial further activation near chromatin, bipolar spindle assembly is robustly induced by cooperativity and pos.-feedback mechanisms within the network of Ran-activated SAFs. The RanGTP gradient is conserved, although its roles vary among different cell types and species, and much remains to be learned regarding its functions.
- 94Nemergut, M. E.; Mizzen, C. A.; Stukenberg, T.; Allis, C. D.; Macara, I. G. Science 2001, 292, 1540Google Scholar94Chromatin docking and exchange activity enhancement of RCC1 by histones H2A and H2BNemergut, Michael E.; Mizzen, Craig A.; Stukenberg, Todd; Allis, C. David; Macara, Ian G.Science (Washington, DC, United States) (2001), 292 (5521), 1540-1543CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The Ran guanosine triphosphatase (GTPase) controls nucleocytoplasmic transport, mitotic spindle formation, and nuclear envelope assembly. These functions rely on the assocn. of the Ran-specific exchange factor, RCC1 (regulator of chromosome condensation 1), with chromatin. We find that RCC1 binds directly to mononucleosomes and to histones H2A and. H2B. RCC1 utilizes these histones to bind Xenopus sperm chromatin, and the binding of RCC1 to nucleosomes or histones stimulates the catalytic activity of RCC1. We propose that the docking of RCC1 to H2A/H2B establishes the polarity of the Ran-GTP gradient that drives nuclear envelope assembly, nuclear transport, and other nuclear events.
- 95England, J. R.; Huang, J.; Jennings, M. J.; Makde, R. D.; Tan, S. J. Mol. Biol. 2010, 398, 518Google Scholar95RCC1 Uses a Conformationally Diverse Loop Region to Interact with the Nucleosome: A Model for the RCC1-Nucleosome ComplexEngland, Joseph R.; Huang, Jiehuan; Jennings, Matthew J.; Makde, Ravindra D.; Tan, SongJournal of Molecular Biology (2010), 398 (4), 518-529CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)The binding of RCC1 (regulator of chromosome condensation 1) to chromatin is crit. for cellular processes such as mitosis, nucleocytoplasmic transport, and nuclear envelope formation because RCC1 recruits the small GTPase Ran (Ras-related nuclear protein) to chromatin and sets up a Ran-GTP gradient around the chromosomes. However, the mol. mechanism by which RCC1 binds to nucleosomes, the repeating unit of chromatin, is not known. Biochem. approaches were used to test structural models for how the RCC1 β-propeller protein could bind to the nucleosome. In contrast to the prevailing model, RCC1 does not appear to use the β-propeller face opposite to its Ran-binding face to interact with nucleosomes. Instead, it is found that RCC1 uses a conformationally flexible loop region, termed the switchback loop, in addn. to its N-terminal tail to bind to the nucleosome. The juxtaposition of the RCC1 switchback loop to its Ran binding surface suggests a novel mechanism for how nucleosome-bound RCC1 recruits Ran to chromatin. Furthermore, this model accounts for previously unexplained observations for how Ran can interact with the nucleosome both dependent and independent of RCC1 and how binding of the nucleosome can enhance RCC1's Ran nucleotide exchange activity.
- 96Rusche, L. N.; Kirchmaier, A. L.; Rine, J. Annu. Rev. Biochem. 2003, 72, 481Google Scholar96The establishment, inheritance, and function of silenced chromatin in Saccharomyces cerevisiaeRusche, Laura N.; Kirchmaier, Ann L.; Rine, JasperAnnual Review of Biochemistry (2003), 72 (), 481-516CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)A review. Genomes are organized into active regions known as euchromatin and inactive regions known as heterochromatin, or silenced chromatin. This review describes contemporary knowledge and models for how silenced chromatin in Saccharomyces cerevisiae forms, functions, and is inherited. In S. cerevisiae, Sir proteins are the key structural components of silenced chromatin. Sir proteins interact first with silencers, which dictate which regions are silenced, and then with histone tails in nucleosomes as the Sir proteins spread from silencers along chromosomes. Importantly, the spreading of silenced chromatin requires the histone deacetylase activity of Sir2p. This requirement leads to a general model for the spreading and inheritance of silenced chromatin or other special chromatin states. Such chromatin domains are marked by modifications of the nucleosomes or DNA, and this mark is able to recruit an enzyme that makes further marks. Thus, among different organisms, multiple forms of repressive chromatin can be formed using similar strategies but completely different proteins. We also describe emerging evidence that mutations that cause global changes in the modification of histones can alter the balance between euchromatin and silenced chromatin within a cell.
- 97McBryant, S. J.; Krause, C.; Woodcock, C. L.; Hansen, J. C. Mol. Cell. Biol. 2008, 28, 3563Google Scholar97The silent information regulator 3 protein, SIR3p, binds to chromatin fibers and assembles a hypercondensed chromatin architecture in the presence of saltMcBryant, Steven J.; Krause, Christine; Woodcock, Christopher L.; Hansen, Jeffrey C.Molecular and Cellular Biology (2008), 28 (11), 3563-3572CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)The telomeres and mating-type loci of budding yeast adopt a condensed, heterochromatin-like state through recruitment of the silent information regulator (SIR) proteins SIR2p, SIR3p, and SIR4p. In this study we characterize the chromatin binding determinants of recombinant SIR3p and identify how SIR3p mediates chromatin fiber condensation in vitro. Purified full-length SIR3p was incubated with naked DNA, nucleosome core particles, or defined nucleosomal arrays, and the resulting complexes were analyzed by electrophoretic shift assays, sedimentation velocity, and electron microscopy. SIR3p bound avidly to all three types of templates. SIR3p loading onto its nucleosomal sites in chromatin produced thickened condensed fibers that retained a beaded morphol. At higher SIR3p concns., individual nucleosomal arrays formed oligomeric suprastructures bridged by SIR3p oligomers. When condensed SIR3p-bound chromatin fibers were incubated in Mg2+, they folded and oligomerized even further to produce hypercondensed higher-order chromatin structures. Collectively, these results define how SIR3p may function as a chromatin architectural protein and provide new insight into the interplay between endogenous and protein-mediated chromatin fiber condensation pathways.
- 98Johnson, A.; Li, G.; Sikorski, T. W.; Buratowski, S.; Woodcock, C. L.; Moazed, D. Mol. Cell 2009, 35, 769Google Scholar98Reconstitution of heterochromatin-dependent transcriptional gene silencingJohnson, Aaron; Li, Geng; Sikorski, Timothy W.; Buratowski, Stephen; Woodcock, Christopher L.; Moazed, DaneshMolecular Cell (2009), 35 (6), 769-781CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)Heterochromatin assembly in budding yeast requires the SIR complex, which contains the NAD-dependent deacetylase Sir2 and the Sir3 and Sir4 proteins. Sir3 binds to nucleosomes contg. deacetylated histone H4 lysine 16 (H4K16) and, with Sir4, promotes spreading of Sir2 and deacetylation along the chromatin fiber. Combined action of histone modifying and binding activities is a conserved hallmark of heterochromatin, but the relative contribution of each activity to silencing has remained unclear. Here, we reconstitute SIR-chromatin complexes using purified components and show that the SIR complex efficiently deacetylates chromatin templates and promotes the assembly of altered structures that silence Gal4-VP16-activated transcription. Silencing requires all three Sir proteins, even with fully deacetylated chromatin, and involves the specific assocn. of Sir3 with deacetylated H4K16. These results define a minimal set of components that mediate heterochromatic gene silencing and demonstrate distinct contributions for histone deacetylation and nucleosome binding in the silencing mechanism.
- 99Martino, F.; Kueng, S.; Robinson, P.; Tsai-Pflugfelder, M.; van Leeuwen, F.; Ziegler, M.; Cubizolles, F.; Cockell, M. M.; Rhodes, D.; Gasser, S. M. Mol. Cell 2009, 33, 323Google Scholar99Reconstitution of yeast silent chromatin: multiple contact sites and O-AADPR binding load SIR complexes onto nucleosomes in vitroMartino, Fabrizio; Kueng, Stephanie; Robinson, Philip; Tsai-Pflugfelder, Monika; van Leeuwen, Fred; Ziegler, Mathias; Cubizolles, Fabien; Cockell, Moira M.; Rhodes, Daniela; Gasser, Susan M.Molecular Cell (2009), 33 (3), 323-334CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)At yeast telomeres and silent mating-type loci, chromatin assumes a higher-order structure that represses transcription by means of the histone deacetylase Sir2 and structural proteins Sir3 and Sir4. Here, we present a fully reconstituted system to analyze SIR holocomplex binding to nucleosomal arrays. Purified Sir2-3-4 heterotrimers bind chromatin, cooperatively yielding a stable complex of homogeneous mol. wt. Remarkably, Sir2-3-4 also binds naked DNA, reflecting the strong, albeit nonspecific, DNA-binding activity of Sir4. The binding of Sir3 to nucleosomes is sensitive to histone H4 N-terminal tail removal, while that of Sir2-4 is not. Dot1-mediated methylation of histone H3K79 reduces the binding of both Sir3 and Sir2-3-4. Addnl., a byproduct of Sir2-mediated NAD hydrolysis, O-acetyl-ADP-ribose, increases the efficiency with which Sir3 and Sir2-3-4 bind nucleosomes. Thus, in small cumulative steps, each Sir protein, unmodified histone domains, and contacts with DNA contribute to the stability of the silent chromatin complex.
- 100Norris, A.; Bianchet, M. A.; Boeke, J. D. PLoS Genet. 2008, 4, e1000301Google ScholarThere is no corresponding record for this reference.
- 101Singer, M. S.; Kahana, A.; Wolf, A. J.; Meisinger, L. L.; Peterson, S. E.; Goggin, C.; Mahowald, M.; Gottschling, D. E. Genetics 1998, 150, 613Google Scholar101Identification of high-copy disruptors of telomeric silencing in Saccharomyces cerevisiaeSinger, Miriam S.; Kahana, Alon; Wolf, Alexander J.; Meisinger, Lia L.; Peterson, Suzanne E.; Goggin, Colin; Mahowald, Maureen; Gottschling, Daniel E.Genetics (1998), 150 (2), 613-632CODEN: GENTAE; ISSN:0016-6731. (Genetics Society of America)The ends of chromosomes in Saccharomyces cerevisiae initiate a repressive chromatin structure that spreads internally and inhibits the transcription of nearby genes, a phenomenon termed telomeric silencing. To investigate the mol. basis of this process, we carried out a genetic screen to identify genes whose overexpression disrupts telomeric silencing. We thus isolated 10 DOT genes (disruptor of telomeric silencing). Among these were genes encoding chromatin component Sir4p, DNA helicase Dna2p, ribosomal protein L32, and two proteins of unknown function, Asf1p and Ifh1p. The collection also included genes that had not previously been identified: DOT1, DOT4, DOT5, DOT6, and TLC1, which encodes the RNA template component of telomerase. With the exception of TLC1, all these genes, particularly DOT1 and DOT4, also reduced silencing at other repressed loci (HM loci and rDNA) when overexpressed. Moreover, deletion of the latter two genes weakened silencing as well, suggesting that DOT1 and DOT4 normally play important roles in gene repression. DOT1 deletion also affected telomere tract length. The function of Dot1p is not known. The sequence of Dot4p suggests that it is a ubiquitin-processing protease. Taken together, the DOT genes include both components and regulators of silent chromatin.
- 102Ng, H. H.; Ciccone, D. N.; Morshead, K. B.; Oettinger, M. A.; Struhl, K. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 1820Google Scholar102Lysine-79 of histone H3 is hypomethylated at silenced loci in yeast and mammalian cells: A potential mechanism for position-effect variegationNg, Huck Hui; Ciccone, David N.; Morshead, Katrina B.; Oettinger, Marjorie A.; Struhl, KevinProceedings of the National Academy of Sciences of the United States of America (2003), 100 (4), 1820-1825CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Methylation of lysine-79 (K79) within the globular domain of histone H3 by Dot1 methylase is important for transcriptional silencing and for assocn. of the Sir silencing proteins in yeast. Here, we show that the level of H3-K79 methylation is low at all Sir-dependent silenced loci but not at other transcriptionally repressed regions. Hypomethylation of H3-K79 at the telomeric and silent mating-type loci, but not the ribosomal DNA, requires the Sir proteins. Overexpression of Sir3 concomitantly extends the domain of Sir protein assocn. and H3-K79 hypomethylation at telomeres. In mammalian cells, H3-K79 methylation is found at loci that are active for V(D)J recombination, but not at recombinationally inactive loci that are heterochromatic. These results suggest that H3-K79 methylation is an evolutionarily conserved marker of active chromatin regions, and that silencing proteins block the ability of Dot1 to methylate histone H3. Further, they suggest that Sir proteins preferentially bind chromatin with hypomethylated H3-K79 and then block H3-K79 methylation. This pos. feedback loop, and the reverse loop in which H3-K79 methylation weakens Sir protein assocn. and leads to further methylation, suggests a model for position-effect variegation.
- 103van Leeuwen, F.; Gafken, P. R.; Gottschling, D. E. Cell 2002, 109, 745Google Scholar103Dot1p modulates silencing in yeast by methylation of the nucleosome coreVan Leeuwen, Fred; Gafken, Philip R.; Gottschling, Daniel E.Cell (Cambridge, MA, United States) (2002), 109 (6), 745-756CODEN: CELLB5; ISSN:0092-8674. (Cell Press)DOT1 was originally identified as a gene affecting telomeric silencing in S. cerevisiae. We now find that Dot1p methylates histone H3 on lysine 79, which maps to the top and bottom of the nucleosome core. Methylation occurs only when histone H3 is assembled in chromatin. In vivo, Dot1p is solely responsible for this methylation and methylates ∼90% of histone H3. In dot1Δ cells, silencing is compromised and silencing proteins become redistributed at the expense of normally silenced loci. We suggest that methylation of histone H3 lysine 79 limits silencing to discrete loci by preventing the binding of Sir proteins elsewhere along the genome. Because Dot1p and histone H3 are conserved, similar mechanisms are likely at work in other eukaryotes.
- 104Arnaudo, N.; Fernández, I. S.; McLaughlin, S. H.; Peak-Chew, S. Y.; Rhodes, D.; Martino, F. Nat. Struct. Mol. Biol. 2013, 20, 1119Google Scholar104The N-terminal acetylation of Sir3 stabilizes its binding to the nucleosome core particleArnaudo, Nadia; Fernandez, Israel S.; McLaughlin, Stephen H.; Peak-Chew, Sew Y.; Rhodes, Daniela; Martino, FabrizioNature Structural & Molecular Biology (2013), 20 (9), 1119-1121CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)The N-terminal acetylation of Sir3 is essential for heterochromatin establishment and maintenance in yeast, but its mechanism of action is unknown. The crystal structure of the N-terminally acetylated BAH domain of Saccharomyces cerevisiae Sir3 bound to the nucleosome core particle reveals that the N-terminal acetylation stabilizes the interaction of Sir3 with the nucleosome. Addnl., we present a new method for the prodn. of protein-nucleosome complexes for structural anal.
- 105Yang, D.; Fang, Q.; Wang, M.; Ren, R.; Wang, H.; He, M.; Sun, Y.; Yang, N.; Xu, R.-M. Nat. Struct. Mol. Biol. 2013, 20, 1116Google Scholar105Nα-acetylated Sir3 stabilizes the conformation of a nucleosome-binding loop in the BAH domainYang, Dongxue; Fang, Qianglin; Wang, Mingzhu; Ren, Ren; Wang, Hong; He, Meng; Sun, Youwei; Yang, Na; Xu, Rui-MingNature Structural & Molecular Biology (2013), 20 (9), 1116-1118CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)In Saccharomyces cerevisiae, acetylation of the Sir3 N terminus is important for transcriptional silencing. This covalent modification promotes the binding of the Sir3 BAH domain to the nucleosome, but a mechanistic understanding of this phenomenon is lacking. By X-ray crystallog., we show here that the acetylated N terminus of Sir3 does not interact with the nucleosome directly. Instead, it stabilizes a nucleosome-binding loop in the BAH domain.
- 106Connelly, J. J.; Yuan, P.; Hsu, H.-C.; Li, Z.; Xu, R.-M.; Sternglanz, R. Mol. Cell. Biol. 2006, 26, 3256Google Scholar106Structure and function of the Saccharomyces cerevisiae Sir3 BAH domainConnelly, Jessica J.; Yuan, Peihua; Hsu, Hao-Chi; Li, Zhizhong; Xu, Rui-Ming; Sternglanz, RolfMolecular and Cellular Biology (2006), 26 (8), 3256-3265CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)Previous work has shown that the N terminus of the Saccharomyces cerevisiae Sir3 protein is crucial for the function of Sir3 in transcriptional silencing. Here, we show that overexpression of N-terminal fragments of Sir3 in strains lacking the full-length protein can lead to some silencing of HML and HMR. Sir3 contains a BAH (bromo-adjacent homol.) domain at its N terminus. Overexpression of this domain alone can lead to silencing as long as Sir1 is overexpressed and Sir2 and Sir4 are present. Overexpression of the closely related Orc1 BAH domain can also silence in the absence of any Sir3 protein. A previously characterized hypermorphic sir3 mutation, D205N, greatly improves silencing by the Sir3 BAH domain and allows it to bind to DNA and oligonucleosomes in vitro. A previously uncharacterized region in the Sir1 N terminus is required for silencing by both the Sir3 and Orc1 BAH domains. The structure of the Sir3 BAH domain has been detd. In the crystal, the mol. multimerizes in the form of a left-handed superhelix. This superhelix may be relevant to the function of the BAH domain of Sir3 in silencing.
- 107Kato, H.; Jiang, J.; Zhou, B.-R.; Rozendaal, M.; Feng, H.; Ghirlando, R.; Xiao, T. S.; Straight, A. F.; Bai, Y. Science 2013, 340, 1110Google Scholar107A conserved mechanism for centromeric nucleosome recognition by centromere protein CENP-CKato, Hidenori; Jiang, Jiansheng; Zhou, Bing-Rui; Rozendaal, Marieke; Feng, Hanqiao; Ghirlando, Rodolfo; Xiao, T. Sam; Straight, Aaron F.; Bai, YawenScience (Washington, DC, United States) (2013), 340 (6136), 1110-1113CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Chromosome segregation during mitosis requires assembly of the kinetochore complex at the centromere. Kinetochore assembly depends on specific recognition of the histone variant CENP-A in the centromeric nucleosome by centromere protein C (CENP-C). Here, the authors defined the determinants of this recognition mechanism and discovered that CENP-C binds a hydrophobic region in the CENP-A tail and docks onto the acidic patch of histone H2A and H2B. The authors further found that the more broadly conserved CENP-C motif used the same mechanism for CENP-A nucleosome recognition. These findings revealed a conserved mechanism for protein recruitment to centromeres and a histone recognition mode whereby a disordered peptide binds the histone tail through hydrophobic interactions facilitated by nucleosome docking.
- 108Henikoff, S.; Ahmad, K.; Malik, H. S. Science 2001, 293, 1098Google Scholar108The centromere paradox: Stable inheritance with rapidly evolving DNAHenikoff, Steven; Ahmad, Kami; Malik, Harmit S.Science (Washington, DC, United States) (2001), 293 (5532), 1098-1102CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The review. Every eukaryotic chromosome has a centromere, the locus responsible for poleward movement at mitosis and meiosis. Although conventional loci are specified by their DNA sequences, current evidence favors a chromatin-based inheritance mechanism for centromeres. The chromosome segregation machinery is highly conserved across all eukaryotes, but the DNA and protein components specific to centromeric chromatin are evolving rapidly. Incompatibilities between rapidly evolving centromeric components may be responsible for both the organization of centromeric regions and the reproductive isolation of emerging species.
- 109Verdaasdonk, J. S.; Bloom, K. Nat. Rev. Mol. Cell Biol. 2011, 12, 320Google Scholar109Centromeres: unique chromatin structures that drive chromosome segregationVerdaasdonk, Jolien S.; Bloom, KerryNature Reviews Molecular Cell Biology (2011), 12 (5), 320-332CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. Fidelity during chromosome segregation is essential to prevent aneuploidy. The proteins and chromatin at the centromere form a unique site for kinetochore attachment and allow the cell to sense and correct errors during chromosome segregation. Centromeric chromatin is characterized by distinct chromatin organization, epigenetics, centromere-assocd. proteins, and histone variants. These include histone H3 variant centromeric protein A (CENP-A), the compn. and deposition of which have been widely investigated. Studies have examd. the structural and biophys. properties of the centromere and have suggested that the centromere is not simply a 'landing pad' for kinetochore formation, but plays an essential role in mitosis by assembling and directing the organization of the kinetochore.
- 110Hori, T.; Amano, M.; Suzuki, A.; Backer, C. B.; Welburn, J. P.; Dong, Y.; McEwen, B. F.; Shang, W.-H.; Suzuki, E.; Okawa, K.; Cheeseman, I. M.; Fukagawa, T. Cell 2008, 135, 1039Google Scholar110CCAN makes multiple contacts with centromeric DNA to provide distinct pathways to the outer kinetochoreHori, Tetsuya; Amano, Miho; Suzuki, Aussie; Backer, Chelsea B.; Welburn, Julie P.; Dong, Yimin; McEwen, Bruce F.; Shang, Wei-Hao; Suzuki, Emiko; Okawa, Katsuya; Cheeseman, Iain M.; Fukagawa, TatsuoCell (Cambridge, MA, United States) (2008), 135 (6), 1039-1052CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Kinetochore specification and assembly requires the targeted deposition of specialized nucleosomes contg. the histone H3 variant CENP-A at centromeres. However, CENP-A is not sufficient to drive full-kinetochore assembly, and it is not clear how centromeric chromatin is established. Here, we identify CENP-W as a component of the DNA-proximal constitutive centromere-assocd. network (CCAN) of proteins. We demonstrate that CENP-W forms a DNA-binding complex together with the CCAN component CENP-T. This complex directly assocs. with nucleosomal DNA and with canonical histone H3, but not with CENP-A, in centromeric regions. CENP-T/CENP-W functions upstream of other CCAN components with the exception of CENP-C, an addnl. putative DNA-binding protein. Our anal. indicates that CENP-T/CENP-W and CENP-C provide distinct pathways to connect the centromere with outer kinetochore assembly. In total, our results suggest that the CENP-T/CENP-W complex is directly involved in establishment of centromere chromatin structure coordinately with CENP-A.
- 111McGinty, R. K.; Henrici, R. C.; Tan, S. Nature 2014, 514, 591Google Scholar111Crystal structure of the PRC1 ubiquitylation module bound to the nucleosomeMcGinty, Robert K.; Henrici, Ryan C.; Tan, SongNature (London, United Kingdom) (2014), 514 (7524), 591-596CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)The Polycomb group of epigenetic enzymes represses expression of developmentally regulated genes in many eukaryotes. This group includes the Polycomb repressive complex 1 (PRC1), which ubiquitylates nucleosomal histone H2A Lys 119 using its E3 ubiquitin ligase subunits, Ring1B and Bmi1, together with an E2 ubiquitin-conjugating enzyme, UbcH5c. However, the mol. mechanism of nucleosome substrate recognition by PRC1 or other chromatin enzymes is unclear. Here we present the crystal structure of the human Ring1B-Bmi1-UbcH5c E3-E2 complex (the PRC1 ubiquitylation module) bound to its nucleosome core particle substrate. The structure shows how a chromatin enzyme achieves substrate specificity by interacting with several nucleosome surfaces spatially distinct from the site of catalysis. Our structure further reveals an unexpected role for the ubiquitin E2 enzyme in substrate recognition, and provides insight into how the related histone H2A E3 ligase, BRCA1, interacts with and ubiquitylates the nucleosome.
- 112Cao, R.; Tsukada, Y.-I.; Zhang, Y. Mol. Cell 2005, 20, 845Google Scholar112Role of Bmi-1 and Ring1A in H2A ubiquitylation and Hox gene silencingCao, Ru; Tsukada, Yu-ichi; Zhang, YiMolecular Cell (2005), 20 (6), 845-854CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)Polycomb group (PcG) proteins exist in at least two biochem. distinct protein complexes, the EED-EZH2 complex and the PRC1 complex, that resp. possess H3-K27 methyltransferase and H2A-K119 ubiquitin E3 ligase activities. How the enzymic activities are regulated and what their role is in Hox gene silencing are not clear. Here, the authors demonstrate that Bmi-1 and Ring1A, two components of the PRC1 complex, play important roles in H2A ubiquitylation and Hox gene silencing. Both proteins pos. regulate H2A ubiquitylation. Chromatin immunopptn. (ChIP) assays demonstrate that Bmi-1 and other components of the two PcG complexes bind to the promoter of HoxC13. Knockout of Bmi-1 results in significant loss of H2A ubiquitylation and upregulation of Hoxc13 expression, whereas EZH2-mediated H3-K27 methylation is not affected. These results suggest that EZH2-mediated H3-K27 methylation functions upstream of PRC1 and establishes a crit. role for Bmi-1 and Ring1A in H2A ubiquitylation and Hox gene silencing.
- 113Grau, D. J.; Chapman, B. A.; Garlick, J. D.; Borowsky, M.; Francis, N. J.; Kingston, R. E. Genes Dev. 2011, 25, 2210Google Scholar113Compaction of chromatin by diverse polycomb group proteins requires localized regions of high chargeGrau, Daniel J.; Chapman, Brad A.; Garlick, Joe D.; Borowsky, Mark; Francis, Nicole J.; Kingston, Robert E.Genes & Development (2011), 25 (20), 2210-2221CODEN: GEDEEP; ISSN:0890-9369. (Cold Spring Harbor Laboratory Press)Polycomb group (PcG) proteins are required for the epigenetic maintenance of developmental genes in a silent state. Proteins in the Polycomb-repressive complex 1 (PRC1) class of the PcG are conserved from flies to humans and inhibit transcription. One hypothesis for PRC1 mechanism is that it compacts chromatin, based in part on electron microscopy expts. demonstrating that Drosophila PRC1 compacts nucleosomal arrays. We show that this function is conserved between Drosophila and mouse PRC1 complexes and requires a region with an overrepresentation of basic amino acids. While the active region is found in the Posterior Sex Combs (PSC) subunit in Drosophila, it is unexpectedly found in a different PRC1 subunit, a Polycomb homolog called M33, in mice. We provide exptl. support for the general importance of a charged region by predicting the compacting capability of PcG proteins from species other than Drosophila and mice and by testing several of these proteins using soln. assays and microscopy. We infer that the ability of PcG proteins to compact chromatin in vitro can be predicted by the presence of domains of high pos. charge and that PRC1 components from a variety of species conserve this highly charged region. This supports the hypothesis that compaction is a key aspect of PcG function.
- 114Simon, J. A.; Kingston, R. E. Nat. Rev. Mol. Cell Biol. 2009, 10, 697Google Scholar114Mechanisms of Polycomb gene silencing: Knowns and unknownsSimon, Jeffrey A.; Kingston, Robert E.Nature Reviews Molecular Cell Biology (2009), 10 (10), 697-708CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. Polycomb proteins form chromatin-modifying complexes that implement transcriptional silencing in higher eukaryotes. Hundreds of genes are silenced by Polycomb proteins, including dozens of genes that encode crucial developmental regulators in organisms ranging from plants to humans. Two main families of complexes, called Polycomb repressive complex 1 (PRC1) and PRC2, are targeted to repressed regions. Recent studies have advanced our understanding of these complexes, including their potential mechanisms of gene silencing, the roles of chromatin modifications, their means of delivery to target genes and the functional distinctions among variant complexes. Emerging concepts include the existence of a Polycomb barrier to transcription elongation and the involvement of non-coding RNAs in the targeting of Polycomb complexes. These findings have an impact on the epigenetic programming of gene expression in many biol. systems.
- 115Buchwald, G.; van der Stoop, P.; Weichenrieder, O.; Perrakis, A.; van Lohuizen, M.; Sixma, T. K. EMBO J. 2006, 25, 2465Google Scholar115Structure and E3-ligase activity of the Ring-Ring complex of Polycomb proteins Bmi1 and Ring1bBuchwald, Gretel; van der Stoop, Petra; Weichenrieder, Oliver; Perrakis, Anastassis; van Lohuizen, Maarten; Sixma, Titia K.EMBO Journal (2006), 25 (11), 2465-2474CODEN: EMJODG; ISSN:0261-4189. (Nature Publishing Group)Polycomb group (PcG) proteins Ring1b and Bmi1 (B-cell-specific Moloney murine leukemia virus integration site 1) are crit. components of the chromatin-modulating PRC1 complex. Histone H2A ubiquitination by the PRC1 complex strongly depends on the Ring1b protein. Here we show that the E3-ligase activity of Ring1b on histone H2A is enhanced by Bmi1 in vitro. The N-terminal Ring-domains are sufficient for this activity and Ring1a can replace Ring1b. E2 enzymes UbcH5a, b, c or UbcH6 support this activity with varying processivity and selectivity. All four E2s promote autoubiquitination of Ring1b without affecting E3-ligase activity. We solved the crystal structure of the Ring-Ring heterodimeric complex of Ring1b and Bmi1. In the structure the arrangement of the Ring-domains is similar to another H2A E3 ligase, the BRCA1/BARD1 complex, but complex formation depends on an N-terminal arm of Ring1b that embraces the Bmi1 Ring-domain. Mutation of a crit. residue in the E2/E3 interface shows that catalytic activity resides in Ring1b and not in Bmi1. These data provide a foundation for understanding the crit. enzymic activity at the core of the PRC1 polycomb complex, which is implicated in stem cell maintenance and cancer.
- 116Bentley, M. L.; Corn, J. E.; Dong, K. C.; Phung, Q.; Cheung, T. K.; Cochran, A. G. EMBO J. 2011, 30, 3285Google Scholar116Recognition of UbcH5c and the nucleosome by the Bmi1/Ring1b ubiquitin ligase complexBentley, Matthew L.; Corn, Jacob E.; Dong, Ken C.; Phung, Qui; Cheung, Tommy K.; Cochran, Andrea G.EMBO Journal (2011), 30 (16), 3285-3297CODEN: EMJODG; ISSN:0261-4189. (Nature Publishing Group)The Polycomb repressive complex 1 (PRC1) mediates gene silencing, in part by monoubiquitination of histone H2A on lysine 119 (uH2A). Bmi1 and Ring1b are crit. components of PRC1 that heterodimerize via their N-terminal RING domains to form an active E3 ubiquitin ligase. We have detd. the crystal structure of a complex between the Bmi1/Ring1b RING-RING heterodimer and the E2 enzyme UbcH5c and find that UbcH5c interacts with Ring1b only, in a manner fairly typical of E2-E3 interactions. However, we further show that the Bmi1/Ring1b RING domains bind directly to duplex DNA through a basic surface patch unique to the Bmi1/Ring1b RING-RING dimer. Mutation of residues on this interaction surface leads to a loss of H2A ubiquitination activity. Computational modeling of the interface between Bmi1/Ring1b-UbcH5c and the nucleosome suggests that Bmi1/Ring1b interacts with both nucleosomal DNA and an acidic patch on histone H4 to achieve specific monoubiquitination of H2A. Our results point to a novel mechanism of substrate recognition, and control of product formation, by Bmi1/Ring1b.
- 117Leung, J. W.; Agarwal, P.; Canny, M. D.; Gong, F.; Robison, A. D.; Finkelstein, I. J.; Durocher, D.; Miller, K. M. PLoS Genet. 2014, 10, e1004178Google Scholar117Nucleosome acidic patch promotes RNF168- and RING1B/BMI1-dependent H2AX and H2A ubiquitination and DNA damage signalingLeung, Justin W.; Agarwal, Poonam; Canny, Marella D.; Gong, Fade; Robison, Aaron D.; Finkelstein, Ilya J.; Durocher, Daniel; Miller, Kyle M.PLoS Genetics (2014), 10 (3), e1004178/1-e1004178/14, 14 pp.CODEN: PGLEB5; ISSN:1553-7404. (Public Library of Science)Histone ubiquitinations are crit. for the activation of the DNA damage response (DDR). In particular, RNF168 and RING1B/BMI1 function in the DDR by ubiquitinating H2A/H2AX on Lys-13/15 and Lys-118/119, resp. However, it remains to be defined how the ubiquitin pathway engages chromatin to provide regulation of ubiquitin targeting of specific histone residues. Here we identify the nucleosome acid patch as a crit. chromatin mediator of H2A/H2AX ubiquitination (ub). The acidic patch is required for RNF168- and RING1B/BMI1-dependent H2A/H2AXub in vivo. The acidic patch functions within the nucleosome as nucleosomes contg. a mutated acidic patch exhibit defective H2A/H2AXub by RNF168 and RING1B/BMI1 in vitro. Furthermore, direct perturbation of the nucleosome acidic patch in vivo by the expression of an engineered acidic patch interacting viral peptide, LANA, results in defective H2AXub and RNF168-dependent DNA damage responses including 53BP1 and BRCA1 recruitment to DNA damage. The acidic patch therefore is a crit. nucleosome feature that may serve as a scaffold to integrate multiple ubiquitin signals on chromatin to compose selective ubiquitinations on histones for DNA damage signaling.
- 118Kato, H.; van Ingen, H.; Zhou, B.-R.; Feng, H.; Bustin, M.; Kay, L. E.; Bai, Y. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 12283Google Scholar118Architecture of the high mobility group nucleosomal protein 2-nucleosome complex as revealed by methyl-based NMRKato, Hidenori; van Ingen, Hugo; Zhou, Bing-Rui; Feng, Hanqiao; Bustin, Michael; Kay, Lewis E.; Bai, YawenProceedings of the National Academy of Sciences of the United States of America (2011), 108 (30), 12283-12288, S12283/1-S12283/15CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Chromatin structure and function are regulated by numerous proteins through specific binding to nucleosomes. The structural basis of many of these interactions is unknown, as in the case of the high mobility group nucleosomal (HMGN) protein family that regulates various chromatin functions, including transcription. Here, we report the architecture of the HMGN2-nucleosome complex detd. by a combination of methyl-transverse relaxation optimized NMR spectroscopy (methyl-TROSY) and mutational anal. We found that HMGN2 binds to both the acidic patch in the H2A-H2B dimer and to nucleosomal DNA near the entry/exit point, "stapling" the histone core and the DNA. These results provide insight into how HMGNs regulate chromatin structure through interfering with the binding of linker histone H1 to the nucleosome as well as a structural basis of how phosphorylation induces dissocn. of HMGNs from chromatin during mitosis. Importantly, our approach is generally applicable to the study of nucleosome-binding interactions in chromatin.
- 119Zhou, B.-R.; Feng, H.; Kato, H.; Dai, L.; Yang, Y.; Zhou, Y.; Bai, Y. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 19390Google Scholar119Structural insights into the histone H1-nucleosome complexZhou, Bing-Rui; Feng, Hanqiao; Kato, Hidenori; Dai, Liang; Yang, Yuedong; Zhou, Yaoqi; Bai, YawenProceedings of the National Academy of Sciences of the United States of America (2013), 110 (48), 19390-19395,S19390/1-S19390/27CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Linker H1 histones facilitate formation of higher-order chromatin structures and play important roles in various cell functions. Despite several decades of effort, the structural basis of how H1 interacts with the nucleosome remains elusive. Here, we investigated Drosophila H1 in complex with the nucleosome, using soln. NMR spectroscopy and other biophys. methods. We found that the globular domain of H1 bridges the nucleosome core and one 10-base pair linker DNA asym., with its α3 helix facing the nucleosomal DNA near the dyad axis. Two short regions in the C-terminal tail of H1 and the C-terminal tail of one of the two H2A histones are also involved in the formation of the H1-nucleosome complex. Our results lead to a residue-specific structural model for the globular domain of the Drosophila H1 in complex with the nucleosome, which is different from all previous expt.-based models and has implications for chromatin dynamics in vivo.
- 120Rattner, B. P.; Yusufzai, T.; Kadonaga, J. T. Mol. Cell 2009, 34, 620Google Scholar120HMGN proteins act in opposition to ATP-dependent chromatin remodeling factors to restrict nucleosome mobilityRattner, Barbara P.; Yusufzai, Timur; Kadonaga, James T.Molecular Cell (2009), 34 (5), 620-626CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)The high-mobility group N (HMGN) proteins are abundant nonhistone chromosomal proteins that bind specifically to nucleosomes at two high-affinity sites. Here we report that purified recombinant human HMGN1 (HMG14) and HMGN2 (HMG17) potently repress ATP-dependent chromatin remodeling by four different mol. motor proteins. In contrast, mutant HMGN proteins with double Ser-to-Glu mutations in their nucleosome-binding domains are unable to inhibit chromatin remodeling. The HMGN-mediated repression of chromatin remodeling is reversible and dynamic. With the ACF chromatin remodeling factor, HMGN2 does not directly inhibit the ATPase activity but rather appears to reduce the affinity of the factor to chromatin. These findings suggest that HMGN proteins serve as a counterbalance to the action of the many ATP-dependent chromatin remodeling activities in the nucleus.
- 121Lim, J.-H.; West, K. L.; Rubinstein, Y.; Bergel, M.; Postnikov, Y. V.; Bustin, M. EMBO J. 2005, 24, 3038Google Scholar121Chromosomal protein HMGN1 enhances the acetylation of lysine 14 in histone H3Lim, Jae-Hwan; West, Katherine L.; Rubinstein, Yaffa; Bergel, Michael; Postnikov, Yuri V.; Bustin, MichaelEMBO Journal (2005), 24 (17), 3038-3048CODEN: EMJODG; ISSN:0261-4189. (Nature Publishing Group)The acetylation levels of lysine residues in nucleosomes, which are detd. by the opposing activities of histone acetyltransferases (HATs) and deacetylases, play an important role in regulating chromatin-related processes, including transcription. We report that HMGN1, a nucleosomal binding protein that reduces the compaction of the chromatin fiber, increases the levels of acetylation of K14 in H3. The levels of H3K14ac in Hmgn1-/- cells are lower than in Hmgn1+/+ cells. Induced expression of wild-type HMGN1, but not of a mutant that does not bind to chromatin, in Hmgn1-/- cells elevates the levels of H3K14ac. In vivo, HMGN1 elevates the levels of H3K14ac by enhancing the action of HAT. In vitro, HMGN1 enhances the ability of PCAF to acetylate nucleosomal, but not free, H3. Thus, HMGN1 modulates the levels of H3K14ac by binding to chromatin. We suggest that HMGN1, and perhaps similar architectural proteins, modulates the levels of acetylation in chromatin by altering the equil. generated by the opposing enzymic activities that continuously modify and de-modify the histone tails in nucleosomes.
- 122Lim, J.-H.; Catez, F.; Birger, Y.; West, K. L.; Prymakowska-Bosak, M.; Postnikov, Y. V.; Bustin, M. Mol. Cell 2004, 15, 573Google Scholar122Chromosomal protein HMGN1 modulates histone H3 phosphorylationLim, Jae-Hwan; Catez, Frederic; Birger, Yehudit; West, Katherine L.; Prymakowska-Bosak, Marta; Postnikov, Yuri V.; Bustin, MichaelMolecular Cell (2004), 15 (4), 573-584CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)Here we demonstrate that HMGN1, a nuclear protein that binds to nucleosomes and reduces the compaction of the chromatin fiber, modulates histone posttranslational modifications. In Hmgn1-/- cells, loss of HMGN1 elevates the steady-state levels of phospho-S10-H3 and enhances the rate of stress-induced phosphorylation of S10-H3. In vitro, HMGN1 reduces the rate of phospho-S10-H3 by hindering the ability of kinases to modify nucleosomal, but not free, H3. During anisomycin treatment, the phosphorylation of HMGN1 precedes that of H3 and leads to a transient weakening of the binding of HMGN1 to chromatin. We propose that the reduced binding of HMGN1 to nucleosomes, or the absence of the protein, improves access of anisomysin-induced kinases to H3. Thus, the levels of posttranslational modifications in chromatin are modulated by nucleosome binding proteins that alter the ability of enzymic complexes to access and modify their nucleosomal targets.
- 123Postnikov, Y.; Bustin, M. Biochim. Biophys. Acta, Gene Regul. Mech. 2010, 1799, 62Google Scholar123Regulation of chromatin structure and function by HMGN proteinsPostnikov, Yuri; Bustin, MichaelBiochimica et Biophysica Acta, Gene Regulatory Mechanisms (2010), 1799 (1-2), 62-68CODEN: BBAGC6; ISSN:1874-9399. (Elsevier B.V.)A review. High mobility group nucleosome-binding (HMGN) proteins are architectural non-histone chromosomal proteins that bind to nucleosomes and modulate the structure and function of chromatin. The interaction of HMGN proteins with nucleosomes is dynamic and the proteins compete with the linker histone H1 chromatin-binding sites. HMGNs reduce the H1-mediated compaction of the chromatin fiber and facilitate the targeting of regulatory factors to chromatin. They modulate the cellular epigenetic profile, affect gene expression, and impact biol. processes such as development and the cellular response to environmental and hormonal signals. Here, the authors review the role of HMGN in chromatin structure, the link between HMGN proteins and histone modifications, and discuss the consequence of this link on nuclear processes and cellular phenotype.
- 124Prymakowska-Bosak, M.; Misteli, T.; Herrera, J. E.; Shirakawa, H.; Birger, Y.; Garfield, S.; Bustin, M. Mol. Cell. Biol. 2001, 21, 5169Google Scholar124Mitotic phosphorylation prevents the binding of HMGN proteins to chromatinPrymakowska-Bosak, Marta; Misteli, Tom; Herrera, Julio E.; Shirakawa, Hitoshi; Birger, Yehudit; Garfield, Susan; Bustin, MichaelMolecular and Cellular Biology (2001), 21 (15), 5169-5178CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)Condensation of the chromatin fiber and transcriptional inhibition during mitosis is assocd. with the redistribution of many DNA- and chromatin-binding proteins, including members of the high-mobility-group N (HMGN) family. Here we study the mechanism governing the organization of HMGN proteins in mitosis. Using site-specific antibodies and quant. gel anal. with proteins extd. from synchronized HeLa cells, we demonstrate that, during mitosis, the conserved serine residues in the nucleosomal binding domain (NBD) of this protein family are highly and specifically phosphorylated. Nucleosome mobility shift assays with both in vitro-phosphorylated proteins and with point mutants bearing neg. charges in the NBD demonstrate that the neg. charge abolishes the ability of the proteins to bind to nucleosomes. Fluorescence loss of photobleaching demonstrates that, in living cells, the neg. charge in the NBD increases the intranuclear mobility of the protein and significantly decreases the relative time that it is bound to chromatin. Expression of wild-type and mutant proteins in HmgN1-/- cells indicates that the neg. charged protein is not bound to chromosomes. We conclude that during mitosis the NBD of HMGN proteins is highly phosphorylated and that this modification regulates the interaction of the proteins with chromatin.
- 125Mattiroli, F.; Uckelmann, M.; Sahtoe, D. D.; van Dijk, W. J.; Sixma, T. K. Nat. Commun. 2014, 5, 3291Google Scholar125The nucleosome acidic patch plays a critical role in RNF168-dependent ubiquitination of histone H2AMattiroli Francesca; Uckelmann Michael; Sahtoe Danny D; van Dijk Willem J; Sixma Titia KNature communications (2014), 5 (), 3291 ISSN:.During DNA damage response, the RING E3 ligase RNF168 ubiquitinates nucleosomal H2A at K13-15. Here we show that the ubiquitination reaction is regulated by its substrate. We define a region on the RING domain important for target recognition and identify the H2A/H2B dimer as the minimal substrate to confer lysine specificity to the RNF168 reaction. Importantly, we find an active role for the substrate in the reaction. H2A/H2B dimers and nucleosomes enhance the E3-mediated discharge of ubiquitin from the E2 and redirect the reaction towards the relevant target, in a process that depends on an intact acidic patch. This active contribution of a region distal from the target lysine provides regulation of the specific K13-15 ubiquitination reaction during the complex signalling process at DNA damage sites.
- 126Carriere, V.; Roussel, L.; Ortega, N.; Lacorre, D.-A.; Americh, L.; Aguilar, L.; Bouche, G.; Girard, J.-P. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 282Google Scholar126IL-33, the IL-1-like cytokine ligand for ST2 receptor, is a chromatin-associated nucleus factor in vivoCarriere, Virginie; Roussel, Lucie; Ortega, Nathalie; Lacorre, Delphine-Armelle; Americh, Laure; Aguilar, Luc; Bouche, Gerard; Girard, Jean-PhilippeProceedings of the National Academy of Sciences of the United States of America (2007), 104 (1), 282-287CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Recent studies indicate that IL-1α functions intracellularly in path ways independent of its cell surface receptors by translocating to the nucleus and regulating transcription. Similarly, the chromatin-assocd. protein HMGB1 acts as both a nuclear factor and a secreted proinflammatory cytokine. Here, we show that IL-33, an IL-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-assocd. cytokines, is an endothelium-derived, chromatin-assocd. nuclear factor with transcriptional repressor properties. We found that IL-33 is identical to NF-HEV, a nuclear factor preferentially expressed in high endothelial venules (HEV), that we previously characterized. Accordingly, in situ hybridization demonstrated that endothelial cells constitute a major source of IL-33 mRNA in chronically inflamed tissues from patients with rheumatoid arthritis and Crohn's disease. Immunostaining with three distinct antisera, directed against the N-terminal part and IL-1-like C-terminal domain, revealed that IL-33 is a heterochromatin-assocd. nuclear factor in HEV endothelial cells in vivo. Assocn. of IL-33 with heterochromatin was also obsd. in human and mouse cells under living conditions. In addn., colocalization of IL-33 with mitotic chromatin was noted. Nuclear localization, heterochromatin-assocn., and targeting to mitotic chromosomes were all found to be mediated by an evolutionarily conserved homeodomain-like helix-turn-helix motif within the IL-33 N-terminal part. Finally, IL-33 was found to possess transcriptional repressor properties, assocd. with the homeodomain-like helix-turn-helix motif. Together, these data suggest that, similarly to IL1α and HMGB1, IL-33 is a dual function protein that may function as both a proinflammatory cytokine and an intracellular nuclear factor with transcriptional regulatory properties.
- 127Kalashnikova, A. A.; Porter-Goff, M. E.; Muthurajan, U. M.; Luger, K.; Hansen, J. C. J. R. Soc., Interface 2013, 10, 20121022Google Scholar127The role of the nucleosome acidic patch in modulating higher order chromatin structureKalashnikova, Anna A.; Porter-Goff, Mary E.; Muthurajan, Uma M.; Luger, Karolin; Hansen, Jeffrey C.Journal of the Royal Society, Interface (2013), 10 (82), 20121022/1-20121022/9CODEN: JRSICU; ISSN:1742-5689. (Royal Society)A review. Higher order folding of chromatin fiber is mediated by interactions of the histone H4 N-terminal tail domains with neighboring nucleosomes. Mechanistically, the H4 tails of one nucleosome bind to the acidic patch region on the surface of adjacent nucleosomes, causing fiber compaction. The functionality of the chromatin fiber can be modified by proteins that interact with the nucleosome. The co-structures of five different proteins with the nucleosome (LANA, IL-33, RCC1, Sir3 and HMGN2) recently have been examd. by exptl. and computational studies. Interestingly, each of these proteins displays steric, ionic and hydrogen bond complementarity with the acidic patch, and therefore will compete with each other for binding to the nucleosome. We first review the mol. details of each interface, focusing on the key non-covalent interactions that stabilize the protein-acidic patch interactions. We then propose a model in which binding of proteins to the nucleosome disrupts interaction of the H4 tail domains with the acidic patch, preventing the intrinsic chromatin folding pathway and leading to assembly of alternative higher order chromatin structures with unique biol. functions.
- 128Clapier, C. R.; Cairns, B. R. Annu. Rev. Biochem. 2009, 78, 273Google Scholar128The biology of chromatin remodeling complexesClapier, Cedric R.; Cairns, Bradley R.Annual Review of Biochemistry (2009), 78 (), 273-304CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)A review. The packaging of chromosomal DNA by nucleosomes condenses and organizes the genome, but occludes many regulatory DNA elements. However, this constraint also allows nucleosomes and other chromatin components to actively participate in the regulation of transcription, chromosome segregation, DNA replication, and DNA repair. To enable dynamic access to packaged DNA and to tailor nucleosome compn. in chromosomal regions, cells have evolved a set of specialized chromatin remodeling complexes (remodelers). Remodelers use the energy of ATP hydrolysis to move, destabilize, eject, or restructure nucleosomes. Here, we address many aspects of remodeler biol.: their targeting, mechanism, regulation, shared and unique properties, and specialization for particular biol. processes. We also address roles for remodelers in development, cancer, and human syndromes.
- 129Chaban, Y.; Ezeokonkwo, C.; Chung, W.-H.; Zhang, F.; Kornberg, R. D.; Maier-Davis, B.; Lorch, Y.; Asturias, F. J. Nat. Struct. Mol. Biol. 2008, 15, 1272Google Scholar129Structure of a RSC-nucleosome complex and insights into chromatin remodelingChaban, Yuriy; Ezeokonkwo, Chukwudi; Chung, Wen-Hsiang; Zhang, Fan; Kornberg, Roger D.; Maier-Davis, Barbara; Lorch, Yahli; Asturias, Francisco J.Nature Structural & Molecular Biology (2008), 15 (12), 1272-1277CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)ATP-dependent chromatin-remodeling complexes, such as RSC, can reposition, evict, or restructure nucleosomes. A structure of a RSC-nucleosome complex with a nucleosome detd. by cryo-EM shows the nucleosome bound in a central RSC cavity. Extensive interaction of RSC with histones and DNA seems to destabilize the nucleosome and lead to an overall ATP-independent rearrangement of its structure. Nucleosomal DNA appears disordered and largely free to bulge out into soln. as required for remodeling, but the structure of the RSC-nucleosome complex indicates that RSC is unlikely to displace the octamer from the nucleosome to which it is bound. Consideration of the RSC-nucleosome structure and published biochem. information suggests that ATP-dependent DNA translocation by RSC may result in the eviction of histone octamers from adjacent nucleosomes.
- 130Asturias, F. J.; Chung, W.-H.; Kornberg, R. D.; Lorch, Y. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 13477Google Scholar130Structural analysis of the RSC chromatin-remodeling complexAsturias, Francisco J.; Chung, Wen-Hsiang; Kornberg, Roger D.; Lorch, YahliProceedings of the National Academy of Sciences of the United States of America (2002), 99 (21), 13477-13480CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Electron microscopy of the RSC chromatin-remodeling complex reveals a ring of protein densities around a central cavity. The size and shape of the cavity correspond closely to those of a nucleosome. Results of nuclease protection anal. are consistent with nucleosome binding in the cavity. Such binding could explain the ability of RSC to expose nucleosomal DNA in the presence of ATP without loss of assocd. histones.
- 131Saha, A.; Wittmeyer, J.; Cairns, B. R. Nat. Struct. Mol. Biol. 2005, 12, 747Google Scholar131Chromatin remodeling through directional DNA translocation from an internal nucleosomal siteSaha, Anjanabha; Wittmeyer, Jacqueline; Cairns, Bradley R.Nature Structural & Molecular Biology (2005), 12 (9), 747-755CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)The RSC chromatin remodeler contains Sth1, an ATP-dependent DNA translocase. On DNA substrates, RSC/Sth1 tracks along one strand of the duplex with a 3' → 5' polarity and a tracking requirement of one base, properties that may enable directional DNA translocation on nucleosomes. The binding of RSC or Sth1 elicits a DNase I-hypersensitive site approx. two DNA turns from the nucleosomal dyad, and the binding of Sth1 requires intact DNA at this location. Results with various nucleosome substrates suggest that RSC/Sth1 remains at a fixed position on the histone octamer and that Sth1 conducts directional DNA translocation from a location about two turns from the nucleosomal dyad, drawing in DNA from one side of the nucleosome and pumping it toward the other. These studies suggest that nucleosome mobilization involves directional DNA translocation initiating from a fixed internal site on the nucleosome.
- 132Leschziner, A. E.; Saha, A.; Wittmeyer, J.; Zhang, Y.; Bustamante, C.; Cairns, B. R.; Nogales, E. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 4913Google Scholar132Conformational flexibility in the chromatin remodeler RSC observed by electron microscopy and the orthogonal tilt reconstruction methodLeschziner, Andres E.; Saha, Anjanabha; Wittmeyer, Jacqueline; Zhang, Yongli; Bustamante, Carlos; Cairns, Bradley R.; Nogales, EvaProceedings of the National Academy of Sciences of the United States of America (2007), 104 (12), 4913-4918CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Chromatin remodeling complexes (remodelers) are large, multi-subunit macromol. assemblies that use ATP hydrolysis to alter the structure and positioning of nucleosomes. The mechanisms proposed for remodeler action on nucleosomes are diverse, and require structural evaluation and insights. Previous reconstructions of remodelers using electron microscopy revealed interesting features, but also significant discrepancies, prompting new approaches. Here, we use the orthogonal tilt reconstruction method, which is well suited for heterogeneous samples, to provide a reconstruction of the yeast RSC (_remodel the _structure of _chromatin) complex. Two interesting features are revealed: first, we observe a deep central cavity within RSC, displaying a remarkable surface complementarity for the nucleosome. Second, we are able to visualize two distinct RSC conformers, revealing a major conformational change in a large protein "arm," which may shift to further envelop the nucleosome. We present a model of the RSC-nucleosome complex that rationalizes the single mol. results obtained by using optical tweezers and also discuss the mechanistic implications of our structures.
- 133Racki, L. R.; Yang, J. G.; Naber, N.; Partensky, P. D.; Acevedo, A.; Purcell, T. J.; Cooke, R.; Cheng, Y.; Narlikar, G. J. Nature 2009, 462, 1016Google Scholar133The chromatin remodeller ACF acts as a dimeric motor to space nucleosomesRacki, Lisa R.; Yang, Janet G.; Naber, Nariman; Partensky, Peretz D.; Acevedo, Ashley; Purcell, Thomas J.; Cooke, Roger; Cheng, Yifan; Narlikar, Geeta J.Nature (London, United Kingdom) (2009), 462 (7276), 1016-1021CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Evenly spaced nucleosomes directly correlate with condensed chromatin and gene silencing. The ATP-dependent chromatin assembly factor (ACF) forms such structures in vitro and is required for silencing in vivo. ACF generates and maintains nucleosome spacing by constantly moving a nucleosome towards the longer flanking DNA faster than the shorter flanking DNA. How the enzyme rapidly moves back and forth between both sides of a nucleosome to accomplish bidirectional movement is unknown. Here, we show that nucleosome movement depends cooperatively on two ACF mols., indicating that ACF functions as a dimer of ATPases. Further, the nucleotide state dets. whether the dimer closely engages one or both sides of the nucleosome. Three-dimensional reconstruction by single-particle electron microscopy of the ATPase-nucleosome complex in an activated ATP state reveals a dimer architecture in which the two ATPases face each other. Our results indicate a model in which the two ATPases work in a coordinated manner, taking turns to engage either side of a nucleosome, thereby allowing processive bidirectional movement. This novel dimeric motor mechanism differs from that of dimeric motors such as kinesin and dimeric helicases that processively translocate unidirectionally and reflects the unique challenges faced by motors that move nucleosomes.
- 134Yamada, K.; Frouws, T. D.; Angst, B.; Fitzgerald, D. J.; DeLuca, C.; Schimmele, K.; Sargent, D. F.; Richmond, T. J. Nature 2011, 472, 448Google Scholar134Structure and mechanism of the chromatin remodelling factor ISW1aYamada, Kazuhiro; Frouws, Timothy D.; Angst, Brigitte; Fitzgerald, Daniel J.; DeLuca, Carl; Schimmele, Kyoko; Sargent, David F.; Richmond, Timothy J.Nature (London, United Kingdom) (2011), 472 (7344), 448-453CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Site-specific recognition of DNA in eukaryotic organisms depends on the arrangement of nucleosomes in chromatin. In the yeast Saccharomyces cerevisiae, ISW1a and related chromatin remodeling factors are implicated in establishing the nucleosome repeat during replication and altering nucleosome position to affect gene activity. Here we have solved the crystal structures of S. cerevisiae ISW1a lacking its ATPase domain both alone and with DNA bound at resolns. of 3.25 Å and 3.60 Å, resp., and we have visualized two different nucleosome-contg. remodeling complexes using cryo-electron microscopy. The composite x-ray and electron microscopy structures combined with site-directed photocrosslinking analyses of these complexes suggest that ISW1a uses a dinucleosome substrate for chromatin remodeling. Results from a remodeling assay corroborate the dinucleosome model. We show how a chromatin remodeling factor could set the spacing between two adjacent nucleosomes acting as a ' protein ruler'.
- 135Saravanan, M.; Wuerges, J.; Bose, D.; McCormack, E. A.; Cook, N. J.; Zhang, X.; Wigley, D. B. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 20883Google Scholar135Interactions between the nucleosome histone core and Arp8 in the INO80 chromatin remodeling complexSaravanan, Matheshwaran; Wuerges, Jochen; Bose, Daniel; McCormack, Elizabeth A.; Cook, Nicola J.; Zhang, Xiaodong; Wigley, Dale B.Proceedings of the National Academy of Sciences of the United States of America (2012), 109 (51), 20883-20888, S20883/1-S20883/29CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Actin-related protein Arp8 is a component of the INO80 chromatin remodeling complex. Yeast Arp8 (yArp8) comprises two domains: a 25-KDa N-terminal domain, found only in yeast, and a 75-KDa C-terminal domain (yArp8CTD) that contains the actin fold and is conserved across other species. The crystal structure shows that yArp8CTD contains three insertions within the actin core. Using a combination of biochem. and EM, we show that Arp8 forms a complex with nucleosomes, and that the principal interactions are via the H3 and H4 histones, mediated through one of the yArp8 insertions. We show that recombinant yArp8 exists in monomeric and dimeric states, but the dimer is the biol. relevant form required for stable interactions with histones that exploits the twofold symmetry of the nucleosome core. Taken together, these data provide unique insight into the stoichiometry, architecture, and mol. interactions between components of the INO80 remodeling complex and nucleosomes, providing a first step toward building up the structure of the complex.
- 136Tosi, A.; Haas, C.; Herzog, F.; Gilmozzi, A.; Berninghausen, O.; Ungewickell, C.; Gerhold, C. B.; Lakomek, K.; Aebersold, R.; Beckmann, R.; Hopfner, K.-P. Cell 2013, 154, 1207Google Scholar136Structure and Subunit Topology of the INO80 Chromatin Remodeler and Its Nucleosome ComplexTosi, Alessandro; Haas, Caroline; Herzog, Franz; Gilmozzi, Andrea; Berninghausen, Otto; Ungewickell, Charlotte; Gerhold, Christian B.; Lakomek, Kristina; Aebersold, Ruedi; Beckmann, Roland; Hopfner, Karl-PeterCell (Cambridge, MA, United States) (2013), 154 (6), 1207-1219CODEN: CELLB5; ISSN:0092-8674. (Cell Press)INO80/SWR1 family chromatin remodelers are complexes composed of >15 subunits and mol. masses exceeding 1 MDa. Their important role in transcription and genome maintenance is exchanging the histone variants H2A and H2A. Z. We report the architecture of S. cerevisiae INO80 using an integrative approach of electron microscopy, crosslinking and mass spectrometry. INO80 has an embryo-shaped head-neck-body-foot architecture and shows dynamic open and closed conformations. We can assign an Rvb1/Rvb2 heterododecamer to the head in close contact with the Ino80 Snf2 domain, Ies2, and the Arp5 module at the neck. The high-affinity nucleosome-binding Nhp10 module localizes to the body, whereas the module that contains actin, Arp4, and Arp8 maps to the foot. Structural and biochem. analyses indicate that the nucleosome is bound at the concave surface near the neck, flanked by the Rvb1/2 and Arp8 modules. Our anal. establishes a structural and functional framework for this family of large remodelers.
- 137Chittuluru, J. R.; Chaban, Y.; Monnet-Saksouk, J.; Carrozza, M. J.; Sapountzi, V.; Selleck, W.; Huang, J.; Utley, R. T.; Cramet, M.; Allard, S.; Cai, G.; Workman, J. L.; Fried, M. G.; Tan, S.; Côté, J.; Asturias, F. J. Nat. Struct. Mol. Biol. 2011, 18, 1196Google Scholar137Structure and nucleosome interaction of the yeast NuA4 and Piccolo-NuA4 histone acetyltransferase complexesChittuluru, Johnathan R.; Chaban, Yuriy; Monnet-Saksouk, Julie; Carrozza, Michael J.; Sapountzi, Vasileia; Selleck, William; Huang, Jiehuan; Utley, Rhea T.; Cramet, Myriam; Allard, Stephane; Cai, Gang; Workman, Jerry L.; Fried, Michael G.; Tan, Song; Cote, Jacques; Asturias, Francisco J.Nature Structural & Molecular Biology (2011), 18 (11), 1196-1203CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)We have used EM and biochem. to characterize the structure of NuA4, an essential yeast histone acetyltransferase (HAT) complex conserved throughout eukaryotes, and we have detd. the interaction of NuA4 with the nucleosome core particle (NCP). The ATM-related Tra1 subunit, which is shared with the SAGA coactivator complex, forms a large domain joined to a second region that accommodates the catalytic subcomplex Piccolo and other NuA4 subunits. EM anal. of a NuA4-NCP complex shows the NCP bound at the periphery of NuA4. EM characterization of Piccolo and Piccolo-NCP provided further information about subunit organization and confirmed that histone acetylation requires minimal contact with the NCP. A small conserved region at the N terminus of Piccolo subunit enhancer of Polycomb-like 1 (Epl1) is essential for NCP interaction, whereas the subunit yeast homolog of mammalian Ing1 2 (Yng2) apparently positions Piccolo for efficient acetylation of histone H4 or histone H2A tails. Taken together, these results provide an understanding of the NuA4 subunit organization and the NuA4-NCP interactions.
- 138Boudreault, A. A.; Cronier, D.; Selleck, W.; Lacoste, N.; Utley, R. T.; Allard, S.; Savard, J.; Lane, W. S.; Tan, S.; Côté, J. Genes Dev. 2003, 17, 1415Google Scholar138Yeast Enhancer of Polycomb defines global Esa1-dependent acetylation of chromatinBoudreault, Alexandre A.; Cronier, Dominique; Selleck, William; Lacoste, Nicolas; Utley, Rhea T.; Allard, Stephane; Savard, Julie; Lane, William S.; Tan, Song; Cote, JacquesGenes & Development (2003), 17 (11), 1415-1428CODEN: GEDEEP; ISSN:0890-9369. (Cold Spring Harbor Laboratory Press)Drosophila Enhancer of Polycomb, E(Pc), is a suppressor of position-effect variegation and an enhancer of both Polycomb and trithorax mutations. A homologous yeast protein, Epl1, is a subunit of the NuA4 histone acetyltransferase complex. Epl1 depletion causes cells to accumulate in G2/M and global loss of acetylated histones H4 and H2A. In relation to the Drosophila protein, mutation of Epl1 suppresses gene silencing by telomere position effect. Epl1 protein is found in the NuA4 complex and a novel highly active smaller complex named Piccolo NuA4 (picNuA4). The picNuA4 complex contains Esa1, Epl1, and Yng2 as subunits and strongly prefers chromatin over free histones as substrate. Epl1 conserved N-terminal domain bridges Esa1 and Yng2 together, stimulating Esa1 catalytic activity and enabling acetylation of chromatin substrates. A recombinant picNuA4 complex shows characteristics similar to the native complex, including strong chromatin preference. Cells expressing only the N-terminal half of Epl1 lack NuA4 HAT activity, but possess picNuA4 complex and activity. These results indicate that the essential aspect of Esa1 and Epl1 resides in picNuA4 function. We propose that picNuA4 represents a nontargeted histone H4/H2A acetyltransferase activity responsible for global acetylation, whereas the NuA4 complex is recruited to specific genomic loci to perturb locally the dynamic acetylation/deacetylation equil.
- 139Huang, J.; Tan, S. Mol. Cell. Biol. 2013, 33, 159Google Scholar139Piccolo NuA4-catalyzed acetylation of nucleosomal histones: critical roles of an Esa1 tudor/chromo barrel loop and an Epl1 enhancer of polycomb A (EPcA) basic regionHuang, Jiehuan; Tan, SongMolecular and Cellular Biology (2013), 33 (1), 159-169CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)Piccolo NuA4 is an essential yeast histone acetyltransferase (HAT) complex that targets histones H4 and H2A in nucleosome substrates. While Piccolo NuA4's catalytic subunit Esa1 alone is unable to acetylate nucleosomal histones, its accessory subunits, Yng2 and Epl1, enable Esa1 to bind to and to act on nucleosomes. We previously detd. that the Tudor domain of Esa1 and the EPcA homol. domain of Epl1 play crit. roles in Piccolo NuA4's ability to act on the nucleosome. In this work, we pinpoint a loop within the Esa1 Tudor domain and a short basic region at the N terminus of the Epl1 EPcA domain as necessary for this nucleosomal HAT activity. We also show that this Esa1 Tudor domain loop region is positioned close to nucleosomal DNA and that the Epl1 EPcA basic region is in proximity to the N-terminal histone H2A tail, the globular region of histone H4, and also to nucleosomal DNA when Piccolo NuA4 interacts with the nucleosome. Since neither region identified is required for Piccolo NuA4 to bind to nucleosomes and yet both are needed to acetylate nucleosomes, these regions may function after the enzyme binds nucleosomes to disengage substrate histone tails from nucleosomal DNA.
- 140Harshman, S. W.; Young, N. L.; Parthun, M. R.; Freitas, M. A. Nucleic Acids Res. 2013, 41, 9593Google Scholar140H1 histones: current perspectives and challengesHarshman, Sean W.; Young, Nicolas L.; Parthun, Mark R.; Freitas, Michael A.Nucleic Acids Research (2013), 41 (21), 9593-9609CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)A review. H1 and related linker histones are important both for maintenance of higher-order chromatin structure and for the regulation of gene expression. The biol. of the linker histones is complex, as they are evolutionarily variable, exist in multiple isoforms and undergo a large variety of posttranslational modifications in their long, unstructured, NH2- and C-terminal tails. We review recent progress in understanding the structure, genetics and posttranslational modifications of linker histones, with an emphasis on the dynamic interactions of these proteins with DNA and transcriptional regulators. We also discuss various exptl. challenges to the study of H1 and related proteins, including limitations of immunol. reagents and practical difficulties in the anal. of posttranslational modifications by mass spectrometry.
- 141Allan, J.; Hartman, P. G.; Crane-Robinson, C.; Aviles, F. X. Nature 1980, 288, 675Google Scholar141The structure of histone H1 and its location in chromatinAllan, J.; Hartman, P. G.; Crane-Robinson, C.; Aviles, F. X.Nature (London, United Kingdom) (1980), 288 (5792), 675-9CODEN: NATUAS; ISSN:0028-0836.The lysine-rich histones were shown to be a unified family of proteins on the basis of their primary structure. Each histone had an amino acid chain which fell into 3 distinct domains. Only the central domain (∼80 residues) was in a folded conformation. It was protected from trypsin digestion in chromatin and corresponded to the segment of highest sequence conservation. Without the flanking domains, the central domain was able to close 2 full turns of DNA in the nucleosome and could thus locate the H1 mol. The central domain alone could protect the extra ∼20 base pairs of DNA present in the chromatosome above that in the core particle. Full compaction of chromatin by H1 may require the intact mol.
- 142Syed, S. H.; Goutte-Gattat, D.; Becker, N.; Meyer, S.; Shukla, M. S.; Hayes, J. J.; Everaers, R.; Angelov, D.; Bednar, J.; Dimitrov, S. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 9620Google Scholar142Single-base resolution mapping of H1-nucleosome interactions and 3D organization of the nucleosomeSyed, Sajad Hussain; Goutte-Gattat, Damien; Becker, Nils; Meyer, Sam; Shukla, Manu Shubhdarshan; Hayes, Jeffrey J.; Everaers, Ralf; Angelov, Dimitar; Bednar, Jan; Dimitrov, StefanProceedings of the National Academy of Sciences of the United States of America (2010), 107 (21), 9620-9625, S9620/1-S9620/10CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Despite the key role of the linker histone H1 in chromatin structure and dynamics, its location and interactions with nucleosomal DNA have not been elucidated. The authors used a combination of electron cryomicroscopy, hydroxyl radical footprinting, and nanoscale modeling to analyze the structure of precisely positioned mono-, di-, and trinucleosomes contg. physiol. assembled full-length histone H1 or truncated mutants of this protein. Single-base resoln. ·OH footprinting shows that the globular domain of histone H1 (GH1) interacts with the DNA minor groove located at the center of the nucleosome and contacts a 10-bp region of DNA localized sym. with respect to the nucleosomal dyad. In addn., GH1 interacts with and organizes about one helical turn of DNA in each linker region of the nucleosome. Also a seven amino acid residue region (121-127) in the C-terminus of histone H1 was required for the formation of the stem structure of the linker DNA. A mol. model on the basis of these data and coarse-grain DNA mechanics provides novel insights on how the different domains of H1 interact with the nucleosome and predicts a specific H1-mediated stem structure within linker DNA.
- 143Fan, L.; Roberts, V. A. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 8384Google Scholar143Complex of linker histone H5 with the nucleosome and its implications for chromatin packingFan, Li; Roberts, Victoria A.Proceedings of the National Academy of Sciences of the United States of America (2006), 103 (22), 8384-8389CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Linker histones are essential for chromatin filament formation, and they play key roles in the regulation of gene expression. Despite the detn. of structures of the nucleosome and linker histones, the location of the linker histone on the nucleosome is still a matter of debate. Here we show by computational docking that the globular domain of linker histone variant H5 (GH5) has three distinct DNA-binding sites, through which GH5 contacts the DNA at the nucleosome dyad and the linker DNA strands entering and exiting the nucleosome. Our results explain the extensive mutagenesis and crosslinking data showing that side chains spread throughout the GH5 surface interact with nucleosomal DNA. The nucleosome DNA contacts pos. charged side chains that are conserved within the linker histone family, indicating that our model extends to linker histone-nucleosome interactions in general. Furthermore, our model provides a structural mechanism for formation of a dinucleosome complex specific to the linker histone H5, explaining its efficiency in chromatin compaction and transcription regulation. Thus, this work provides a basis for understanding how structural differences within the linker histone family result in functional differences, which in turn are important for gene regulation.
- 144Meyer, S.; Becker, N. B.; Syed, S. H.; Goutte-Gattat, D.; Shukla, M. S.; Hayes, J. J.; Angelov, D.; Bednar, J.; Dimitrov, S.; Everaers, R. Nucleic Acids Res. 2011, 39, 9139Google Scholar144From crystal and NMR structures, footprints and cryo-electron-micrographs to large and soft structures: nanoscale modeling of the nucleosomal stemMeyer, Sam; Becker, Nils B.; Syed, Sajad Hussain; Goutte-Gattat, Damien; Shukla, Manu Shubhdarshan; Hayes, Jeffrey J.; Angelov, Dimitar; Bednar, Jan; Dimitrov, Stefan; Everaers, RalfNucleic Acids Research (2011), 39 (21), 9139-9154CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)The interaction of histone H1 with linker DNA results in the formation of the nucleosomal stem structure, with considerable influence on chromatin organization. In a recent paper, we published results of biochem. footprinting and cryo-electron-micrographs of reconstituted mono-, di- and tri-nucleosomes, for H1 variants with different lengths of the cationic C-terminus. Here, we present a detailed account of the anal. of the exptl. data on the stem structure including thermal fluctuations of the stem structure. By combining (i) crystal and NMR structures of the nucleosome core particle and H1, (ii) the known nano-scale structure and elasticity of DNA, (iii) footprinting information on the location of protected sites on the DNA backbone and (iv) cryo-electron micrographs of reconstituted tri-nucleosomes, we arrive at a description of a polymorphic, hierarchically organized stem with a typical length of 20±2 base pairs. A comparison to linker conformations inferred for poly-601 fibers with different linker lengths suggests that intra-stem interactions stabilize and facilitate the formation of dense chromatin fibers.
- 145Zhou, Y. B.; Gerchman, S. E.; Ramakrishnan, V.; Travers, A.; Muyldermans, S. Nature 1998, 395, 402Google Scholar145Position and orientation of the globular domain of linker histone H5 on the nucleosomeZhou, Y.-B.; Gerchman, Sue Ellen; Ramakrishnan, V.; Travers, Andrew; Muyldermans, SergeNature (London) (1998), 395 (6700), 402-405CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)It is essential to identify the exact location of the linker histone within nucleosomes, the fundamental packing units of chromatin, in order to understand how condensed, transcriptionally inactive chromatin forms. Here, using a site-specific protein-DNA photocrosslinking method1, we map the binding site and the orientation of the globular domain of linker histone H5 on mixed-sequence chicken nucleosomes. We show, in contrast to an earlier model2, that the globular domain forms a bridge between one terminus of chromatosomal DNA and the DNA in the vicinity of the dyad axis of sym. of the core particle. Helix III of the globular domain binds in the major groove of the first helical turn of the chromatosomal DNA, whereas the secondary DNA-binding site on the opposite face of the globular domain of histone H5 makes contact with the nucleosomal DNA close to its midpoint. We also infer that helix I and helix II of the globular domain of histone H5 probably face, resp., the solvent and the nucleosome. This location places the basic carboxy-terminal region of the globular domain in a position from which it could simultaneously bind the nucleosome-linking DNA strands that exit and enter the nucleosome.
- 146Bharath, M. M. S.; Chandra, N. R.; Rao, M. R. S. Nucleic Acids Res. 2003, 31, 4264Google Scholar146Molecular modeling of the chromatosome particleBharath, M. M. Srinivas; Chandra, Nagasuma R.; Rao, M. R. S.Nucleic Acids Research (2003), 31 (14), 4264-4274CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)In an effort to understand the role of the linker histone in chromatin folding, its structure and location in the nucleosome has been studied by mol. modeling methods. The structure of the globular domain of the rat histone H1d, a highly conserved part of the linker histone, built by homol. modeling methods, revealed a three-helical bundle fold that could be described as a helix-turn-helix variant with its characteristic properties of binding to DNA at the major groove. Using the information of its preferential binding to four-way Holliday junction (HJ) DNA, a model of the domain complexed to HJ was built, which was subsequently used to position the globular domain onto the nucleosome. The model revealed that the primary binding site of the domain interacts with the extra 20 bp of DNA of the entering duplex at the major groove while the secondary binding site interacts with the minor groove of the central gyre of the DNA superhelix of the nucleosomal core. The positioning of the globular domain served as an anchor to locate the C-terminal domain onto the nucleosome to obtain the structure of the chromatosome particle. The resulting structure had a stem-like appearance, resembling that obsd. by electron microscopic studies. The C-terminal domain which adopts a high mobility group (HMG)-box-like fold, has the ability to bend DNA, causing DNA condensation or compaction. It was obsd. that the three S/TPKK motifs in the C-terminal domain interact with the exiting duplex, thus defining the path of linker DNA in the chromatin fiber. This study has provided an insight into the probable individual roles of globular and the C-terminal domains of histone H1 in chromatin organization.
- 147Wong, J.; Li, Q.; Levi, B. Z.; Shi, Y. B.; Wolffe, A. P. EMBO J. 1997, 16, 7130Google Scholar147Structural and functional features of a specific nucleosome containing a recognition element for the thyroid hormone receptorWong, Jiemin; Li, Qiao; Levi, Ben-Zion; Shi, Yun-Bo; Wolffe, Alan P.EMBO Journal (1997), 16 (23), 7130-7145CODEN: EMJODG; ISSN:0261-4189. (Oxford University Press)The Xenopus thyroid hormone receptor βA (TRβA) gene contains an important thyroid hormone response element (TRE) that is assembled into a positioned nucleosome. We det. the translational position of the nucleosome contg. the TRE and the rotational positioning of the double helix with respect to the histone surface. Histone H1 is incorporated into the nucleosome leading to an asym. protection to micrococcal nuclease cleavage of linker DNA relative to the nucleosome core. Histone H1 assocn. is without significant consequence for the binding of the heterodimer of thyroid hormone receptor and 9-cis retinoic acid receptor (TR/RXR) to nucleosomal DNA in vitro, or for the regulation of TRβA gene transcription following microinjection into the oocyte nucleus. Small alterations of 3 and 6 bp in the translational positioning of the TRE in chromatin are also without effect on the transcriptional activity of the TRβA gene, whereas a small change in the rotational position of the TRE (3 bp) relative to the histone surface significantly reduces the binding of TR/RXR to the nucleosome and decreases transcriptional activation directed by TR/RXR. Our results indicate that the specific architecture of the nucleosome contg. the TRE may have regulatory significance for expression of the TRβA gene.
- 148Pachov, G. V.; Gabdoulline, R. R.; Wade, R. C. Nucleic Acids Res. 2011, 39, 5255Google Scholar148On the structure and dynamics of the complex of the nucleosome and the linker histonePachov, Georgi V.; Gabdoulline, Razif R.; Wade, Rebecca C.Nucleic Acids Research (2011), 39 (12), 5255-5263CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Several different models of the linker histone (LH)-nucleosome complex have been proposed, but none of them has unambiguously revealed the position and binding sites of the LH on the nucleosome. Using Brownian dynamics-based docking together with normal mode anal. of the nucleosome to account for the flexibility of two flanking 10 bp long linker DNAs (L-DNA), we identified binding modes of the H5-LH globular domain (GH5) to the nucleosome. For a wide range of nucleosomal conformations with the L-DNA ends less than 65 Å apart, one dominant binding mode was identified for GH5 and found to be consistent with fluorescence recovery after photobleaching (FRAP) expts. GH5 binds asym. with respect to the nucleosomal dyad axis, fitting between the nucleosomal DNA and one of the L-DNAs. For greater distances between L-DNA ends, docking of GH5 to the L-DNA that is more restrained and less open becomes favored. These results suggest a selection mechanism by which GH5 preferentially binds one of the L-DNAs and thereby affects DNA dynamics and accessibility and contributes to formation of a particular chromatin fiber structure. The two binding modes identified would, resp., favor a tight zigzag chromatin structure or a loose solenoid chromatin fiber.
- 149An, W.; Leuba, S. H.; van Holde, K.; Zlatanova, J. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3396Google Scholar149Linker histone protects linker DNA on only one side of the core particle and in a sequence-dependent mannerAn, Woojin; Leuba, Sanford H.; Van Holde, Kensal; Zlatanova, JordankaProceedings of the National Academy of Sciences of the United States of America (1998), 95 (7), 3396-3401CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The protection against micrococcal nuclease digestion afforded to chromatosomal DNA by the presence of a linker histone (H1°) has been quant. measured in two reconstituted systems. We have used chromatosomes reconstituted at two distinct positions on a DNA fragment contg. the 5S rRNA gene from Lytechinus variegatus and at a specific position on a sequence contg. Gal4- and USF-binding sites. In all cases, we find asym. protection, with ≈20 bp protected on one side of the core particle and no protection on the other. We demonstrated through crosslinking expts. that the result is not due to any sliding of the histone core caused by either linker histone addn. or micrococcal nuclease cleavage. Because the core particle is itself a sym. object, the preferred asym. location of a linker histone must be dictated by unknown elements in the DNA sequence.
- 150Vogler, C.; Huber, C.; Waldmann, T.; Ettig, R.; Braun, L.; Izzo, A.; Daujat, S.; Chassignet, I.; Lopez-Contreras, A. J.; Fernandez-Capetillo, O.; Dundr, M.; Rippe, K.; Längst, G.; Schneider, R. PLoS Genet. 2010, 6, e1001234Google ScholarThere is no corresponding record for this reference.
- 151Thakar, A.; Gupta, P.; Ishibashi, T.; Finn, R.; Silva-Moreno, B.; Uchiyama, S.; Fukui, K.; Tomschik, M.; Ausio, J.; Zlatanova, J. Biochemistry 2009, 48, 10852Google Scholar151H2A.Z and H3.3 Histone Variants Affect Nucleosome Structure: Biochemical and Biophysical StudiesThakar, Amit; Gupta, Pooja; Ishibashi, Toyotaka; Finn, Ron; Silva-Moreno, Begonia; Uchiyama, Susumu; Fukui, Kiichi; Tomschik, Miroslav; Ausio, Juan; Zlatanova, JordankaBiochemistry (2009), 48 (46), 10852-10857CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Histone variants play important roles in regulation of chromatin structure and function. To understand the structural role played by histone variants H2A.Z and H3.3, both of which are implicated in transcription regulation, we conducted extensive biochem. and biophys. anal. on mononucleosomes reconstituted from either random-sequence DNA derived from native nucleosomes or a defined DNA nucleosome positioning sequence and recombinant human histones. Using established electrophoretic and sedimentation anal. methods, we compared the properties of nucleosomes contg. canonical histones and histone variants H2A.Z and H3.3 (in isolation or in combination). We find only subtle differences in the compaction and stability of the particles. Interestingly, both H2A.Z and H3.3 affect nucleosome positioning, either creating new positions or altering the relative occupancy of the existing nucleosome position space. On the other hand, only H2A.Z-contg. nucleosomes exhibit altered linker histone binding. These properties could be physiol. significant as nucleosome positions and linker histone binding partly det. factor binding accessibility.
- 152McGhee, J. D.; Nickol, J. M.; Felsenfeld, G.; Rau, D. C. Cell 1983, 33, 831Google Scholar152Higher order structure of chromatin: orientation of nucleosomes within the 30 nm chromatin solenoid is independent of species and spacer lengthMcGhee, James D.; Nickol, Joanne M.; Felsenfeld, Gary; Rau, Donald C.Cell (Cambridge, MA, United States) (1983), 33 (3), 831-41CODEN: CELLB5; ISSN:0092-8674.Elec. dichroism was used to study the arrangement of nucleosomes in 30-nm chromatin solenoidal fibers prepd. from a variety of sources (CHO cells, HeLa cells, rat liver, chicken erythrocytes, and sea urchin sperm) in which the nucleosome spacer length varies in the ∼10-∼80-base-pair range. Field-free relaxation times are consistent only with structures contg. 6 ± 1 nucleosomes for every 11 nm of solenoidal length. With very few assumptions about the arrangement of the spacer DNA, the dichroism data are consistent with the same orientation of the chromatosomes for every chromatin sample examd. The orientation, which maintains the faces of the radially arranged chromatosomes inclined at an angle of 20-33° to the solenoid axis, thus appears to be a general structural feature of the higher-order chromatin fiber.
- 153Widom, J.; Klug, A. Cell 1985, 43, 207Google Scholar153Structure of the 300Å chromatin filament: x-ray diffraction from oriented samplesWidom, J.; Klug, A.Cell (Cambridge, MA, United States) (1985), 43 (1), 207-13CODEN: CELLB5; ISSN:0092-8674.X-ray diffraction patterns have been obtained from partially oriented samples of 300-Å chromatin filaments. The chromatin was prepd. by methods that preserve its structure, and conditions were found in which the 300-Å filaments spontaneously form ordered aggregates, so that it was not necessary to pull fibers. The diffraction patterns show a meridional band at 110 Å, and equatorial bands at 340, 57, 37, and 27 Å. These patterns, together with patterns calcd. from the known 7-Å electron d. map of the nucleosome core particle, imply side-to-side packing of nucleosomes in the direction of the 300-Å filament and radial packing around it. These results are consistent with the solenoid model of J. R. Finch and A. Klug (1976) and are inconsistent with many other proposed models.
- 154Thoma, F.; Koller, T.; Klug, A. J. Cell Biol. 2003, 83, 403Google ScholarThere is no corresponding record for this reference.
- 155Worcel, A.; Strogatz, S.; Riley, D. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 1461Google Scholar155Structure of chromatin and the linking number of DNAWorcel, Abraham; Strogatz, Steven; Riley, DonaldProceedings of the National Academy of Sciences of the United States of America (1981), 78 (3), 1461-5CODEN: PNASA6; ISSN:0027-8424.Recent observations suggest that the basic supranucleosomal structure of chromatin is a zigzag helical ribbon with a repeat unit made of 2 nucleosomes connected by a relaxed spacer DNA. A remarkable feature of 1 particular ribbon is that it solves the apparent paradox between the no. of DNA turns/nucleosome and the total linking no. of a nucleosome-contg. closed circular DNA mol. The repeat unit of the proposed structure, which contains 2 nucleosomes with -1.75 DNA turns/nucleosome and 1 spacer crossover/repeat, contributes -2 to the linking no. of closed circular DNA. Space-filling models show that the cylindrical 250-Å chromatin fiber can be generated by twisting the ribbon.
- 156Woodcock, C. L.; Frado, L. L.; Rattner, J. B. J. Cell Biol. 2002, 99, 42Google ScholarThere is no corresponding record for this reference.
- 157Williams, S. P.; Athey, B. D.; Muglia, L. J.; Schappe, R. S.; Gough, A. H.; Langmore, J. P. Biophys. J. 1986, 49, 233Google Scholar157Chromatin fibers are left-handed double helixes with diameter and mass per unit length that depend on linker lengthWilliams, Shawn P.; Athey, Brian D.; Muglia, Louis J.; Schappe, R. Scott; Gough, Albert H.; Langmore, John P.Biophysical Journal (1986), 49 (1), 233-48, 373CODEN: BIOJAU; ISSN:0006-3495.Four classes of models were proposed for the internal structure of eukaryotic chromosome fibers: the solenoid, twisted-ribbon, crossed-linker, and superbead models. Electron image and x-ray scattering data were collected from nuclei and isolated chromatin fibers of 7 different tissues to distinguish between these models. The fiber diams. were related to the linker lengths by the equation: D(N) = 19.3 + 0.23 N, where D(N) is the external diam. (nm) and N is the linker length (base pairs). The no. of nucleosomes per unit length of the fibers was also related to linker length. Detailed studies were done on the highly regular chromatin from erythrocytes of Necturus (mud puppy) and sperm of Thyone (sea cucumber). Necturus Chromatin fibers [N = 48 base pairs (bp)] had diams. of 31 nm and had 7.5 nucleosomes per 10 nm along the axis. Thyone Chromatin fibers (N = 87 bp) had diams. of 39 nm and had 12 nucleosomes per 10 nm along the axis. Fourier transforms of electron micrographs of Necturus fibers showed left-handed helical symmetry with a pitch of 25.8 nm and pitch angle of 32°, consistent with a double helix. Comparable conclusions were drawn from the Thyone data. The data did not support the solenoid, twisted-ribbon, or supranucleosomal particle models. The data did support 2 crossed-linker models having left-handed double-helical symmetry and conserved nucleosome interactions.
- 158Wong, H.; Victor, J.-M.; Mozziconacci, J. PLoS One 2007, 2, e877Google Scholar158An all-atom model of the chromatin fiber containing linker histones reveals a versatile structure tuned by the nucleosomal repeat lengthWong Hua; Victor Jean-Marc; Mozziconacci JulienPloS one (2007), 2 (9), e877 ISSN:.In the nucleus of eukaryotic cells, histone proteins organize the linear genome into a functional and hierarchical architecture. In this paper, we use the crystal structures of the nucleosome core particle, B-DNA and the globular domain of H5 linker histone to build the first all-atom model of compact chromatin fibers. In this 3D jigsaw puzzle, DNA bending is achieved by solving an inverse kinematics problem. Our model is based on recent electron microscopy measurements of reconstituted fiber dimensions. Strikingly, we find that the chromatin fiber containing linker histones is a polymorphic structure. We show that different fiber conformations are obtained by tuning the linker histone orientation at the nucleosomes entry/exit according to the nucleosomal repeat length. We propose that the observed in vivo quantization of nucleosomal repeat length could reflect nature's ability to use the DNA molecule's helical geometry in order to give chromatin versatile topological and mechanical properties.
- 159Eltsov, M.; MacLellan, K. M.; Maeshima, K.; Frangakis, A. S.; Dubochet, J. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 19732Google Scholar159Analysis of cryo-electron microscopy images does not support the existence of 30-nm chromatin fibers in mitotic chromosomes in situEltsov, Mikhail; MacLellan, Kirsty M.; Maeshima, Kazuhiro; Frangakis, Achilleas S.; Dubochet, JacquesProceedings of the National Academy of Sciences of the United States of America (2008), 105 (50), 19732-19737CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Although the formation of 30-nm chromatin fibers is thought to be the most basic event of chromatin compaction, it remains controversial because high-resoln. imaging of chromatin in living eukaryotic cells had not been possible until now. Cryo-electron microscopy of vitreous sections is a relatively new technique, which enables direct high-resoln. observation of cell structures in a close-to-native state. We used cryo-electron microscopy and image processing to further investigate the presence of 30-nm chromatin fibers in human mitotic chromosomes. HeLa S3 cells were vitrified by high-pressure freezing, thin-sectioned, and then imaged under the cryo-electron microscope without any further chem. treatment or staining. For an unambiguous interpretation of the images, the effects of the contrast transfer function were computationally cor. The mitotic chromosomes of the HeLa S3 cells appeared as compact structures with a homogeneous grainy texture, in which there were no visible 30-nm fibers. Power spectra of the chromosome images also gave no indication of 30-nm chromatin folding. These results, together with our observations of the effects of chromosome swelling, strongly suggest that, within the bulk of compact metaphase chromosomes, the nucleosomal fiber does not undergo 30-nm folding, but exists in a highly disordered and interdigitated state, which is, on the local scale, comparable with a polymer melt.
- 160Maeshima, K.; Hihara, S.; Eltsov, M. Curr. Opin. Cell Biol. 2010, 22, 291Google Scholar160Chromatin structure: does the 30-nm fiber exist in vivo?Maeshima, Kazuhiro; Hihara, Saera; Eltsov, MikhailCurrent Opinion in Cell Biology (2010), 22 (3), 291-297CODEN: COCBE3; ISSN:0955-0674. (Elsevier B.V.)A review. A long strand of DNA is wrapped around the core histone and forms a nucleosome. Although the nucleosome has long been assumed to be folded into 30-nm chromatin fibers, their structural details and how such fibers are organized into a nucleus or mitotic chromosome remain unclear. When the authors obsd. frozen hydrated (vitrified) human mitotic cells using cryo-electron microscopy, which enables direct high-resoln. imaging of the cellular structures in a close-to-native state, they found no higher-order structures including 30-nm chromatin fibers in the chromosome. Therefore, the authors have proposed that the nucleosome fibers exist in a highly disordered, interdigitated state like a 'polymer melt' that undergoes dynamic movement. The authors have postulated that a similar state exists in active interphase nuclei, resulting in several advantages in the transcription and DNA replication processes.
- 161Joti, Y.; Hikima, T.; Nishino, Y.; Kamada, F.; Hihara, S.; Takata, H.; Ishikawa, T.; Maeshima, K. Nucleus 2012, 3, 404Google Scholar161Chromosomes without a 30-nm chromatin fiberJoti Yasumasa; Hikima Takaaki; Nishino Yoshinori; Kamada Fukumi; Hihara Saera; Takata Hideaki; Ishikawa Tetsuya; Maeshima KazuhiroNucleus (Austin, Tex.) (2012), 3 (5), 404-10 ISSN:.How is a long strand of genomic DNA packaged into a mitotic chromosome or nucleus? The nucleosome fiber (beads-on-a-string), in which DNA is wrapped around core histones, has long been assumed to be folded into a 30-nm chromatin fiber, and a further helically folded larger fiber. However, when frozen hydrated human mitotic cells were observed using cryoelectron microscopy, no higher-order structures that included 30-nm chromatin fibers were found. To investigate the bulk structure of mitotic chromosomes further, we performed small-angle X-ray scattering (SAXS), which can detect periodic structures in noncrystalline materials in solution. The results were striking: no structural feature larger than 11 nm was detected, even at a chromosome-diameter scale (~1 μm). We also found a similar scattering pattern in interphase nuclei of HeLa cells in the range up to ~275 nm. Our findings suggest a common structural feature in interphase and mitotic chromatins: compact and irregular folding of nucleosome fibers occurs without a 30-nm chromatin structure.
- 162Nishino, Y.; Eltsov, M.; Joti, Y.; Ito, K.; Takata, H.; Takahashi, Y.; Hihara, S.; Frangakis, A. S.; Imamoto, N.; Ishikawa, T.; Maeshima, K. EMBO J. 2012, 31, 1644Google Scholar162Human mitotic chromosomes consist predominantly of irregularly folded nucleosome fibers without a 30-nm chromatin structureNishino, Yoshinori; Eltsov, Mikhail; Joti, Yasumasa; Ito, Kazuki; Takata, Hideaki; Takahashi, Yukio; Hihara, Saera; Frangakis, Achilleas S.; Imamoto, Naoko; Ishikawa, Tetsuya; Maeshima, KazuhiroEMBO Journal (2012), 31 (7), 1644-1653CODEN: EMJODG; ISSN:0261-4189. (Nature Publishing Group)How a long strand of genomic DNA is compacted into a mitotic chromosome remains one of the basic questions in biol. The nucleosome fiber, in which DNA is wrapped around core histones, has long been assumed to be folded into a 30-nm chromatin fiber and further hierarchical regular structures to form mitotic chromosomes, although the actual existence of these regular structures is controversial. Here, the authors show that human mitotic HeLa chromosomes are mainly composed of irregularly folded nucleosome fibers rather than 30-nm chromatin fibers. The authors' comprehensive and quant. study using cryo-electron microscopy and synchrotron x-ray scattering resolved the long-standing contradictions regarding the existence of 30-nm chromatin structures and detected no regular structure >11 nm. This finding suggests that the mitotic chromosome consists of irregularly arranged nucleosome fibers, with a fractal nature, which permits a more dynamic and flexible genome organization than would be allowed by static regular structures.
- 163Hansen, J. C. EMBO J. 2012, 31, 1621Google Scholar163Human mitotic chromosome structure: what happened to the 30-nm fiber?Hansen, Jeffrey C.EMBO Journal (2012), 31 (7), 1621-1623CODEN: EMJODG; ISSN:0261-4189. (Nature Publishing Group)A review. The long-standing view of chromosome packaging is that 10-nm beads-on-a-string chromatin fibers fold into higher-order 30-nm fibers, which further twist and coil to form highly condensed chromosomes. After 4 decades of intense pursuit of the structure and properties of 30-nm chromatin fibers, the work of Y. Nishino et al. (2012) demonstrates that regular 30-nm fibers are absent from human mitotic chromosomes. The emerging view is that chromosome-level condensation can be achieved through packaging of 10-nm fibers in a fractal manner.
- 164Baker, N. A.; Sept, D.; Joseph, S.; Holst, M. J.; McCammon, J. A. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 10037Google Scholar164Electrostatics of nanosystems: application to microtubules and the ribosomeBaker, Nathan A.; Sept, David; Joseph, Simpson; Holst, Michael J.; McCammon, J. AndrewProceedings of the National Academy of Sciences of the United States of America (2001), 98 (18), 10037-10041CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Evaluation of the electrostatic properties of biomols. has become a std. practice in mol. biophysics. Foremost among the models used to elucidate the electrostatic potential is the Poisson-Boltzmann equation; however, existing methods for solving this equation have limited the scope of accurate electrostatic calcns. to relatively small biomol. systems. Here we present the application of numerical methods to enable the trivially parallel soln. of the Poisson-Boltzmann equation for supramol. structures that are orders of magnitude larger in size. As a demonstration of this methodol., electrostatic potentials have been calcd. for large microtubule and ribosome structures. The results point to the likely role of electrostatics in a variety of activities of these structures.
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Abstract
Figure 1
Figure 1. Nucleosome core particle structure and the histone-fold heterodimers. (a) Nucleosome core particle structure (PDB ID 1KX5). Histones and DNA are depicted in cartoon and sticks representations, respectively, and colored as indicated. (b) H3/H4 histone-fold heterodimer. (c) H2A/H2B histone-fold heterodimer. Structures (top) and schemes (bottom) with secondary structure elements indicated. All molecular graphics in this review were prepared using PyMOL software (The PyMOL Molecular Graphics System, version 1.6, Schrodinger, LLC). All structures of NCP using high-resolution structure (17) (PDB ID 1KX5) unless indicated otherwise.
Figure 2
Figure 2. Histone octamer constructed with four helix bundles. (a) Nucleosome core particle structure highlighting H3–H3 four helix bundle (blue). Remainder of H3 and H4 are shown in light blue and light green, respectively. (b) Nucleosome core particle structure highlighting one H4–H2B four helix bundle (green for H4 and red for H2B). Remainder of H4 and H2B are shown in light green and pink, respectively.
Figure 3
Figure 3. Histone-fold heterodimers in the nucleosome core particle structure. (a) Nucleosome core particle structure with central H3/H4 histone-fold tetramer shown in blue (H3) and green (H4). H3 and H4 extensions are shown in light blue and light green, respectively. (b) Nucleosome core particle structure with one H2A/H2B histone-fold dimer shown in yellow (H2A) and red (H2B). H2A and H2B extensions are shown in light yellow and pink, respectively.
Figure 4
Figure 4. Histone-fold heterodimers form a ramp for nucleosomal DNA. (a) H2A/H2B histone-fold heterodimers interact with DNA in two different parallel planes. Structure of NCP viewed from opposite dyad, highlighting H2A and H2B in yellow and red, respectively (left) and scheme of DNA planes (right). (b) H3/H4 tetramer forms a diagonal ramp for DNA connecting two parallel planes. Structure of NCP view from dyad (black oval and orange base pair) with H3 and H4 in blue and green, respectively, (left) and scheme of diagonal DNA ramp (right). Arrows point away from central dyad base pair.
Figure 5
Figure 5. Surface topology and charge of the nucleosome core particle. (a) Surface of nucleosome core particle viewed down the DNA superhelical axis in space-filling representation. (b) Surface electrostatic potential of nucleosome core particle contoured from −5 to +5 kT/e calculated with ABPS. (164) Location of acidic patch is indicated.
Figure 6
Figure 6. Scheme of asymmetric and symmetric 601 sequences. Sequences of 601R symmetric, (canonical) 601 asymmetric, and 601L symmetric sequences with H3/H4 TA steps highlighted in red for left half and blue for right half. (61) Nucleosome salt stability values (molar monovalent salt) are listed at right and indicate stability as follows: 601L > 601 > 601R. This trend correlates with the number of H3/H4 TA steps: 601L (6), 601 (4), 601R (2). The dyad position is indicated (purple).
Figure 7
Figure 7. Location of TA steps in 601L nucleosome core particle structure. (a) 601L NCP structure viewed down the DNA superhelical axis with TA steps interacting with H3/H4 and H2A/H2B colored red and orange, respectively. The dyad is indicated (purple). Histones H3, H4, H2A, and H2B are shown in cartoon representation and colored blue, green, yellow, and red, respectively. Nucleosomal DNA is shown as sticks (light blue). (b) Enlarged view showing one H3/H4 heterodimer bound to DNA containing three TA steps (other histones are not shown for clarity purposes). Backbone phosphates bound to the H3/H4 histone folds are shown in space-filling representation as indicated. Secondary structure elements of dimer are shown.
Figure 8
Figure 8. Minimal base stacking in TA and CA compared to other base pair steps. TA, CA, AA, and AT base pair steps colored as follows: T = yellow, A = blue, G = green, C = red. The thymine methyl groups are shown highlighted in space-filling representation (dark yellow), all other non-hydrogen atoms shown in sticks representation. The minimal base stacking and the absence of atoms close to the thymine methyl group permit greater flexibility of the TA and CA base pair steps.
Figure 9
Figure 9. RNA polymerase II blocking by nucleosome positioning sequences. Sequences of NCP601, NCP603, and NCP605 sequences and their reversed counterparts together with ability to block RNA polymerase II. (73) Multiple TA steps bound to the H3/H4 tetramer downstream (red) of the dyad (purple) blocks RNA polymerase II passage as compared with upstream (blue) of the dyad. TA steps bound to the H2A/H2B dimers are shown in orange. The sequence shown for the 601 sequence is the reverse complement of what is shown in Figure 6 to be consistent with ref 73.
Figure 10
Figure 10. DNA end-to-end packing in nucleosome core particle crystals. Three nucleosome core particles from one plane of the high resolution NCP crystal structure (PDB ID 1KX5) colored yellow, red and blue. (a) Full and (b) enlarged views of the alignment of the DNA ends from adjacent NCP in the structure. The DNA end-to-end packing exists in all crystals of the nucleosome core particle on its own.
Figure 11
Figure 11. DNA stretching in nucleosome core particle structures. Cartoon representation of structure of approximately half of the nucleosomal DNA for (a) 146 bp human alpha-satellite (HAS146) (PDB ID 1AOI, blue) and (b) 145 bp 601 (PDB ID 3LZO, red) nucleosome positioning sequences relative to the HAS147 sequence (PDB ID 1KX5, yellow) (top). Stretching of 1 bp is observed at superhelical location (SHL) −2 with the HAS146 sequence and 1 bp each at SHL ± 5 with the 145 bp 601 sequence. SHLs and the dyad = SHL 0 are indicated. The length of DNA wrapped on each side of the NCP for each of the sequences is also shown (bottom).
Figure 12
Figure 12. Nucleosome recognition using the acidic patch arginine-anchor. From top to bottom, structures of RCC1 (PDB ID 3MVD), (52) Sir3 (PDB ID 3TU4), (78) PRC1 (PDB ID 4R8P), (111) LANA peptide (PDB ID 1ZLA), (79) and CENP-C peptide (PDB ID 4INM) (107) bound to the nucleosome core particle. Overview of structures as viewed from opposite the dyad (right) and zoomed view of acidic patch (left) with arginine-anchor in space-filling representation and key H2A residues shown as sticks. Locations of RCC1 switchback loop (1), DNA binding loop (2), and N-terminus (N) and Sir3 loop 3 (3) and N-terminus (N) are indicated. Histones H3, H4, H2A, and H2B are shown in cartoon representation and colored cornflower blue, light green, wheat, and pink, respectively. DNA (light pink) is shown as sticks.
Figure 13
Figure 13. Models of the 30 nm fiber. Orthogonal views perpendicular to the 30 nm fiber axis (top) and down the axis (bottom) of the Richmond two-start model (left), Rhodes one-start model (center) and Li-Zhu tetranucleosome-unit repeat two-start model (right). The sequence of nucleosomes in each model is indicated. In the Richmond model, each sequential pair of nucleosomes across the fiber is colored similarly. For the Rhodes model, all nucleosomes in the same turn of the solenoid are colored similarly. In the Li-Zhu model, each tetranucleosome repeating unit is colored similarly. Unlabeled nucleosomes in the two-start models are not shown in the bottom views for figure clarity. Linker DNA is not present in the Rhodes model but, given the nature of the solenoidal structure, must be bent. The B-form DNA double helix is shown for comparison (far right). All models shown in space-filling representation and scaled as indicated.
Robert K. McGinty
Robert K. McGinty received his Ph.D. from The Rockefeller University in 2010 and M.D. from Weill Cornell Medical College as a member of the Tri-Institutional M.D.–Ph.D. program. During his graduate studies under the supervision of Prof. Tom Muir, McGinty used protein chemistry to study crosstalk between histone modifications. In 2011, he joined the laboratory of Prof. Song Tan in the Center for Eukaryotic Gene Regulation at Penn State, where he is using X-ray crystallography to study the molecular recognition of the nucleosome by histone modifying enzymes. He is currently a Damon Runyon postdoctoral fellow.
Song Tan
Song Tan studied physics as an undergraduate at Cornell University (1985) before pursuing his Ph.D. at the MRC Laboratory of Molecular Biology as a Marshall Scholar (1989). He continued his training as a postdoctoral fellow and project leader under Tim Richmond at the ETH-Zürich (Swiss Federal Institute of Technology) where he determined crystal structures of several transcription factor/DNA complexes. Dr. Tan joined the Center for Eukaryotic Gene Regulation and the faculty of the Department of Biochemistry and Molecular Biology in 1998. He was named a Pew Scholar in the Biomedical Sciences in 2001. Dr. Tan’s laboratory investigates how chromatin enzymes and factors interact with their nucleosome substrates through biochemical and structural approaches. His laboratory determined the first chromatin factor-nucleosome crystal structure (RCC1-nucleosome) in 2010 and, with Dr. McGinty, the first chromatin enzyme-nucleosome crystal structure (PRC1-nucleosome) in 2014.
References
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- 2Simpson, R. T. Biochemistry 1978, 17, 55242Structure of the chromatosome, a chromatin particle containing 160 base pairs of DNA and all the histonesSimpson, Robert T.Biochemistry (1978), 17 (25), 5524-31CODEN: BICHAW; ISSN:0006-2960.Chromatin particles were prepd. from chicken erythrocytes which contain a 160 base pair length of DNA, an octamer of the 4 smaller histones, and a mol. of lysine-rich histone, H1 or H5, and some physicochem. properties of these particles were compared with those of core particles, which lack H1 or H5 and contain 20 base pairs less of DNA. The former particles, called chromatosomes, contain all the major components necessary for organization of the structure of long stretches of chromatin. Assuming that the structure of the 140 base pairs of DNA common to the core particle and the chromatosome is similar in these 2 particles, physicochem. data suggest that (1) the addnl. DNA in the chromatosome is folded around the histone core, (2) the addnl. DNA is not in the same conformation as the 140 base pairs of DNA in the core particle, and (3) both the 20 addnl. base pairs of DNA in the chromatosomes and some DNA segments of the core particle itself are markedly stabilized to thermal denaturation, presumably by interaction with the lysine-rich histone. Using 5'-end labeling, the relative susceptibilities of DNase I cleavage sites in the chromatosomes were mapped. Sites at 20, 50, 60, and 100 bases from the ends are highly susceptible; the sites 30, 40, and 70-90 bases from the ends are cleaved with lower frequency. These data suggest that the chromatosome likely contains 2 full turns of DNA around the histone nucleus of the core particle, with a mol. of lysine-rich histone bound outside the DNA-inner histone complex.
- 3Taverna, S. D.; Li, H.; Ruthenburg, A. J.; Allis, C. D.; Patel, D. J. Nat. Struct. Mol. Biol. 2007, 14, 10253How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickersTaverna, Sean D.; Li, Haitao; Ruthenburg, Alexander J.; Allis, C. David; Patel, Dinshaw J.Nature Structural & Molecular Biology (2007), 14 (11), 1025-1040CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)A review. Histones comprise the major protein component of chromatin, the scaffold in which the eukaryotic genome is packaged, and are subject to many types of post-translational modifications (PTMs), esp. on their flexible tails. These modifications may constitute a 'histone code' and could be used to manage epigenetic information that helps extend the genetic message beyond DNA sequences. This proposed code, read in part by histone PTM-binding 'effector' modules and their assocd. complexes, has been predicted to define unique functional states of chromatin and/or regulate various chromatin-templated processes. A wealth of structural and functional data show how chromatin effector modules target their cognate covalent histone modifications. Here, the authors summarize key features in the mol. recognition of histone PTMs by a diverse family of 'reader pockets' (including bromodomains, Royal superfamily modules, PHD fingers, WD40 protein repeats, 14-3-3 proteins, and BRCT domains), highlighting specific readout mechanisms for individual marks, common themes, and insights into the downstream functional consequences of the interactions. Changes in these interactions may have far-reaching implications for human biol. and disease, notably cancer.
- 4Luger, K.; Dechassa, M. L.; Tremethick, D. J. Nat. Rev. Mol. Cell Biol. 2012, 13, 4364New insights into nucleosome and chromatin structure: an ordered state or a disordered affair?Luger, Karolin; Dechassa, Mekonnen L.; Tremethick, David J.Nature Reviews Molecular Cell Biology (2012), 13 (7), 436-447CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. The compaction of genomic DNA into chromatin has profound implications for the regulation of key processes such as transcription, replication and DNA repair. Nucleosomes, the repeating building blocks of chromatin, vary in the compn. of their histone protein components. This is the result of the incorporation of variant histones and post-translational modifications of histone amino acid side chains. The resulting changes in nucleosome structure, stability and dynamics affect the compaction of nucleosomal arrays into higher-order structures. It is becoming clear that chromatin structures are not nearly as uniform and regular as previously assumed. This implies that chromatin structure must also be viewed in the context of specific biol. functions.
- 5Shogren-Knaak, M.; Ishii, H.; Sun, J.-M.; Pazin, M. J.; Davie, J. R.; Peterson, C. L. Science 2006, 311, 8445Histone H4-K16 Acetylation Controls Chromatin Structure and Protein InteractionsShogren-Knaak, Michael; Ishii, Haruhiko; Sun, Jian-Min; Pazin, Michael J.; Davie, James R.; Peterson, Craig L.Science (Washington, DC, United States) (2006), 311 (5762), 844-847CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Acetylation of histone H4 on lysine 16 (H4-K16Ac) is a prevalent and reversible posttranslational chromatin modification in eukaryotes. To characterize the structural and functional role of this mark, we used a native chem. ligation strategy to generate histone H4 that was homogeneously acetylated at K16. The incorporation of this modified histone into nucleosomal arrays inhibits the formation of compact 30-nm-like fibers and impedes the ability of chromatin to form cross-fiber interactions. H4-K16Ac also inhibits the ability of the ATP-utilizing chromatin assembly and remodeling enzyme ACF to mobilize a mononucleosome, indicating that this single histone modification modulates both higher order chromatin structure and functional interactions between a nonhistone protein and the chromatin fiber.
- 6Lu, X.; Simon, M. D.; Chodaparambil, J. V.; Hansen, J. C.; Shokat, K. M.; Luger, K. Nat. Struct. Mol. Biol. 2008, 15, 11226The effect of H3K79 dimethylation and H4K20 trimethylation on nucleosome and chromatin structureLu, Xu; Simon, Matthew D.; Chodaparambil, Jayanth V.; Hansen, Jeffrey C.; Shokat, Kevan M.; Luger, KarolinNature Structural & Molecular Biology (2008), 15 (10), 1122-1124CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)Histone methylation regulates chromatin function dependent on the site and degree of the modification. In addn. to creating binding sites for proteins, methylated lysine residues are likely to influence chromatin structure directly. Here we present crystal structures of nucleosomes reconstituted with methylated histones and investigate the folding behavior of resulting arrays. We demonstrate that dimethylation of histone H3 at lysine residue 79 locally alters the nucleosomal surface, whereas trimethylation of H4 at lysine residue 20 affects higher-order structure.
- 7Fierz, B.; Chatterjee, C.; McGinty, R. K.; Bar-Dagan, M.; Raleigh, D. P.; Muir, T. W. Nat. Chem. Biol. 2011, 7, 1137Histone H2B ubiquitylation disrupts local and higher-order chromatin compactionFierz, Beat; Chatterjee, Champak; McGinty, Robert K.; Bar-Dagan, Maya; Raleigh, Daniel P.; Muir, Tom W.Nature Chemical Biology (2011), 7 (2), 113-119CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Regulation of chromatin structure involves histone post-translational modifications that can modulate intrinsic properties of the chromatin fiber to change the chromatin state. We used chem. defined nucleosome arrays to demonstrate that H2B ubiquitylation (uH2B), a modification assocd. with transcription, interferes with chromatin compaction and leads to an open and biochem. accessible fiber conformation. Notably, these effects were specific for ubiquitin, as compaction of chromatin modified with a similar ubiquitin-sized protein, Hub1, was only weakly affected. Applying a fluorescence-based method, we found that uH2B acts through a mechanism distinct from H4 tail acetylation, a modification known to disrupt chromatin folding. Finally, incorporation of both uH2B and acetylated H4 resulted in synergistic inhibition of higher-order chromatin structure formation, possibly a result of their distinct modes of action.
- 8Robinson, P. J.; Rhodes, D. Curr. Opin. Struct. Biol. 2006, 16, 3368Structure of the '30 nm' chromatin fiber: A key role for the linker histoneRobinson, Philip J. J.; Rhodes, DanielaCurrent Opinion in Structural Biology (2006), 16 (3), 336-343CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)A review. The structure of the '30 nm' chromatin fiber has eluded investigators for 30 yr and remains a major unsolved problem in biol. Progress during the past year has led to the proposal of 2 significantly different models: one derived from the crystal structure of a 4-nucleosome core array lacking the linker histone and the other, much more compact structure, derived from electron microscopy anal. of long nucleosome arrays contg. the linker histone. The 1st model is of the 2-start helix type, and the 2nd model is of the 1-start helix type with interdigitated nucleosomes. These models provide new evidence that the topol. and compactness of the '30 nm' chromatin fiber structure are regulated by the linker histone. The structural information also provides insights into the mechanisms by which the degree of chromatin compaction might be regulated by histone compn. and post-transcriptional modifications.
- 9Luger, K.; Mäder, A. W.; Richmond, R. K.; Sargent, D. F.; Richmond, T. J. Nature 1997, 389, 2519Crystal structure of the nucleosome core particle at 2.8 Å resolutionLuger, Karolin; Mader, Armin W.; Richmond, Robin K.; Sargent, David F.; Richmond, Timothy J.Nature (London) (1997), 389 (6648), 251-260CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)The x-ray crystal structure of the nucleosome core particle of chromatin shows in at. detail how the histone protein octamer is assembled and how 146 base pairs of DNA are organized into a superhelix around it. Both histone/histone and histone/DNA interactions depend on the histone fold domains and addnl., well ordered structure elements extending from this motif. Histone amino-terminal tails pass over and between the gyres of the DNA superhelix to contact neighboring particles. The lack of uniformity between multiple histone/DNA-binding sites causes the DNA to deviate from ideal superhelix geometry.
- 10Lowary, P. T.; Widom, J. J. Mol. Biol. 1998, 276, 1910New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioningLowary, P. T.; Widom, J.Journal of Molecular Biology (1998), 276 (1), 19-42CODEN: JMOBAK; ISSN:0022-2836. (Academic Press Ltd.)DNA sequences that position nucleosomes are of increasing interest because of their relationship to gene regulation in vivo and because of their utility in studies of nucleosome structure and function in vitro. However, at present our understanding of the rules for DNA sequence-directed nucleosome positioning is fragmentary, and existing positioning sequences have many limitations. We carried out a SELEX expt. starting with a large pool of chem. synthetic random DNA mols. to identify those individuals having the highest affinity for histone octamer. A set of highest-affinity mols. were selected, cloned, and sequenced, their affinities (free energies) for histone octamer in nucleosome reconstitution measured, and their ability to position nucleosomes in vitro assessed by native gel electrophoresis. The selected sequences have higher affinity than previously known natural or non-natural sequences, and have a correspondingly strong nucleosome positioning ability. A variety of analyses including Fourier transform, real-space correlation, and direct counting computations were carried out to assess non-random features in the selected sequences. The results reveal sequence rules that were already identified in earlier studies of natural nucleosomal DNA, together with a large set of new rules having even stronger statistical significance. Possible phys. origins of the selected mols.' high affinities are discussed. The sequences isolated in this study should prove valuable for studies of chromatin structure and function in vitro and, potentially, for studies in vivo.
- 11Song, F.; Chen, P.; Sun, D.; Wang, M.; Dong, L.; Liang, D.; Xu, R.-M.; Zhu, P.; Li, G. Science 2014, 344, 37611Cryo-EM Study of the Chromatin Fiber Reveals a Double Helix Twisted by Tetranucleosomal UnitsSong, Feng; Chen, Ping; Sun, Dapeng; Wang, Mingzhu; Dong, Liping; Liang, Dan; Xu, Rui-Ming; Zhu, Ping; Li, GuohongScience (Washington, DC, United States) (2014), 344 (6182), 376-380CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The hierarchical packaging of eukaryotic chromatin plays a central role in transcriptional regulation and other DNA-related biol. processes. Here, we report the 11-angstrom-resoln. cryogenic electron microscopy (cryo-EM) structures of 30-nm chromatin fibers reconstituted in the presence of linker histone H1 and with different nucleosome repeat lengths. The structures show a histone H1-dependent left-handed twist of the repeating tetranucleosomal structural units, within which the four nucleosomes zigzag back and forth with a straight linker DNA. The asym. binding and the location of histone H1 in chromatin play a role in the formation of the 30-nm fiber. Our results provide mechanistic insights into how nucleosomes compact into higher-order chromatin fibers.
- 12Robinson, P. J. J.; Fairall, L.; Huynh, V. A. T.; Rhodes, D. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 650612EM measurements define the dimensions of the "30-nm" chromatin fiber: evidence for a compact, interdigitated structureRobinson, Philip J. J.; Fairall, Louise; Huynh, Van A. T.; Rhodes, DanielaProceedings of the National Academy of Sciences of the United States of America (2006), 103 (17), 6506-6511CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Chromatin structure plays a fundamental role in the regulation of nuclear processes such as DNA transcription, replication, recombination, and repair. Despite considerable efforts during three decades, the structure of the 30-nm chromatin fiber remains controversial. To define fiber dimensions accurately, we have produced very long and regularly folded 30-nm fibers from in vitro reconstituted nucleosome arrays contg. the linker histone and with increasing nucleosome repeat lengths (10 to 70 bp of linker DNA). EM measurements show that the dimensions of these fully folded fibers do not increase linearly with increasing linker length, a finding that is inconsistent with two-start helix models. Instead, we find that there are two distinct classes of fiber structure, both with unexpectedly high nucleosome d.: arrays with 10 to 40 bp of linker DNA all produce fibers with a diam. of 33 nm and 11 nucleosomes per 11 nm, whereas arrays with 50 to 70 bp of linker DNA all produce 44-nm-wide fibers with 15 nucleosomes per 11 nm. Using the phys. constraints imposed by these measurements, we have built a model in which tight nucleosome packing is achieved through the interdigitation of nucleosomes from adjacent helical gyres. Importantly, the model closely matches raw image projections of folded chromatin arrays recorded in the soln. state by using electron cryo-microscopy.
- 13Schalch, T.; Duda, S.; Sargent, D. F.; Richmond, T. J. Nature 2005, 436, 13813X-ray structure of a tetranucleosome and its implications for the chromatin fiberSchalch, Thomas; Duda, Sylwia; Sargent, David F.; Richmond, Timothy J.Nature (London, United Kingdom) (2005), 436 (7047), 138-141CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)DNA in eukaryotic chromosomes is organized in arrays of nucleosomes compacted into chromatin fibers. This higher-order structure of nucleosomes is the substrate for DNA replication, recombination, transcription and repair. Although the structure of the nucleosome core is known at near-at. resoln., even the most fundamental information about the organization of nucleosomes in the fiber is controversial. Here we report the crystal structure of an oligonucleosome (a compact tetranucleosome) at 9 Å resoln., solved by mol. replacement using the nucleosome core structure. The structure shows that linker DNA zigzags back and forth between two stacks of nucleosome cores, which form a truncated two-start helix, and does not follow a path compatible with a one-start solenoidal helix. The length of linker DNA is most probably buffered by stretching of the DNA contained in the nucleosome cores. We have built continuous fiber models by successively stacking tetranucleosomes one on another. The resulting models are nearly fully compacted and most closely resemble the previously described crossed-linker model. They suggest that the interfaces between nucleosomes along a single helix start are polymorphic.
- 14Flaus, A.; Luger, K.; Tan, S.; Richmond, T. J. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 137014Mapping nucleosome position at single base-pair resolution by using site-directed hydroxyl radicalsFlaus A; Luger K; Tan S; Richmond T JProceedings of the National Academy of Sciences of the United States of America (1996), 93 (4), 1370-5 ISSN:0027-8424.A base-pair resolution method for determining nucleosome position in vitro has been developed to com- plement existing, less accurate methods. Cysteaminyl EDTA was tethered to a recombinant histone octamer via a mutant histone H4 with serine 47 replaced by cysteine. When assembled into nucleosome core particles, the DNA could be cut site specifically by hydroxyl radical-catalyzed chain scission by using the Fenton reaction. Strand cleavage occurs mainly at a single nucleotide close to the dyad axis of the core particle, and assignment of this location via the symmetry of the nucleosome allows base-pair resolution mapping of the histone octamer position on the DNA. The positions of the histone octamer and H3H4 tetramer were mapped on a 146-bp Lytechinus variegatus 5S rRNA sequence and a twofold-symmetric derivative. The weakness of translational determinants of nucleosome positioning relative to the overall affinity of the histone proteins for this DNA is clearly demonstrated. The predominant location of both histone octamer and H3H4 tetramer assembled on the 5S rDNA is off center. Shifting the nucleosome core particle position along DNA within a conserved rotational phase could be induced under physiologically relevant conditions. Since nucleosome shifting has important consequences for chromatin structure and gene regulation, an approach to the thermodynamic characterization of this movement is proposed. This mapping method is potentially adaptable for determining nucleosome position in chromatin in vivo.
- 15Arents, G.; Burlingame, R. W.; Wang, B. C.; Love, W. E.; Moudrianakis, E. N. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 1014815The nucleosomal core histone octamer at 3.1 Å resolution: a tripartite protein assembly and a left-handed superhelixArents, Gina; Burlingame, Rufus W.; Wang, Bi Cheng; Love, Warner E.; Moudrianakis, Evangelos N.Proceedings of the National Academy of Sciences of the United States of America (1991), 88 (22), 10148-52CODEN: PNASA6; ISSN:0027-8424.The structure of the octameric histone core of the nucleosome has been detd. by x-ray crystallog. to a resoln. of 3.1 Å. The histone octamer is a tripartite assembly in which a centrally located (H3-H4)2 tetramer is flanked by 2 H2A-H2B dimers. It has a complex outer surface; depending on the perspective, the structure appears as a wedge or as a flat disk. The disk represents the planar projection of a left-handed proteinaceous superhelix with ≈28 Å pitch. The diam. of the particle is 65 Å and the length is 60 Å at its max. and ≈10 Å at its min. extension; these dimensions are in agreement with those reported earlier by A. Klug et al. (1980). The folded histone chains are elongated rather than globular and are assembled in a characteristic handshake motif. The individual polypeptides share a common central structural element of the helix-loop-helix type, which is named the histone fold.
- 16Arents, G.; Moudrianakis, E. N. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 1117016The histone fold: a ubiquitous architectural motif utilized in DNA compaction and protein dimerizationArents, Gina; Moudrianakis, Evangelos N.Proceedings of the National Academy of Sciences of the United States of America (1995), 92 (24), 11170-4CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The histones of all eukaryotes show only a low degree of primary structure homol., but our earlier crystallog. results defined a three-dimensional structural motif, the histone fold, common to all core histones. We now examine the specific architectural patterns within the fold and analyze the nature of the amino acid residues within its functional segments. The histone fold emerges as a fundamental protein dimerization motif while the differentiations of the tips of the histone dimers appear to provide the rules of core octamer assembly and the basis for nucleosome regulation. We present evidence for the occurrence of the fold from archaebacteria to mammals and propose the use of this structural motif to define a distinct family of proteins, the histone fold superfamily. It appears that evolution has conserved the conformation of the fold even through variations in primary structure and among proteins with various functional roles.
- 17Davey, C. A.; Sargent, D. F.; Luger, K.; Maeder, A. W.; Richmond, T. J. J. Mol. Biol. 2002, 319, 109717Solvent Mediated Interactions in the Structure of the Nucleosome Core Particle at 1.9 ResolutionDavey, Curt A.; Sargent, David F.; Luger, Karolin; Maeder, Armin W.; Richmond, Timothy J.Journal of Molecular Biology (2002), 319 (5), 1097-1113CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Science Ltd.)Solvent binding in the nucleosome core particle contg. a 147 base pair, defined-sequence DNA is characterized from the X-ray crystal structure at 1.9 resoln. A single-base-pair increase in DNA length over that used previously results in substantially improved clarity of the electron d. and accuracy for the histone protein and DNA at. coordinates. The reduced disorder has allowed for the first time extensive modeling of water mols. and ions. Over 3000 water mols. and 18 ions have been identified. Water mols. acting as hydrogen-bond bridges between protein and DNA are approx. equal in no. to the direct hydrogen bonds between these components. Bridging water mols. have a dual role in promoting histone-DNA assocn. not only by providing further stability to direct protein-DNA interactions, but also by enabling formation of many addnl. interactions between more distantly related elements. Water mols. residing in the minor groove play an important role in facilitating insertion of arginine side-chains. Water structure at the interface of the histones and DNA provides a means of accommodating intrinsic DNA conformational variation, thus limiting the sequence dependency of nucleosome positioning while enhancing mobility. Monovalent anions are bound near the N termini of histone α-helixes that are not occluded by DNA phosphate groups. Their location in proximity to the DNA phosphodiester backbone suggests that they damp the electrostatic interaction between the histone proteins and the DNA. Divalent cations are bound at specific sites in the nucleosome core particle and contribute to histone-histone and histone-DNA interparticle interactions. These interactions may be relevant to nucleosome assocn. in arrays.
- 18McGinty, R. K.; Tan, S. In Fundamentals of Chromatin; Springer: New York: New York, NY, 2013; pp 1– 28.There is no corresponding record for this reference.
- 19Preez, du L. L.; Patterton, H.-G. Subcell. Biochem. 2013, 61, 37There is no corresponding record for this reference.
- 20Zheng, C.; Hayes, J. J. J. Biol. Chem. 2003, 278, 2421720Intra- and Inter-nucleosomal Protein-DNA Interactions of the Core Histone Tail Domains in a Model SystemZheng, Chunyang; Hayes, Jeffrey J.Journal of Biological Chemistry (2003), 278 (26), 24217-24224CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)The core histone tail domains are key regulators of eukaryotic chromatin structure and function, and alterations in the tail-directed folding of chromatin fibers and higher order structures are the probable outcome of much of the post-translational modifications occurring in these domains. The functions of the tail domains are likely to involve complex intra- and inter-nucleosomal histone-DNA interactions, yet little is known about either the structures or interactions of these domains. Here the authors introduce a method for examg. inter-nucleosome interactions of the tail domains in a model dinucleosome and det. the propensity of each of the four N-terminal tail domains to mediate such interactions in this system. Using a strong nucleosome "positioning" sequence, the authors reconstituted a nucleosome contg. a single histone site specifically modified with a photoinducible crosslinker within the histone tail domain, and a second nucleosome contg. a radiolabeled DNA template. These two nucleosomes were then ligated together and crosslinking induced by brief UV irradn. under various soln. conditions. After crosslinking, the two templates were again sepd. so that crosslinking representing inter-nucleosomal histone-DNA interactions could be unambiguously distinguished from intra-nucleosomal crosslinks. The results show that the N-terminal tails of H2A and H2B, but not of H3 and H4, make internucleosomal histone-DNA interactions within the dinucleosome. The relative extent of intra- to inter-nucleosome interactions was not strongly dependent on ionic strength. Addnl., the authors find that binding of a linker histone to the dinucleosome increased the assocn. of the H3 and H4 tails with the linker DNA region.
- 21Kan, P. Y.; Lu, X.; Hansen, J. C.; Hayes, J. J. Mol. Cell. Biol. 2007, 27, 208421The H3 tail domain participates in multiple interactions during folding and self-association of nucleosome arraysKan, Pu-Yeh; Lu, Xu; Hansen, Jeffrey C.; Hayes, Jeffrey J.Molecular and Cellular Biology (2007), 27 (6), 2084-2091CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)The core histone tail domains play a central role in chromatin structure and epigenetic processes controlling gene expression. Although little is known regarding the mol. details of tail interactions, it is likely that they participate in both short-range and long-range interactions between nucleosomes. Previously, we demonstrated that the H3 tail domain participates in internucleosome interactions during MgCl2-dependent condensation of model nucleosome arrays. However, these studies did not distinguish whether these internucleosome interactions represented short-range intra-array or longer-range interarray interactions. To better understand the complex interactions of the H3 tail domain during chromatin condensation, we have developed a new site-directed crosslinking method to identify and quantify interarray interactions mediated by histone tail domains. Interarray crosslinking was undetectable under salt conditions that induced only local folding, but was detected concomitant with salt-dependent interarray oligomerization at higher MgCl2 concns. Interestingly, lysine-to-glutamine mutations in the H3 tail domain to mimic acetylation resulted in little or no redn. in interarray crosslinking. In contrast, binding of a linker histone caused a much greater enhancement of interarray interactions for unmodified H3 tails compared to "acetylated" H3 tails. Collectively, these results indicate that H3 tail domain performs multiple functions during chromatin condensation via distinct mol. interactions that can be differentially regulated by acetylation or binding of linker histones.
- 22Kan, P. Y.; Caterino, T. L.; Hayes, J. J. Mol. Cell. Biol. 2009, 29, 53822The H4 tail domain participates in intra- and internucleosome interactions with protein and DNA during folding and oligomerization of nucleosome arraysKan, Pu-Yeh; Caterino, Tamara L.; Hayes, Jeffrey J.Molecular and Cellular Biology (2009), 29 (2), 538-546CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)The condensation of nucleosome arrays into higher-order secondary and tertiary chromatin structures likely involves long-range internucleosomal interactions mediated by the core histone tail domains. We have characterized interarray interactions mediated by the H4 tail domain, known to play a predominant role in the formation of such structures. We find that the N-terminal end of the H4 tail mediates interarray contacts with DNA during self-assocn. of oligonucleosome arrays similar to that found previously for the H3 tail domain. However, a site near the histone fold domain of H4 participates in a distinct set of interactions, contacting both DNA and H2A in condensed structures. Moreover, we also find that H4-H2A interactions occur via an intra- as well as an internucleosomal fashion, supporting an addnl. intranucleosomal function for the tail. Interestingly, acetylation of the H4 tail has little effect on interarray interactions by itself but overrides the strong stimulation of interarray interactions induced by linker histones. Our results indicate that the H4 tail facilitates secondary and tertiary chromatin structure formation via a complex array of potentially exclusive interactions that are distinct from those of the H3 tail domain.
- 23White, C. L.; Suto, R. K.; Luger, K. EMBO J. 2001, 20, 520723Structure of the yeast nucleosome core particle reveals fundamental changes in internucleosome interactionsWhite, Cindy L.; Suto, Robert K.; Luger, KarolinEMBO Journal (2001), 20 (18), 5207-5218CODEN: EMJODG; ISSN:0261-4189. (Oxford University Press)Chromatin is composed of nucleosomes, the universally repeating protein-DNA complex in eukaryotic cells. The crystal structure of the nucleosome core particle from Saccharomyces cerevisiae reveals that the structure and function of this fundamental complex is conserved between single-cell organisms and metazoans. Our results show that yeast nucleosomes are likely to be subtly destabilized as compared with nucleosomes from higher eukaryotes, consistent with the idea that much of the yeast genome remains constitutively open during much of its life cycle. Importantly, minor sequence variations lead to dramatic changes in the way in which nucleosomes pack against each other within the crystal lattice. This has important implications for our understanding of the formation of higher-order chromatin structure and its modulation by post-translational modifications. Finally, the yeast nucleosome core particle provides a structural context by which to interpret genetic data obtained from yeast. Coordinates have been deposited with the Protein Data Bank under accession no. 1ID3.
- 24Clapier, C. R.; Chakravarthy, S.; Petosa, C.; Fernández-Tornero, C.; Luger, K.; Müller, C. W. Proteins 2008, 71, 124Structure of the Drosophila nucleosome core particle highlights evolutionary constraints on the H2A-H2B histone dimerClapier, Cedric R.; Chakravarthy, Srinivas; Petosa, Carlo; Fernandez-Tornero, Carlos; Luger, Karolin; Muller, Christoph W.Proteins: Structure, Function, and Bioinformatics (2008), 71 (1), 1-7CODEN: PSFBAF ISSN:. (Wiley-Liss, Inc.)We detd. the 2.45 Å crystal structure of the nucleosome core particle from Drosophila melanogaster and compared it to that of Xenopus laevis bound to the identical 147 base-pair DNA fragment derived from human α-satellite DNA. Differences between the two structures primarily reflect 16 amino acid substitutions between species, 15 of which are in histones H2A and H2B. Four of these involve histone tail residues, resulting in subtly altered protein-DNA interactions that exemplify the structural plasticity of these tails. Of the 12 substitutions occurring within the histone core regions, five involve small, solvent-exposed residues not involved in intraparticle interactions. The remaining seven involve buried hydrophobic residues, and appear to have coevolved so as to preserve the vol. of side chains within the H2A hydrophobic core and H2A-H2B dimer interface. Thus, apart from variations in the histone tails, amino acid substitutions that differentiate Drosophila from Xenopus histones occur in mutually compensatory combinations. This highlights the tight evolutionary constraints exerted on histones since the vertebrate and invertebrate lineages diverged.
- 25Tsunaka, Y.; Kajimura, N.; Tate, S.-I.; Morikawa, K. Nucleic Acids Res. 2005, 33, 342425Alteration of the nucleosomal DNA path in the crystal structure of a human nucleosome core particleTsunaka, Yasuo; Kajimura, Naoko; Tate, Shin-ichi; Morikawa, KosukeNucleic Acids Research (2005), 33 (10), 3424-3434CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Gene expression in eukaryotes depends upon positioning, mobility and packaging of nucleosomes; thus, we need the detailed information of the human nucleosome core particle (NCP) structure, which could clarify chromatin properties. Here, we report the 2.5 Å crystal structure of a human NCP. The overall structure is similar to those of other NCPs reported previously. However, the DNA path of human NCP is remarkably different from that taken within other NCPs with an identical DNA sequence. A comparison of the structural parameters between human and Xenopus laevis DNA reveals that the DNA path of human NCP consecutively shifts by 1 bp in the regions of superhelix axis location -5.0 to -2.0 and 5.0 to 7.0. This alteration of the human DNA path is caused predominantly by tight DNA-DNA contacts within the crystal. It is also likely that the conformational change in the human H2B tail induces the local alteration of the DNA path. In human NCP, the region with the altered DNA path lacks Mn2+ ions and the B-factors of the DNA phosphate groups are substantially high. Therefore, in contrast to the histone octamer, the nucleosomal DNA is sufficiently flexible and mobile and can undergo drastic conformational changes, depending upon the environment.
- 26Sugiyama, M.; Arimura, Y.; Shirayama, K.; Fujita, R.; Oba, Y.; Sato, N.; Inoue, R.; Oda, T.; Sato, M.; Heenan, R. K.; Kurumizaka, H. Biophys. J. 2014, 106, 220626Distinct features of the histone core structure in nucleosomes containing the histone H2A.B variantSugiyama, Masaaki; Arimura, Yasuhiro; Shirayama, Kazuyoshi; Fujita, Risa; Oba, Yojiro; Sato, Nobuhiro; Inoue, Rintaro; Oda, Takashi; Sato, Mamoru; Heenan, Richard K.; Kurumizaka, HitoshiBiophysical Journal (2014), 106 (10), 2206-2213CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)Nucleosomes contg. a human histone variant, H2A.B, were analyzed in aq. soln. by SANS utilizing a contrast variation technique. Comparisons with the canonical H2A nucleosome structure revealed that the DNA termini of the H2A.B nucleosome were detached from the histone core surface, and flexibly expanded toward the solvent. In contrast, the histone tails were compacted in H2A.B nucleosomes compared to those in canonical H2A nucleosomes, suggesting that they bind to the surface of the histone core and/or DNA. Thus, the histone tail dynamics may function to regulate the flexibility of the DNA termini in the nucleosomes.
- 27Urahama, T.; Horikoshi, N.; Osakabe, A.; Tachiwana, H.; Kurumizaka, H. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2014, 70, 44427Structure of human nucleosome containing the testis-specific histone variant TSH2BUrahama, Takashi; Horikoshi, Naoki; Osakabe, Akihisa; Tachiwana, Hiroaki; Kurumizaka, HitoshiActa Crystallographica, Section F: Structural Biology Communications (2014), 70 (4), 444-449CODEN: ACSFEN; ISSN:2053-230X. (International Union of Crystallography)The human histone H2B variant TSH2B is highly expressed in testis and may function in the chromatin transition during spermatogenesis. In the present study, the crystal structure of the human testis-specific nucleosome contg. TSH2B was detd. at 2.8 Å resoln. A local structural difference between TSH2B and canonical H2B in nucleosomes was detected around the TSH2B-specific amino-acid residue Ser85. The TSH2B Ser85 residue does not interact with H4 in the nucleosome, but in the canonical nucleosome the H2B Asn84 residue (corresponding to the TSH2B Ser85 residue) forms water-mediated hydrogen bonds with the H4 Arg78 residue. In contrast, the other TSH2B-specific amino-acid residues did not induce any significant local structural changes in the TSH2B nucleosome. These findings may provide important information for understanding how testis-specific histone variants form nucleosomes during spermatogenesis.
- 28Tachiwana, H.; Kagawa, W.; Shiga, T.; Osakabe, A.; Miya, Y.; Saito, K.; Hayashi-Takanaka, Y.; Oda, T.; Sato, M.; Park, S.-Y.; Kimura, H.; Kurumizaka, H. Nature 2011, 476, 23228Crystal structure of the human centromeric nucleosome containing CENP-ATachiwana, Hiroaki; Kagawa, Wataru; Shiga, Tatsuya; Osakabe, Akihisa; Miya, Yuta; Saito, Kengo; Hayashi-Takanaka, Yoko; Oda, Takashi; Sato, Mamoru; Park, Sam-Yong; Kimura, Hiroshi; Kurumizaka, HitoshiNature (London, United Kingdom) (2011), 476 (7359), 232-235CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)In eukaryotes, accurate chromosome segregation during mitosis and meiosis is coordinated by kinetochores, which are unique chromosomal sites for microtubule attachment. Centromeres specify the kinetochore formation sites on individual chromosomes, and are epigenetically marked by the assembly of nucleosomes contg. the centromere-specific histone H3 variant, CENP-A. Although the underlying mechanism is unclear, centromere inheritance is probably dictated by the architecture of the centromeric nucleosome. Here we report the crystal structure of the human centromeric nucleosome contg. CENP-A and its cognate α-satellite DNA deriv. (147 base pairs). In the human CENP-A nucleosome, the DNA is wrapped around the histone octamer, consisting of two each of histones H2A, H2B, H4 and CENP-A, in a left-handed orientation. However, unlike the canonical H3 nucleosome, only the central 121 base pairs of the DNA are visible. The thirteen base pairs from both ends of the DNA are invisible in the crystal structure, and the αN helix of CENP-A is shorter than that of H3, which is known to be important for the orientation of the DNA ends in the canonical H3 nucleosome. A structural comparison of the CENP-A and H3 nucleosomes revealed that CENP-A contains two extra amino acid residues (Arg 80 and Gly 81) in the loop 1 region, which is completely exposed to the solvent. Mutations of the CENP-A loop 1 residues reduced CENP-A retention at the centromeres in human cells. Therefore, the CENP-A loop 1 may function in stabilizing the centromeric chromatin contg. CENP-A, possibly by providing a binding site for trans-acting factors. The structure provides the first at.-resoln. picture of the centromere-specific nucleosome.
- 29Tachiwana, H.; Osakabe, A.; Shiga, T.; Miya, Y.; Kimura, H.; Kagawa, W.; Kurumizaka, H. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 57829Structures of human nucleosomes containing major histone H3 variantsTachiwana, Hiroaki; Osakabe, Akihisa; Shiga, Tatsuya; Miya, Yuta; Kimura, Hiroshi; Kagawa, Wataru; Kurumizaka, HitoshiActa Crystallographica, Section D: Biological Crystallography (2011), 67 (6), 578-583CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)The nucleosome is the fundamental repeating unit of chromatin, via which genomic DNA is packaged into the nucleus in eukaryotes. In the nucleosome, 2 copies of each core histone (H2A, H2B, H3, and H4) form a histone octamer which wraps 146 base pairs of DNA around itself. All of the core histones except for histone H4 have nonallelic isoforms called histone variants. In humans, 8 histone H3 variants (H3.1, H3.2, H3.3, H3T, H3.5, H3.X, H3.Y, and CENP-A) have been reported to date. Previous studies have suggested that histone H3 variants possess distinct functions in the formation of specific chromosome regions and/or in the regulation of transcription and replication. Histones H3.1, H3.2, and H3.3 are the most abundant H3 variants. Here, crystal structures of human HeLa cell nucleosomes contg. either H3.2 or H3.3 were solved. The structures were essentially the same as that of the H3.1 nucleosome. Since the amino acid residues specific for H3.2 and H3.3 are located on the accessible surface of the H3/H4 tetramer, they may be potential interaction sites for H3.2- and H3.3-specific chaperones.
- 30Tachiwana, H.; Kagawa, W.; Osakabe, A.; Kawaguchi, K.; Shiga, T.; Hayashi-Takanaka, Y.; Kimura, H.; Kurumizaka, H. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 1045430Structural basis of instability of the nucleosome containing a testis-specific histone variant, human H3TTachiwana, Hiroaki; Kagawa, Wataru; Osakabe, Akihisa; Kawaguchi, Koichiro; Shiga, Tatsuya; Hayashi-Takanaka, Yoko; Kimura, Hiroshi; Kurumizaka, HitoshiProceedings of the National Academy of Sciences of the United States of America (2010), 107 (23), 10454-10459, S10454/1-S10454/5CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A histone H3 variant, H3T, is highly expressed in the testis, suggesting that it may play an important role in the chromatin reorganization required for. meiosis and/or spermatogenesis. In the present study, the authors found that the nucleosome contg. human H3T is significantly unstable both in vitro and in vivo, as compared to the conventional nucleosome contg. H3.1. The crystal structure of the H3T nucleosome revealed structural differences in the H3T regions on both ends of the central αa2 helix, as compared to those of H3.1. The H3T-specific residues (Met71 and Val111) are the source of the structural differences obsd. between H3T and H3.1. A mutational anal. revealed that these residues are responsible for the reduced stability of the H3T-contg. nucleosome. These phys. and structural properties of the H3T-contg. nucleosome may provide the basis of chromatin reorganization during spermatogenesis.
- 31Arimura, Y.; Kimura, H.; Oda, T.; Sato, K.; Osakabe, A.; Tachiwana, H.; Sato, Y.; Kinugasa, Y.; Ikura, T.; Sugiyama, M.; Sato, M.; Kurumizaka, H. Sci. Rep. 2013, 3, 351031Structural basis of a nucleosome containing histone H2A.B/H2A.Bbd that transiently associates with reorganized chromatinArimura Yasuhiro; Sato Koichi; Osakabe Akihisa; Tachiwana Hiroaki; Kurumizaka Hitoshi; Kimura Hiroshi; Sato Yuko; Oda Takashi; Kinugasa Yasuha; Ikura Tsuyoshi; Sugiyama Masaaki; Sato MamoruScientific reports (2013), 3 (), 3510 ISSN:.Human histone H2A.B (formerly H2A.Bbd), a non-allelic H2A variant, exchanges rapidly as compared to canonical H2A, and preferentially associates with actively transcribed genes. We found that H2A.B transiently accumulated at DNA replication and repair foci in living cells. To explore the biochemical function of H2A.B, we performed nucleosome reconstitution analyses using various lengths of DNA. Two types of H2A.B nucleosomes, octasome and hexasome, were formed with 116, 124, or 130 base pairs (bp) of DNA, and only the octasome was formed with 136 or 146 bp DNA. In contrast, only hexasome formation was observed by canonical H2A with 116 or 124 bp DNA. A small-angle X-ray scattering analysis revealed that the H2A.B octasome is more extended, due to the flexible detachment of the DNA regions at the entry/exit sites from the histone surface. These results suggested that H2A.B rapidly and transiently forms nucleosomes with short DNA segments during chromatin reorganization.
- 32Chakravarthy, S.; Bao, Y.; Roberts, V. A.; Tremethick, D.; Luger, K. Cold Spring Harbor Symp. Quant. Biol. 2004, 69, 22732Structural characterization of histone H2A variantsChakravarthy, S.; Bao, Y.; Roberts, V. A.; Tremethick, D.; Luger, K.Cold Spring Harbor Symposia on Quantitative Biology (2004), 69 (), 227-234CODEN: CSHSAZ; ISSN:0091-7451. (Cold Spring Harbor Laboratory Press)A review on available structural information on nucleosomes and chromatin contg. histone H2A variants and how structure relates to their varied function. A hypothesis why "true" histone variants have been identified for only histone H2A and H3 is presented, and data in support of this hypothesis that particular regions in the H2A amino acid sequence appeared to have been targets during the evolution of H2A histone variants is shown.
- 33Chakravarthy, S.; Gundimella, S. K. Y.; Caron, C.; Perche, P.-Y.; Pehrson, J. R.; Khochbin, S.; Luger, K. Mol. Cell. Biol. 2005, 25, 761633Structural characterization of the histone variant macroH2AChakravarthy, Srinivas; Gundimella, Sampath Kumar Y.; Caron, Cecile; Perche, Pierre-Yves; Pehrson, John R.; Khochbin, Saadi; Luger, KarolinMolecular and Cellular Biology (2005), 25 (17), 7616-7624CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)MacroH2A is an H2A variant with a highly unusual structural organization. It has a C-terminal domain connected to the N-terminal histone domain by a linker. Crystallog. and biochem. studies show that changes in the L1 loop in the histone fold region of macroH2A impact the structure and potentially the function of nucleosomes. The 1.6-A x-ray structure of the nonhistone region reveals an α/β fold which has previously been found in a functionally diverse group of proteins. This region assocs. with histone deacetylases and affects the acetylation status of nucleosomes contg. macroH2A. Thus, the unusual domain structure of macroH2A integrates independent functions that are instrumental in establishing a structurally and functionally unique chromatin domain.
- 34Chakravarthy, S.; Luger, K. J. Biol. Chem. 2006, 281, 2552234The hstone variant macro-H2A preferentially forms "hybrid nucleosomes"Chakravarthy, Srinivas; Luger, KarolinJournal of Biological Chemistry (2006), 281 (35), 25522-25531CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)The histone domain of macro-H2A, which constitutes the N-terminal one-third of this histone variant, is only 64% identical to major H2A. We have shown previously that the main structural differences in a nucleosome in which both H2A moieties have been replaced by macro-H2A reside in the only point of contact between the two histone dimers, the L1-L1 interface of macro-H2A. Here we show that the L1 loop of macro-H2A is responsible for the increased salt-dependent stability of the histone octamer, with implications for the nucleosome assembly pathway. It is unknown whether only one or both of the H2A-H2B dimers within a nucleosome are replaced with H2A variant contg. nucleosomes in vivo. We demonstrate that macro-H2A preferentially forms hybrid nucleosomes contg. one chain each of major H2A and macro-HA in vitro. The 2.9-Å crystal structure of such a hybrid nucleosome shows significant structural differences in the L1-L1 interface when comparing with homotypic major H2A- and macro-H2A-contg. nucleosomes. Both homotypic and hybrid macro-nucleosome core particles (NCPs) are resistant to chaperone-assisted H2A-H2B dimer exchange. Together, our findings suggest that the histone domain of macro-H2A modifies the dynamic properties of the nucleosome. We propose that the possibility of forming hybrid macro-NCP adds yet another level of complexity to variant nucleosome structure and function.
- 35Abbott, D. W.; Laszczak, M.; Lewis, J. D.; Su, H.; Moore, S. C.; Hills, M.; Dimitrov, S.; Ausio, J. Biochemistry 2004, 43, 135235Structural Characterization of MacroH2A Containing ChromatinAbbott, D. Wade; Laszczak, Mario; Lewis, John D.; Su, Harvey; Moore, Susan C.; Hills, Melissa; Dimitrov, Stefan; Ausio, JuanBiochemistry (2004), 43 (5), 1352-1359CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)MacroH2A (mH2A) is one of the most recently identified members of the heteromorphous histone variant family. It is unique among the members of this group because it contains an unusually large non-histone C-terminal end, from where its name derives, and appears to be restricted to subphylum vertebrata. Although a concerted effort has been carried out to characterize the physiol. relevance of mH2A, little is known in comparison about the structural importance of the mol. Elucidating the biophys. and conformational properties of mH2A in chromatin may provide clues into the links between this histone variant and its unique function(s). In this paper, the authors look first at the heterogeneous tissue-specific distribution of this protein in different vertebrate classes. This is followed by a structural comparison between mH2A and H2A protein and by the characterization of the nucleosome core particles with which these histone subtypes are assocd. The authors find that the highly α-helical C-terminus of mH2A confers an asym. conformation to nucleosomes and that this variant is tightly bound to chromatin fragments in a way that does not depend on the overall extent of acetylation of the other core histones.
- 36Eirín-López, J. M.; Ishibashi, T.; Ausio, J. FASEB J. 2008, 22, 31636H2A.Bbd: a quickly evolving hypervariable mammalian histone that destabilizes nucleosomes in an acetylation-independent wayEirin-Lopez, Jose Maria; Ishibashi, Toyotaka; Ausio, JuanFASEB Journal (2008), 22 (1), 316-326, 10.1096/fj.07-9255comCODEN: FAJOEC; ISSN:0892-6638. (Federation of American Societies for Experimental Biology)Mol. evolutionary analyses revealed that histone H2A.Bbd is a highly variable quickly evolving mammalian replacement histone variant, in striking contrast to all other histones. At the nucleotide level, this variability appears to be the result of a larger amt. of nonsynonymous variation, which affects to a lesser extent, the structural domain of the protein comprising the histone fold. The resulting amino acid sequence diversity can be predicted to affect the internucleosomal and intranucleosomal histone interactions. Our phylogenetic anal. has allowed us to identify several of the residues involved. The biophys. characterization of nucleosomes reconstituted with recombinant mouse H2A.Bbd and their comparison to similar data obtained with human H2A.Bbd clearly support this notion. Despite the high interspecific amino acid sequence variability, all of the H2A.Bbd variants exert similar structural effects at the nucleosome level, which result in an unfolded highly unstable nucleoprotein complex. Such structure resembles that previously described for the highly dynamically acetylated nucleosomes assocd. with transcriptionally active regions of the genome. Nevertheless, the structure of nucleosome core particles reconstituted from H2A.Bbd is not affected by the presence of a hyperacetylated histone complement. This suggests that replacement by H2A.Bbd provides an alternative mechanism to unfold chromatin structure, possibly in euchromatic regions, in a way that is not dependent on acetylation.
- 37Horikoshi, N.; Sato, K.; Shimada, K.; Arimura, Y.; Osakabe, A.; Tachiwana, H.; Hayashi-Takanaka, Y.; Iwasaki, W.; Kagawa, W.; Harata, M.; Kimura, H.; Kurumizaka, H. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2013, 69, 243137Structural polymorphism in the L1 loop regions of human H2A.Z.1 and H2A.Z.2Horikoshi, Naoki; Sato, Koichi; Shimada, Keisuke; Arimura, Yasuhiro; Osakabe, Akihisa; Tachiwana, Hiroaki; Hayashi-Takanaka, Yoko; Iwasaki, Wakana; Kagawa, Wataru; Harata, Masahiko; Kimura, Hiroshi; Kurumizaka, HitoshiActa Crystallographica, Section D: Biological Crystallography (2013), 69 (12), 2431-2439CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)The histone H2A.Z variant is widely conserved among eukaryotes. Two isoforms, H2A.Z.1 and H2A.Z.2, have been identified in vertebrates and may have distinct functions in cell growth and gene expression. However, no structural differences between H2A.Z.1 and H2A.Z.2 have been reported. In the present study, the crystal structures of nucleosomes contg. human H2A.Z.1 and H2A.Z.2 were detd. The structures of the L1 loop regions were found to clearly differ between H2A.Z.1 and H2A.Z.2, although their amino-acid sequences in this region are identical. This structural polymorphism may have been induced by a substitution that evolutionarily occurred at the position of amino acid 38 and by the flexible nature of the L1 loops of H2A.Z.1 and H2A.Z.2. It was also found that in living cells nucleosomal H2A.Z.1 exchanges more rapidly than H2A.Z.2. A mutational anal. revealed that the amino-acid difference at position 38 is at least partially responsible for the distinctive dynamics of H2A.Z.1 and H2A.Z.2. These findings provide important new information for understanding the differences in the regulation and functions of H2A.Z.1 and H2A.Z.2 in cells.
- 38Suto, R. K.; Clarkson, M. J.; Tremethick, D. J.; Luger, K. Nat. Struct. Biol. 2000, 7, 112138Crystal structure of a nucleosome core particle containing the variant histone H2A.ZSuto, Robert K.; Clarkson, Michael J.; Tremethick, David J.; Luger, KarolinNature Structural Biology (2000), 7 (12), 1121-1124CODEN: NSBIEW; ISSN:1072-8368. (Nature America Inc.)Activation of transcription within chromatin has been correlated with the incorporation of the essential histone variant H2A.Z into nucleosomes. H2A.Z and other histone variants may establish structurally distinct chromosomal domains; however, the mol. mechanism by which they function is largely unknown. Here we report the 2.6 Å crystal structure of a nucleosome core particle contg. the histone variant H2A.Z. The overall structure is similar to that of the previously reported 2.8 Å nucleosome structure contg. major histone proteins. However, distinct localized changes result in the subtle destabilization of the interaction between the (H2A.Z-H2B) dimer and the (H3-H4)2 tetramer. Moreover, H2A.Z nucleosomes have an altered surface that includes a metal ion. This altered surface may lead to changes in higher order structure, and/or could result in the assocn. of specific nuclear proteins with H2A.Z. Finally, incorporation of H2A.Z and H2A within the same nucleosome is unlikely, due to significant changes in the interface between the two H2A.Z-H2B dimers.
- 39Dechassa, M. L.; Wyns, K.; Li, M.; Hall, M. A.; Wang, M. D.; Luger, K. Nat. Commun. 2011, 2, 31339Structure and Scm3-mediated assembly of budding yeast centromeric nucleosomesDechassa Mekonnen Lemma; Wyns Katharina; Li Ming; Hall Michael A; Wang Michelle D; Luger KarolinNature communications (2011), 2 (), 313 ISSN:.Much controversy exists regarding the structural organization of the yeast centromeric nucleosome and the role of the nonhistone protein, Scm3, in its assembly and architecture. Here we show that the substitution of H3 with its centromeric variant Cse4 results in octameric nucleosomes that organize DNA in a left-handed superhelix. We demonstrate by single-molecule approaches, micrococcal nuclease digestion and small-angle X-ray scattering that Cse4-nucleosomes exhibit an open conformation with weakly bound terminal DNA segments. The Cse4-octamer does not preferentially form nucleosomes on its cognate centromeric DNA. We show that Scm3 functions as a Cse4-specific nucleosome assembly factor, and that the resulting octameric nucleosomes do not contain Scm3 as a stably bound component. Taken together, our data provide insights into the assembly and structural features of the budding yeast centromeric nucleosome.
- 40Conde e Silva, N.; Black, B. E.; Sivolob, A.; Filipski, J.; Cleveland, D. W.; Prunell, A. J. Mol. Biol. 2007, 370, 55540CENP-A-containing Nucleosomes: Easier Disassembly versus Exclusive Centromeric LocalizationConde e Silva, Natalia; Black, Ben E.; Sivolob, Andrei; Filipski, Jan; Cleveland, Don W.; Prunell, ArielJournal of Molecular Biology (2007), 370 (3), 555-573CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)CENP-A is a histone variant that replaces conventional H3 in nucleosomes of functional centromeres. We report here, from reconstitutions of CENP-A- and H3-contg. nucleosomes on linear DNA fragments and the comparison of their electrophoretic mobility, that CENP-A induces some positioning of its own and some unwrapping at the entry-exit relative to canonical nucleosomes on both 5 S DNA and the α-satellite sequence on which it is normally loaded. This steady-state unwrapping was quantified to 7(±2) bp by nucleosome reconstitutions on a series of DNA minicircles, followed by their relaxation with topoisomerase I. The unwrapping was found to ease nucleosome invasion by exonuclease III, to hinder the binding of a linker histone, and to promote the release of an H2A-H2B dimer by nucleosome assembly protein 1 (NAP-1). The (CENP-A-H4)2 tetramer was also more readily destabilized with heparin than the (H3-H4)2 tetramer, suggesting that CENP-A has evolved to confer its nucleosome a specific ability to disassemble. This dual relative instability is proposed to facilitate the progressive clearance of CENP-A nucleosomes that assemble promiscuously in euchromatin, esp. as is seen following CENP-A transient over-expression.
- 41Sekulic, N.; Bassett, E. A.; Rogers, D. J.; Black, B. E. Nature 2010, 467, 34741The structure of (CENP-AH4)2 reveals physical features that mark centromeresSekulic, Nikolina; Bassett, Emily A.; Rogers, Danielle J.; Black, Ben E.Nature (London, United Kingdom) (2010), 467 (7313), 347-351CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Centromeres are specified epigenetically, and the histone H3 variant CENP-A is assembled into the chromatin of all active centromeres. Divergence from H3 raises the possibility that CENP-A generates unique chromatin features to phys. mark centromere location. Here we report the crystal structure of a subnucleosomal heterotetramer, human (CENP-AH4)2, that reveals three distinguishing properties encoded by the residues that comprise the CENP-A targeting domain (CATD): (1) a CENP-ACENP-A interface that is substantially rotated relative to the H3H3 interface; (2) a protruding loop L1 of the opposite charge compared to that on H3; and (3) strong hydrophobic contacts that rigidify the CENP-AH4 interface. Residues involved in the CENP-ACENP-A rotation are required for efficient incorporation into centromeric chromatin, indicating specificity for an unconventional nucleosome shape. DNA topol. anal. indicates that CENP-A-contg. nucleosomes are octameric with conventional left-handed DNA wrapping, in contrast to other recent proposals. Our results indicate that CENP-A marks centromere location by restructuring the nucleosome from within its folded histone core.
- 42Kingston, I. J.; Yung, J. S. Y.; Singleton, M. R. J. Biol. Chem. 2011, 286, 402142Biophysical Characterization of the Centromere-specific Nucleosome from Budding YeastKingston, Isabel J.; Yung, Jasmine S. Y.; Singleton, Martin R.Journal of Biological Chemistry (2011), 286 (5), 4021-4026CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)The centromeric DNA of all eukaryotes is assembled upon a specialized nucleosome contg. a histone H3 variant known as CenH3. Despite the importance and conserved nature of this protein, the characteristics of the centromeric nucleosome are still poorly understood. In particular, the stoichiometry and DNA-binding properties of the CenH3 nucleosome have been the subject of some debate. We have characterized the budding yeast centromeric nucleosome by biochem. and biophys. methods and show that it forms a stable octamer contg. two copies of the Cse4 protein and wraps DNA in a left-handed supercoil, similar to the canonical H3 nucleosome. The DNA-binding properties of the recombinant nucleosome are identical to those obsd. in vivo, demonstrating that the octameric structure is physiol. relevant.
- 43Panchenko, T.; Sorensen, T. C.; Woodcock, C. L.; Kan, Z.-Y.; Wood, S.; Resch, M. G.; Luger, K.; Englander, S. W.; Hansen, J. C.; Black, B. E. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 1658843Replacement of histone H3 with CENP-A directs global nucleosome array condensation and loosening of nucleosome superhelical terminiPanchenko, Tanya; Sorensen, Troy C.; Woodcock, Christopher L.; Kan, Zhong-yuan; Wood, Stacey; Resch, Michael G.; Luger, Karolin; Englander, S. Walter; Hansen, Jeffrey C.; Black, Ben E.Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (40), 16588-16593, S16588/1-S16588/7CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Centromere protein A (CENP-A) is a histone H3 variant that marks centromere location on the chromosome. To study the subunit structure and folding of human CENP-A-contg. chromatin, we generated a set of nucleosomal arrays with canonical core histones and another set with CENP-A substituted for H3. At the level of quaternary structure and assembly, we find that CENP-A arrays are composed of octameric nucleosomes that assemble in a stepwise mechanism, recapitulating conventional array assembly with canonical histones. At intermediate structural resoln., we find that CENP-A-contg. arrays are globally condensed relative to arrays with the canonical histones. At high structural resoln., using hydrogen-deuterium exchange coupled to mass spectrometry (H/DX-MS), we find that the DNA superhelical termini within each nucleosome are loosely connected to CENP-A, and we identify the key amino acid substitution that is largely responsible for this behavior. Also the C terminus of histone H2A undergoes rapid hydrogen exchange-relative to canonical arrays and does so in a manner that is independent of nucleosomal array folding. These findings have implications for understanding CENP-A-contg. nucleosome structure and higher-order chromatin folding at the centromere.
- 44Doyen, C.-M.; Montel, F.; Gautier, T.; Menoni, H.; Claudet, C.; Delacour-Larose, M.; Angelov, D.; Hamiche, A.; Bednar, J.; Faivre-Moskalenko, C.; Bouvet, P.; Dimitrov, S. EMBO J. 2006, 25, 423444Dissection of the unusual structural and functional properties of the variant H2A.Bbd nucleosomeDoyen, Cecile-Marie; Montel, Fabien; Gautier, Thierry; Menoni, Herve; Claudet, Cyril; Delacour-Larose, Marlene; Angelov, Dimitri; Hamiche, Ali; Bednar, Jan; Faivre-Moskalenko, Cendrine; Bouvet, Philippe; Dimitrov, StefanEMBO Journal (2006), 25 (18), 4234-4244CODEN: EMJODG; ISSN:0261-4189. (Nature Publishing Group)The histone variant H2A.Bbd appeared to be assocd. with active chromatin, but how it functions is unknown. We have dissected the properties of nucleosome contg. H2A.Bbd. At. force microscopy (AFM) and electron cryo-microscopy (cryo-EM) showed that the H2A.Bbd histone octamer organizes only ∼130 bp of DNA, suggesting that 10 bp of each end of nucleosomal DNA are released from the octamer. In agreement with this, the entry/exit angle of the nucleosomal DNA ends formed an angle close to 180° and the physico-chem. anal. pointed to a lower stability of the variant particle. Reconstitution of nucleosomes with swapped-tail mutants demonstrated that the N-terminus of H2A.Bbd has no impact on the nucleosome properties. AFM, cryo-EM and chromatin remodeling expts. showed that the overall structure and stability of the particle, but not its property to interfere with the SWI/SNF induced remodeling, were detd. to a considerable extent by the H2A.Bbd docking domain. These data show that the whole H2A.Bbd histone fold domain is responsible for the unusual properties of the H2A.Bbd nucleosome.
- 45Cosgrove, M. S.; Wolberger, C. Biochem. Cell Biol. 2005, 83, 46845How does the histone code work?Cosgrove, Michael S.; Wolberger, CynthiaBiochemistry and Cell Biology (2005), 83 (4), 468-476CODEN: BCBIEQ; ISSN:0829-8211. (National Research Council of Canada)A review. Patterns of histone post-translational modifications correlate with distinct chromosomal states that regulate access to DNA, leading to the histone-code hypothesis. However, it is not clear how modification of flexible histone tails leads to changes in nucleosome dynamics and, thus, chromatin structure. The recent discovery that, like the flexible histone tails, the structured globular domain of the nucleosome core particle is also extensively modified adds a new and exciting dimension to the histone-code hypothesis, and calls for the re-examn. of current models for the epigenetic regulation of chromatin structure. Here, the authors review these findings and other recent studies that suggest the structured globular domain of the nucleosome core particle plays a key role regulating chromatin dynamics.
- 46Cosgrove, M. S.; Boeke, J. D.; Wolberger, C. Nat. Struct. Mol. Biol. 2004, 11, 103746Regulated nucleosome mobility and the histone codeCosgrove, Michael S.; Boeke, Jef D.; Wolberger, CynthiaNature Structural & Molecular Biology (2004), 11 (11), 1037-1043CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)A review. Post-translational modifications of the histone tails are correlated with distinct chromatin states that regulate access to DNA. Recent proteomic analyses have revealed several new modifications in the globular nucleosome core, many of which lie at the histone-DNA interface. We interpret these modifications in light of previously published data and propose a new and testable model for how cells implement the histone code by modulating nucleosome dynamics.
- 47Mersfelder, E. L.; Parthun, M. R. Nucleic Acids Res. 2006, 34, 265347The tale beyond the tail: histone core domain modifications and the regulation of chromatin structureMersfelder, Erica L.; Parthun, Mark R.Nucleic Acids Research (2006), 34 (9), 2653-2662CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)A review. Histone post-translational modifications occur, not only in the N-terminal tail domains, but also in the core domains. While modifications in the N-terminal tail function largely through the regulation of the binding of non-histone proteins to chromatin, based on their location in the nucleosome, core domain modifications may also function through distinct mechanisms involving structural alterations to the nucleosome. Here, the authors review recent developments with regard to these novel histone modifications and discuss their important role in the regulation of chromatin structure.
- 48North, J. A.; Šimon, M.; Ferdinand, M. B.; Shoffner, M. A.; Picking, J. W.; Howard, C. J.; Mooney, A. M.; van Noort, J.; Poirier, M. G.; Ottesen, J. J. Nucleic Acids Res. 2014, 42, 492248Histone H3 phosphorylation near the nucleosome dyad alters chromatin structureNorth, Justin A.; Simon, Marek; Ferdinand, Michelle B.; Shoffner, Matthew A.; Picking, Jonathan W.; Howard, Cecil J.; Mooney, Alex M.; van Noort, John; Poirier, Michael G.; Ottesen, Jennifer J.Nucleic Acids Research (2014), 42 (8), 4922-4933CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Nucleosomes contain ∼146 bp of DNA wrapped around a histone protein octamer that controls DNA accessibility to transcription and repair complexes. Post-translational modification (PTM) of histone proteins regulates nucleosome function. To date, only modest changes in nucleosome structure have been directly attributed to histone PTMs. Histone residue H3(T118) is located near the nucleosome dyad and can be phosphorylated. This PTM destabilizes nucleosomes and is implicated in the regulation of transcription and repair. Here, we report gel electrophoretic mobility, sucrose gradient sedimentation, thermal disassembly, micrococcal nuclease digestion and at. force microscopy measurements of two DNA-histone complexes that are structurally distinct from nucleosomes. We find that H3(T118ph) facilitates the formation of a nucleosome duplex with two DNA mols. wrapped around two histone octamers, and an altosome complex that contains one DNA mol. wrapped around two histone octamers. The nucleosome duplex complex forms within short ∼150 bp DNA mols., whereas altosomes require at least ∼250 bp of DNA and form repeatedly along 3000 bp DNA mols. These results are the first report of a histone PTM significantly altering the nucleosome structure.
- 49North, J. A.; Javaid, S.; Ferdinand, M. B.; Chatterjee, N.; Picking, J. W.; Shoffner, M.; Nakkula, R. J.; Bartholomew, B.; Ottesen, J. J.; Fishel, R.; Poirier, M. G. Nucleic Acids Res. 2011, 39, 646549Phosphorylation of histone H3(T118) alters nucleosome dynamics and remodelingNorth, Justin A.; Javaid, Sarah; Ferdinand, Michelle B.; Chatterjee, Nilanjana; Picking, Jonathan W.; Shoffner, Matthew; Nakkula, Robin J.; Bartholomew, Blaine; Ottesen, Jennifer J.; Fishel, Richard; Poirier, Michael G.Nucleic Acids Research (2011), 39 (15), 6465-6474CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Nucleosomes, the fundamental units of chromatin structure, are regulators and barriers to transcription, replication, and repair. Post-translational modifications (PTMs) of the histone proteins within nucleosomes regulate these DNA processes. Histone H3(T118) is a site of phosphorylation [H3(T118ph)] and is implicated in regulation of transcription and DNA repair. Here, the authors prepd. H3(T118ph) by expressed protein ligation and detd. its influence on nucleosome dynamics. It was found that H3(T118ph) reduced DNA-histone binding by 2 kcal/mol, increased nucleosome mobility by 28-fold, and increased DNA accessibility near the dyad region by 6-fold. Moreover, H3(T118ph) increased the rate of hMSH2-hMSH6 nucleosome disassembly and enabled nucleosome disassembly by the SWI/SNF chromatin remodeler. These studies suggest that H3(T118ph) directly enhances and may reprogram chromatin remodeling reactions.
- 50Manohar, M.; Mooney, A. M.; North, J. A.; Nakkula, R. J.; Picking, J. W.; Edon, A.; Fishel, R.; Poirier, M. G.; Ottesen, J. J. J. Biol. Chem. 2009, 284, 2331250Acetylation of Histone H3 at the Nucleosome Dyad Alters DNA-Histone BindingManohar, Mridula; Mooney, Alex M.; North, Justin A.; Nakkula, Robin J.; Picking, Jonathan W.; Edon, Annick; Fishel, Richard; Poirier, Michael G.; Ottesen, Jennifer J.Journal of Biological Chemistry (2009), 284 (35), 23312-23321CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Histone post-translational modifications are essential for regulating and facilitating biol. processes such as RNA transcription and DNA repair. Fifteen modifications are located in the DNA-histone dyad interface and include the acetylation of H3-K115 (H3-K115Ac) and H3-K122 (H3-K122Ac), but the functional consequences of these modifications are unknown. We have prepd. semisynthetic histone H3 acetylated at Lys-115 and/or Lys-122 by expressed protein ligation and incorporated them into single nucleosomes. Competitive reconstitution anal. demonstrated that the acetylation of H3-K115 and H3-K122 reduces the free energy of histone octamer binding. Restriction enzyme kinetic anal. suggests that these histone modifications do not alter DNA accessibility near the sites of modification. However, acetylation of H3-K122 increases the rate of thermal repositioning. Remarkably, Lys→Gln substitution mutations, which are used to mimic Lys acetylation, do not fully duplicate the effects of the H3-K115Ac or H3-K122Ac modifications. Our results are consistent with the conclusion that acetylation in the dyad interface reduces DNA-histone interaction(s), which may facilitate nucleosome repositioning and/or assembly/disassembly.
- 51Šimon, M.; North, J. A.; Shimko, J. C.; Forties, R. A.; Ferdinand, M. B.; Manohar, M.; Zhang, M.; Fishel, R.; Ottesen, J. J.; Poirier, M. G. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 1271151Histone fold modifications control nucleosome unwrapping and disassemblySimon, Marek; North, Justin A.; Shimko, John C.; Forties, Robert A.; Ferdinand, Michelle B.; Manohar, Mridula; Zhang, Meng; Fishel, Richard; Ottesen, Jennifer J.; Poirier, Michael G.Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (31), 12711-12716, S12711/1-S12711/9CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Nucleosomes are stable DNA-histone protein complexes that must be unwrapped and disassembled for genome expression, replication, and repair. Histone posttranslational modifications (PTMs) are major regulatory factors of these nucleosome structural changes, but the mol. mechanisms assocd. with PTM function remains poorly understood. Here we demonstrate that histone PTMs within distinct structured regions of the nucleosome directly regulate the inherent dynamic properties of the nucleosome. Precise PTMs were introduced into nucleosomes by chem. ligation. Single mol. magnetic tweezers measurements detd. that only PTMs near the nucleosome dyad increase the rate of histone release in unwrapped nucleosomes. In contrast, FRET and restriction enzyme anal. reveal that only PTMs throughout the DNA entry-exit region increase unwrapping and enhance transcription factor binding to nucleosomal DNA. These results demonstrate that PTMs in sep. structural regions of the nucleosome control distinct dynamic events, where the dyad regulates disassembly while the DNA entry-exit region regulates unwrapping. These studies are consistent with the conclusion that histone PTMs may independently influence nucleosome dynamics and assocd. chromatin functions.
- 52Makde, R. D.; England, J. R.; Yennawar, H. P.; Tan, S. Nature 2010, 467, 56252Structure of RCC1 chromatin factor bound to the nucleosome core particleMakde, Ravindra D.; England, Joseph R.; Yennawar, Hemant P.; Tan, SongNature (London, United Kingdom) (2010), 467 (7315), 562-566CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)The small GTPase Ran enzyme regulates crit. eukaryotic cellular functions including nuclear transport and mitosis through the creation of a RanGTP gradient around the chromosomes. This concn. gradient is created by the chromatin-bound RCC1 (regulator of chromosome condensation) protein, which recruits Ran to nucleosomes and activates Ran's nucleotide exchange activity. Although RCC1 has been shown to bind directly with the nucleosome, the mol. details of this interaction were not known. Here we det. the crystal structure of a complex of Drosophila RCC1 and the nucleosome core particle at 2.9 resoln., providing an at. view of how a chromatin protein interacts with the histone and DNA components of the nucleosome. Our structure also suggests that the Widom601 DNA positioning sequence present in the nucleosomes forms a 145-base-pair nucleosome core particle, not the expected canonical 147-base-pair particle.
- 53Vasudevan, D.; Chua, E. Y. D.; Davey, C. A. J. Mol. Biol. 2010, 403, 153Crystal Structures of Nucleosome Core Particles Containing the 601' Strong Positioning SequenceVasudevan, Dileep; Chua, Eugene Y. D.; Davey, Curt A.Journal of Molecular Biology (2010), 403 (1), 1-10CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)Nucleosome positioning plays a key role in genomic regulation by defining histone-DNA context and by modulating access to specific sites. Moreover, the histone-DNA register influences the double-helix structure, which in turn can affect the assocn. of small mols. and protein factors. Anal. of genomic and synthetic DNA has revealed sequence motifs that direct nucleosome positioning in vitro; thus, establishing the basis for the DNA sequence dependence of positioning would shed light on the mechanics of the double helix and its contribution to chromatin structure in vivo. However, acquisition of well-diffracting nucleosome core particle (NCP) crystals is extremely dependent on the DNA fragment used for assembly, and all previous NCP crystal structures have been based on human α-satellite sequences. Here, we describe the crystal structures of Xenopus NCPs contg. one of the strongest known histone octamer binding and positioning sequences, the so-called 601' DNA. Two distinct 145-bp 601 crystal forms display the same histone-DNA register, which coincides with the occurrence of DNA stretching-overtwisting in both halves of the particle around five double-helical turns from the nucleosome center, giving the DNA aneffective length' of 147 bp. As we have found previously with stretching around two turns from the nucleosome center for a centromere-based sequence, the terminal stretching obsd. in the 601 constructs is assocd. with extreme kinking into the minor groove at purine-purine (pyrimidine-pyrimidine) dinucleotide steps. In other contexts, these step types display an overall nonflexible behavior, which raises the possibility that DNA stretching in the nucleosome or extreme distortions in general have unique sequence dependency characteristics. Our findings indicate that DNA stretching is an intrinsically predisposed site-specific property of the nucleosome and suggest how NCP crystal structures with diverse DNA sequences can be obtained.
- 54Finch, J. T.; Brown, R. S.; Richmond, T.; Rushton, B.; Lutter, L. C.; Klug, A. J. Mol. Biol. 1981, 145, 75754X-ray diffraction study of a new crystal form of the nucleosome core showing higher resolutionFinch J T; Brown R S; Richmond T; Rushton B; Lutter L C; Klug AJournal of molecular biology (1981), 145 (4), 757-69 ISSN:0022-2836.There is no expanded citation for this reference.
- 55Richmond, T. J.; Finch, J. T.; Rushton, B.; Rhodes, D.; Klug, A. Nature 1984, 311, 53255Structure of the nucleosome core particle at 7 Å resolutionRichmond, T. J.; Finch, J. T.; Rushton, B.; Rhodes, D.; Klug, A.Nature (London, United Kingdom) (1984), 311 (5986), 532-7CODEN: NATUAS; ISSN:0028-0836.The crystal structure of the nucleosome core particle was solved to 7 Å resoln. The right-handed B-DNA superhelix on the outside contains several sharp bends and numerous interactions with the histone octamer within. The central turn of the superhelix and the H3-H4 tetramer have dyad symmetry, but the H2A-H2B dimers show departures due to interparticle assocns.
- 56Richmond, T. J.; Finch, J. T.; Klug, A. Cold Spring Harbor Symp. Quant. Biol. 1983, 47, 49356Studies of nucleosome structureRichmond, T. J.; Finch, J. T.; Klug, AaronCold Spring Harbor Symposia on Quantitative Biology (1983), 47 (1), 493-501CODEN: CSHSAZ; ISSN:0091-7451.A review and discussion with 43 refs., of the structure of the nucleosome and its assembly from its constituents. The soln. of the the crystal structure of the nucleosome core particle is emphasized.
- 57Richmond, T. J.; Searles, M. A.; Simpson, R. T. J. Mol. Biol. 1988, 199, 16157Crystals of a nucleosome core particle containing defined sequence DNARichmond, Timothy J.; Searles, M. Alexandra; Simpson, Robert T.Journal of Molecular Biology (1988), 199 (1), 161-70CODEN: JMOBAK; ISSN:0022-2836.Nucleosome core particles were reconstituted from a DNA restriction fragment and histone octamers, crystd., and the crystals examd. by x-ray diffraction. A DNA fragment was engineered by site-directed mutagenesis to obtain a 146 base-pair sequence that takes up a sym. arrangement in the core particle. The resulting DNA sequence was cloned in multiple copies into pUC9 and excised as monomer via EcoRV to produce it in milligram quantities. Nucleosome core particles incorporating the DNA were reconstituted by salt gradient dialysis and purified by anion-exchange HPLC. DNase I digestion was used to demonstrate that the termini of the restriction fragment are located 73 base pairs from the mol. dyad axis of the particle. The diffraction limits of crystals of defined sequence core particles extend along the principal direction to a = ∼4, b = ∼5, and c = ∼3 Å, giving about a 2-fold increase in the no. of measurable x-ray reflections over previous crystals contg. mixed sequence DNA. The methods developed here should be useful in the study of other large protein-DNA complexes.
- 58Simpson, R. T.; Stafford, D. W. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 5158Structural features of a phased nucleosome core particleSimpson, Robert T.; Stafford, Darrell W.Proceedings of the National Academy of Sciences of the United States of America (1983), 80 (1), 51-5CODEN: PNASA6; ISSN:0027-8424.Chicken erythrocyte inner histones assoc. with a cloned 260-base-pair (bp) segment of Lytechinus variegatus DNA in a unique location. The fragment contains a 120-bp segment encoding 5 S rRNA, a 90-bp flanking sequence to the 5'-side of the transcribed segment, and a 50-bp downstream flanking sequence. Assocn. of DNA, uniquely labeled at one end and at either the 3'- or the 5'-terminus of a given strand, with histones at 0.1M strength leads to the formation of a compact complex which sediments at ∼13 S. Anal. of the complex by DNase I cleavage shows that protection from the nuclease is confined to a region beginning 20 bp from the left end of the segment and extending to ∼165 bp from the left end. Within the protected region, the 2 DNA strands differ in susceptibility to nuclease, the precise location of nuclease cleavage sites, and the spacing between these sites, and the relative susceptibility of specific cleavage locations. Information present in DNA and the histone octomer is apparently sufficient to create a precisely phased nucleosome in which interactions of the 2 DNA strands with histone differ. The structure of this unique nucleosome is not predicted by the intellectual model based on studies of mixed population of nucleosome core particles.
- 59Harp, J. M.; Palmer, E. L.; York, M. H.; Gewiess, A.; Davis, M.; Bunick, G. J. Electrophoresis 1995, 16, 186159Preparative separation of nucleosome core particles containing defined-sequence DNA in multiple translational phasesHarp, Joel M.; Palmer, Elise L.; York, Melissa H.; Gewiess, Andreas; Davis, Matthew; Bunick, Gerard J.Electrophoresis (1995), 16 (10), 1861-4CODEN: ELCTDN; ISSN:0173-0835. (VCH)The nucleosome core particle is composed of an octamer of core histone proteins and about 146 bp of DNA. When reconstituted from purified histone octamer and defined-sequence, nucleosome positioning DNA fragments, the DNA will bind to the histone core in a no. of translational phases with respect to the dyad symmetry axis of the histone octamer. Only one of these phases contains sym. bound DNA, and it is this species which is required for crystn. and X-ray diffraction studies. We have developed a technique for sepg. nucleosome core particles, contg. defined-sequence 146 bp DNA, which differ only in translational phasing of the DNA with respect to the histone octamer core.
- 60Satchwell, S. C.; Drew, H. R.; Travers, A. A. J. Mol. Biol. 1986, 191, 65960Sequence periodicities in chicken nucleosome core DNASatchwell, Sandra C.; Drew, Horace R.; Travers, Andrew A.Journal of Molecular Biology (1986), 191 (4), 659-75CODEN: JMOBAK; ISSN:0022-2836.The rotational positioning of DNA about the histone octamer appears to be detd. by certain sequence-dependent modulations of DNA structure. To establish the detailed nature of these interactions, the sequences of 177 different DNA mols. from chicken erythrocyte core particles were analyzed. All variations in the sequence content of these mols., which may be attributed to sequence-dependent preferences for DNA bending, correlate well with the detailed path of the DNA as it wraps around the histone octamer in the crystal structure of the nucleosome core. The sequence-dependent preferences that correlate most closely with the rotational orientation of the DNA, relative to the surface of the protein, are of two kinds: ApApA/TpTpT and ApApT/ApTpT, the minor grooves of which face predominantly in towards the protein; and also GpGpC/GpCpC and ApGpC/GpCpT, whose minor grooves face outward. Fourier anal. has been used to obtain fractional variations in occurrence for all ten dinucleotide and all 32 trinucleotide arrangements. These sequence preferences should apply generally to many other cases of protein-DNA recognition, where the DNA wraps around a protein. In addn., it is obsd. that long runs of homopolymer (dA)·(dT) prefer to occupy the ends of core DNA, five to six turns away from the dyad. These same sequences are apparently excluded from the near-center of core DNA, two to three turns from the dyad. Hence, the translational positioning of any single histone octamer along a DNA mol. of defined sequence may be strongly influenced by the placement of (dA)·(dT) sequences. It may also be influenced by any aversion of the protein for sequences in the linker region, the sequence content of which remains to be detd.
- 61Chua, E. Y. D.; Vasudevan, D.; Davey, G. E.; Wu, B.; Davey, C. A. Nucleic Acids Res. 2012, 40, 633861The mechanics behind DNA sequence-dependent properties of the nucleosomeChua, Eugene Y. D.; Vasudevan, Dileep; Davey, Gabriela E.; Wu, Bin; Davey, Curt A.Nucleic Acids Research (2012), 40 (13), 6338-6352CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Chromatin organization and compn. impart sophisticated regulatory features crit. to eukaryotic genomic function. Although DNA sequence-dependent histone octamer binding is important for nucleosome activity, many aspects of this phenomenon have remained elusive. We studied nucleosome structure and stability with diverse DNA sequences, including Widom 601 derivs. with the highest known octamer affinities, to establish a simple model behind the mechanics of sequence dependency. This uncovers the unique but unexpected role of TA dinucleotides and a propensity for GC-rich sequence elements to conform energetically favorably at most locations around the histone octamer, which rationalizes GC% as the most predictive factor for nucleosome occupancy in vivo. In addn., our findings reveal dominant constraints on double helix conformation by H3-H4 relative to H2A-H2B binding and DNA sequence context-dependency underlying nucleosome structure, positioning and stability. This provides a basis for improved prediction of nucleosomal properties and the design of tailored DNA constructs for chromatin investigations.
- 62Kulaeva, O. I.; Gaykalova, D. A.; Pestov, N. A.; Golovastov, V. V.; Vassylyev, D. G.; Artsimovitch, I.; Studitsky, V. M. Nat. Struct. Mol. Biol. 2009, 16, 127262Mechanism of chromatin remodeling and recovery during passage of RNA polymerase IIKulaeva, Olga I.; Gaykalova, Daria A.; Pestov, Nikolai A.; Golovastov, Viktor V.; Vassylyev, Dmitry G.; Artsimovitch, Irina; Studitsky, Vasily M.Nature Structural & Molecular Biology (2009), 16 (12), 1272-1278CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)Transcription of eukaryotic genes by RNA polymerase II (Pol II) is typically accompanied by nucleosome survival and minimal exchange of histones H3 and H4. The mechanism of nucleosome survival and recovery of chromatin structure remains obscure. Here we show how transcription through chromatin by Pol II is uniquely coupled with nucleosome survival. Structural modeling and functional anal. of the intermediates of transcription through a nucleosome indicated that when Pol II approaches an area of strong DNA-histone interactions, a small intranucleosomal DNA loop (zero-size or O-loop) contg. transcribing enzyme is formed. During formation of the O-loop, the recovery of DNA-histone interactions behind Pol II is tightly coupled with their disruption ahead of the enzyme. This coupling is a distinct feature of the Pol II-type mechanism that allows further transcription through the nucleosome, prevents nucleosome translocation and minimizes displacement of H3 and H4 histones from DNA during enzyme passage.
- 63Cui, F.; Zhurkin, V. B. J. Biomol. Struct. Dyn. 2010, 27, 82163Structure-based analysis of DNA sequence patterns guiding nucleosome positioning in vitroCui Feng; Zhurkin Victor BJournal of biomolecular structure & dynamics (2010), 27 (6), 821-41 ISSN:.Recent studies of genome-wide nucleosomal organization suggest that the DNA sequence is one of the major determinants of nucleosome positioning. Although the search for underlying patterns encoded in nucleosomal DNA has been going on for about 30 years, our knowledge of these patterns still remains limited. Based on our evaluations of DNA deformation energy, we developed new scoring functions to predict nucleosome positioning. There are three principal differences between our approach and earlier studies: (i) we assume that the length of nucleosomal DNA varies from 146 to 147 bp; (ii) we consider the anisotropic flexibility of pyrimidine-purine (YR) dimeric steps in the context of their neighbors (e.g., YYRR versus RYRY); (iii) we postulate that alternating AT-rich and GC-rich motifs reflect sequence-dependent interactions between histone arginines and DNA in the minor groove. Using these functions, we analyzed 20 nucleosome positions mapped in vitro at single nucleotide resolution (including clones 601, 603, 605, the pGUB plasmid, chicken beta-globin and three 5S rDNA genes). We predicted 15 of the 20 positions with 1-bp precision, and two positions with 2-bp precision. The predicted position of the '601' nucleosome (i.e., the optimum of the computed score) deviates from the experimentally determined unique position by no more than 1 bp - an accuracy exceeding that of earlier predictions. Our analysis reveals a clear heterogeneity of the nucleosomal sequences which can be divided into two groups based on the positioning 'rules' they follow. The sequences of one group are enriched by highly deformable YR/YYRR motifs at the minor-groove bending sites SHL+/- 3.5 and +/- 5.5, which is similar to the alpha-satellite sequence used in most crystallized nucleosomes. Apparently, the positioning of these nucleosomes is determined by the interactions between histones H2A/H2B and the terminal parts of nucleosomal DNA. In the other group (that includes the '601' clone) the same YR/YYRR motifs occur predominantly at the sites SHL +/- 1.5. The interaction between the H3/H4 tetramer and the central part of the nucleosomal DNA is likely to be responsible for the positioning of nucleosomes of this group, and the DNA trajectory in these nucleosomes may differ in detail from the published structures. Thus, from the stereochemical perspective, the in vitro nucleosomes studied here follow either an X-ray-like pattern (with strong deformations in the terminal parts of nucleosomal DNA), or an alternative pattern (with the deformations occurring predominantly in the central part of the nucleosomal DNA). The results presented here may be useful for genome-wide classification of nucleosomes, linking together structural and thermodynamic characteristics of nucleosomes with the underlying DNA sequence patterns guiding their positions.
- 64Tolstorukov, M. Y.; Colasanti, A. V.; McCandlish, D. M.; Olson, W. K.; Zhurkin, V. B. J. Mol. Biol. 2007, 371, 72564A Novel Roll-and-Slide Mechanism of DNA Folding in Chromatin: Implications for Nucleosome PositioningTolstorukov, Michael Y.; Colasanti, Andrew V.; McCandlish, David M.; Olson, Wilma K.; Zhurkin, Victor B.Journal of Molecular Biology (2007), 371 (3), 725-738CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)How eukaryotic genomes encode the folding of DNA into nucleosomes and how this intrinsic organization of chromatin guides biol. function are questions of wide interest. The phys. basis of nucleosome positioning lies in the sequence-dependent propensity of DNA to adopt the tightly bent configuration imposed by the binding of the histone proteins. Traditionally, only DNA bending and twisting deformations are considered, while the effects of the lateral displacements of adjacent base pairs are neglected. We demonstrate, however, that these displacements have a much more important structural role than ever imagined. Specifically, the lateral Slide deformations obsd. at sites of local anisotropic bending of DNA define its superhelical trajectory in chromatin. Furthermore, the computed cost of deforming DNA on the nucleosome is sequence-specific: in optimally positioned sequences the most easily deformed base-pair steps (CA:TG and TA) occur at sites of large pos. Slide and neg. Roll (where the DNA bends into the minor groove). These conclusions rest upon a treatment of DNA that goes beyond the conventional ribbon model, incorporating all essential degrees of freedom of "real" duplexes in the estn. of DNA deformation energies. Indeed, only after lateral Slide displacements are considered are we able to account for the sequence-specific folding of DNA found in nucleosome structures. The close correspondence between the predicted and obsd. nucleosome locations demonstrates the potential advantage of our "structural" approach in the computer mapping of nucleosome positioning.
- 65Travers, A. A. Philos. Trans. R. Soc., A 2004, 362, 142365The structural basis of DNA flexibilityTravers, A. A.Philosophical Transactions of the Royal Society of London, Series A: Mathematical, Physical and Engineering Sciences (2004), 362 (1820), 1423-1438CODEN: PTRMAD; ISSN:1364-503X. (Royal Society)Although the av. physico-chem. properties of a long DNA mol. may approx. to those of a thin isotropic homogeneous rod, DNA behaves more locally as an anisotropic heterogeneous rod. This bending anisotropy is sequence dependent and to a first approxn. reflects both the geometry and stability of individual base steps. The biol. manipulation and packaging of the mol. often depend crucially on local variations in both bending and torsional flexibility. However, whereas the probability of DNA untwisting can be strongly correlated with a high bending flexibility, DNA bending, esp. when the mol. is tightly wrapped on a protein surface, may be energetically favored by a less flexible sequence whose preferred configuration conforms more closely to that of the complementary protein surface. In the latter situation the lower bending flexibility may be more than compensated for on binding by a reduced required deformation energy relative to a fully isotropic DNA mol.
- 66Travers, A. A.; Muskhelishvili, G.; Thompson, J. M. T. Philos. Trans. R. Soc., A 2012, 370, 296066DNA information: from digital code to analogue structureTravers, A. A.; Muskhelishvili, G.; Thompson, J. M. T.Philosophical Transactions of the Royal Society, A: Mathematical, Physical & Engineering Sciences (2012), 370 (1969), 2960-2986CODEN: PTRMAD; ISSN:1364-503X. (Royal Society)A review. The digital linear coding carried by the base pairs in the DNA double helix is now known to have an important component that acts by altering, along its length, the natural shape and stiffness of the mol. In this way, one region of DNA is structurally distinguished from another, constituting an addnl. form of encoded information manifest in three-dimensional space. These shape and stiffness variations help in guiding and facilitating the DNA during its three-dimensional spatial interactions. Such interactions with itself allow communication between genes and enhanced wrapping and histone-octamer binding within the nucleosome core particle. Meanwhile, interactions with proteins can have a reduced entropic binding penalty owing to advantageous sequence-dependent bending anisotropy. Sequence periodicity within the DNA, giving a corresponding structural periodicity of shape and stiffness, also influences the supercoiling of the mol., which, in turn, plays an important facilitating role. In effect, the super-helical d. acts as an analog regulatory mode in contrast to the more commonly acknowledged purely digital mode. Many of these ideas are still poorly understood, and represent a fundamental and outstanding biol. question. This review gives an overview of very recent developments, and hopefully identifies promising future lines of enquiry.
- 67Olson, W. K.; Zhurkin, V. B. Curr. Opin. Struct. Biol. 2011, 21, 34867Working the kinks out of nucleosomal DNAOlson, Wilma K.; Zhurkin, Victor B.Current Opinion in Structural Biology (2011), 21 (3), 348-357CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)A review. Condensation of DNA in the nucleosome takes advantage of its double-helical architecture. The DNA deforms at sites where the base pairs face the histone octamer. The largest so-called kink-and-slide deformations occur in the vicinity of Arg residues that penetrate the minor groove. Nucleosome structures formed from the 601 positioning sequence differ subtly from those incorporating an AT-rich human α-satellite DNA. Restraints imposed by the histone Arg residues on the displacement of base pairs can modulate the sequence-dependent deformability of DNA and potentially contribute to the unique features of the different nucleosomes. Steric barriers mimicking constraints found in the nucleosome induce the simulated large-scale rearrangement of canonical B DNA to kink-and-slide states. The pathway to these states shows nonharmonic behavior consistent with bending profiles inferred from AFM measurements.
- 68Richmond, T. J.; Davey, C. A. Nature 2003, 423, 14568The structure of DNA in the nucleosome coreRichmond, Timothy J.; Davey, Curt A.Nature (London, United Kingdom) (2003), 423 (6936), 145-150CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)The 1.9-Å-resoln. crystal structure of the nucleosome core particle contg. 147 DNA base pairs reveals the conformation of nucleosomal DNA with unprecedented accuracy. The DNA structure is remarkably different from that in oligonucleotides and non-histone protein-DNA complexes. The DNA base-pair-step geometry has, overall, twice the curvature necessary to accommodate the DNA superhelical path in the nucleosome. DNA segments bent into the minor groove are either kinked or alternately shifted. The unusual DNA conformational parameters induced by the binding of histone protein have implications for sequence-dependent protein recognition and nucleosome positioning and mobility. Comparison of the 147-base-pair structure with two 146-base-pair structures reveals alterations in DNA twist that are evidently common in bulk chromatin, and which are of probable importance for chromatin fiber formation and chromatin remodelling.
- 69Juo, Z. S.; Chiu, T. K.; Leiberman, P. M.; Baikalov, I.; Berk, A. J.; Dickerson, R. E. J. Mol. Biol. 1996, 261, 23969How proteins recognize the TATA boxJuo, Zong Sean; Chiu, Thang Kien; Lieberman, Paul M.; Baikalov, Igor; Berk, Arnold J.; Dickerson, Richard E.Journal of Molecular Biology (1996), 261 (2), 239-254CODEN: JMOBAK; ISSN:0022-2836. (Academic)The crystal structure of a complex of human TATA-binding protein with TATA-sequence DNA has been solved, complementing earlier TBP/DNA analyses from Saccharomyces cerevisiae and Arabidopsis thaliana. Special insight into TATA box specificity is provided by considering the TBP/DNA complex, not as a protein mol. with bound DNA, but as a DNA duplex with a particularly large minor groove ligand. This point of view provides explanations for: (1) why T·A base-pairs are required rather than C·G; (2) why an alternation of T and A bases is needed; (3) how TBP recognizes the upstream and downstream ends of the TATA box to bind properly; and (4) why the second half of the TATA box can be more variable than the first.
- 70Kim, Y.; Geiger, J. H.; Hahn, S.; Sigler, P. B. Nature 1993, 365, 51270Crystal structure of a yeast TBP/TATA-box complexKim, Youngchang; Geiger, James H.; Hahn, Steven; Sigler, Paul B.Nature (London, United Kingdom) (1993), 365 (6446), 512-20CODEN: NATUAS; ISSN:0028-0836.The 2.5 Å crystal structure of a TATA-box complex with yeast TBP shows that the eight base pairs of the TATA box bind to the concave surface of TBP by bending towards the major groove with unprecedented severity. This produces a wide open, underwound, shallow minor groove which forms a primarily hydrophobic interface with the entire under-surface of the TBP saddle. The severe bend and a pos. writhe radically alter the trajectory of the flanking B-form DNA.
- 71Kim, J. L.; Nikolov, D. B.; Burley, S. K. Nature 1993, 365, 52071Co-crystal structure of TBP recognizing the minor groove of a TATA elementKim, Joseph L.; Nikolov, Dimitar B.; Burley, Stephen K.Nature (London, United Kingdom) (1993), 365 (6446), 520-7CODEN: NATUAS; ISSN:0028-0836.The three-dimensional structure of a TATA-box binding polypeptide (TBP or TFIIDτ) complexed with the TATA element of the adenovirus major late promoter has been detd. by x-ray crystallog. at 2.25 Å resoln. Binding of the saddle-shaped protein induces a conformational change in the DNA, inducing sharp kinks at either end of the sequence TATAAAAG. Between the kinks, the right-handed double helix is smoothly curved and partially unwound, presenting a widened minor groove to TBP's concave, antiparallel β-sheet. Side-chain/base interactions are restricted to the minor groove, and include hydrogen bonds, van der Waals contacts and phenylalanine base stacking interactions.
- 72Olson, W. K.; Gorin, A. A.; Lu, X.-J.; Hock, L. M.; Zhurkin, V. B. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 1116372DNA sequence-dependent deformability deduced from protein-DNA crystal complexesOlson, Wilma K.; Gorin, Andrey A.; Lu, Xiang-Jun; Hock, Lynette M.; Zhurkin, Victor B.Proceedings of the National Academy of Sciences of the United States of America (1998), 95 (19), 11163-11168CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The deformability of double helical DNA is crit. for its packaging in the cell, recognition by other mols., and transient opening during biochem. important processes. Here, a complete set of sequence-dependent empirical energy functions suitable for describing such behavior is extd. from the fluctuations and correlations of structural parameters in DNA-protein crystal complexes. These elastic functions provide useful stereochem. measures of the local base step movements operative in sequence-specific recognition and protein-induced deformations. In particular, the pyrimidine-purine dimers stand out as the most variable steps in the DNA-protein complexes, apparently acting as flexible "hinges" fitting the duplex to the protein surface. In addn. to the angular parameters widely used to describe DNA deformations (i.e., the bend and twist angles), the translational parameters describing the displacements of base pairs along and across the helical axis are analyzed. The obsd. correlations of base pair bending and shearing motions are important for nonplanar folding of DNA in nucleosomes and other nucleoprotein complexes. The knowledge-based energies also offer realistic three-dimensional models for the study of long DNA polymers at the global level, incorporating structural features beyond the scope of conventional elastic rod treatments and adding a new dimension to literal analyses of genomic sequences.
- 73Bondarenko, V. A.; Steele, L. M.; Ujvári, A.; Gaykalova, D. A.; Kulaeva, O. I.; Polikanov, Y. S.; Luse, D. S.; Studitsky, V. M. Mol. Cell 2006, 24, 46973Nucleosomes can form a polar barrier to transcript elongation by RNA polymerase IIBondarenko, Vladimir A.; Steele, Louise M.; Ujvari, Andrea; Gaykalova, Daria A.; Kulaeva, Olga I.; Polikanov, Yury S.; Luse, Donal S.; Studitsky, Vasily M.Molecular Cell (2006), 24 (3), 469-479CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)Nucleosomes uniquely positioned on high-affinity DNA sequences present a polar barrier to transcription by human and yeast RNA polymerase II (Pol II). In one transcriptional orientation, these nucleosomes provide a strong, factor- and salt-insensitive barrier at the entry into the H3/H4 tetramer that can be recapitulated without H2A/H2B dimers. The same nucleosomes transcribed in the opposite orientation form a weaker, more diffuse barrier that is largely relieved by higher salt, TFIIS, or FACT. Barrier properties are therefore dictated by both the local nucleosome structure (influenced by the strength of the histone-DNA interactions) and the location of the high-affinity DNA region within the nucleosome. Pol II transcribes DNA sequences at the entry into the tetramer much less efficiently than the same sequences located distal to the nucleosome dyad. Thus, entry into the tetramer by Pol II facilitates further transcription, perhaps due to partial unfolding of the tetramer from DNA.
- 74Abbott, E. A. Flatland: A romance of many dimensions; Seely & Co.: UK, 1884.There is no corresponding record for this reference.
- 75Makde, R. D.; Tan, S. Anal. Biochem. 2013, 442, 13875Strategies for crystallizing a chromatin protein in complex with the nucleosome core particleMakde, Ravindra D.; Tan, SongAnalytical Biochemistry (2013), 442 (2), 138-145CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)The mol. details of how chromatin factors and enzymes interact with the nucleosome are crit. to understanding fundamental genetic processes including cell division and gene regulation. A structural understanding of such processes has been hindered by the difficulty in producing diffraction-quality crystals of chromatin proteins in complex with the nucleosome. We describe here the steps used to grow crystals of the 300-kDa RCC1 chromatin factor/nucleosome core particle complex that diffract to 2.9-Å resoln. These steps include both pre- and postcrystn. strategies potentially useful to other complexes. We screened multiple variant RCC1/nucleosome core particle complexes assembled using different RCC1 homologs and deletion variants, and nucleosomes contg. nucleosomal DNA with different sequences and lengths, as well as histone deletion variants. We found that using RCC1 from different species produced different crystal forms of the RCC1/nucleosome complex consistent with key crystal packing interactions mediated by RCC1. Optimization of postcrystn. soaks to dehydrate the crystals dramatically improved the diffraction quality of the RCC1/nucleosome crystal from 5.0- to 2.9-Å resoln.
- 76Hock, R.; Furusawa, T.; Ueda, T.; Bustin, M. Trends Cell Biol. 2007, 17, 7276HMG chromosomal proteins in development and diseaseHock, Robert; Furusawa, Takashi; Ueda, Tetsuya; Bustin, MichaelTrends in Cell Biology (2007), 17 (2), 72-79CODEN: TCBIEK; ISSN:0962-8924. (Elsevier B.V.)A review. The high mobility group (HMG) proteins are a superfamily of abundant and ubiquitous nuclear proteins that bind to DNA and nucleosomes and induce structural changes in the chromatin fiber. They are important in chromatin dynamics and influence DNA processing in the context of chromatin. Results emerging from studies of human disease, genetically modified mice and cells with altered HMG expression indicate that the expression of the HMG proteins is developmentally regulated and that changes in HMG protein levels alter the cellular phenotype and can lead to developmental abnormalities and disease. Here, we focus on the biol. function of HMG proteins and highlight their possible roles in cellular differentiation and in the etiol. of various diseases.
- 77Canzio, D.; Liao, M.; Naber, N.; Pate, E.; Larson, A.; Wu, S.; Marina, D. B.; Garcia, J. F.; Madhani, H. D.; Cooke, R.; Schuck, P.; Cheng, Y.; Narlikar, G. J. Nature 2013, 496, 37777A conformational switch in HP1 releases auto-inhibition to drive heterochromatin assemblyCanzio, Daniele; Liao, Maofu; Naber, Nariman; Pate, Edward; Larson, Adam; Wu, Shenping; Marina, Diana B.; Garcia, Jennifer F.; Madhani, Hiten D.; Cooke, Roger; Schuck, Peter; Cheng, Yifan; Narlikar, Geeta J.Nature (London, United Kingdom) (2013), 496 (7445), 377-381CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)A hallmark of histone H3 lysine 9 (H3K9)-methylated heterochromatin, conserved from the fission yeast Schizosaccharomyces pombe to humans, is its ability to spread to adjacent genomic regions. Central to heterochromatin spread is heterochromatin protein 1 (HP1), which recognizes H3K9-methylated chromatin, oligomerizes and forms a versatile platform that participates in diverse nuclear functions, ranging from gene silencing to chromosome segregation. How HP1 proteins assemble on methylated nucleosomal templates and how the HP1-nucleosome complex achieves functional versatility remain poorly understood. Here we show that binding of the key S. pombe HP1 protein, Swi6, to methylated nucleosomes drives a switch from an auto-inhibited state to a spreading-competent state. In the auto-inhibited state, a histone-mimic sequence in one Swi6 monomer blocks methyl-mark recognition by the chromodomain of another monomer. Auto-inhibition is relieved by recognition of two template features, the H3K9 Me mark and nucleosomal DNA. Cryo-electron-microscopy-based reconstruction of the Swi6-nucleosome complex provides the overall architecture of the spreading-competent state in which two unbound chromodomain sticky ends appear exposed. Disruption of the switch between the auto-inhibited and spreading-competent states disrupts heterochromatin assembly and gene silencing in vivo. These findings are reminiscent of other conditionally activated polymn. processes, such as actin nucleation, and open up a new class of regulatory mechanisms that operate on chromatin in vivo.
- 78Armache, K. J.; Garlick, J. D.; Canzio, D.; Narlikar, G. J.; Kingston, R. E. Science 2011, 334, 97778Structural Basis of Silencing: Sir3 BAH Domain in Complex with a Nucleosome at 3.0 Å ResolutionArmache, Karim-Jean; Garlick, Joseph D.; Canzio, Daniele; Narlikar, Geeta J.; Kingston, Robert E.Science (Washington, DC, United States) (2011), 334 (6058), 977-982CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Gene silencing is essential for regulating cell fate in eukaryotes. Altered chromatin architectures contribute to maintaining the silenced state in a variety of species. The silent information regulator (Sir) proteins regulate mating type in Saccharomyces cerevisiae. One of these proteins, Sir3, interacts directly with the nucleosome to help generate silenced domains. We detd. the crystal structure of a complex of the yeast Sir3 BAH (bromo-assocd. homol.) domain and the nucleosome core particle at 3.0 angstrom resoln. We see multiple mol. interactions between the protein surfaces of the nucleosome and the BAH domain that explain numerous genetic mutations. These interactions are accompanied by structural rearrangements in both the nucleosome and the BAH domain. The structure explains how covalent modifications on H4K16 and H3K79 regulate formation of a silencing complex that contains the nucleosome as a central component.
- 79Barbera, A. J.; Chodaparambil, J. V.; Kelley-Clarke, B.; Joukov, V.; Walter, J. C.; Luger, K.; Kaye, K. M. Science 2006, 311, 85679The nucleosomal surface as a docking station for Kaposi's sarcoma herpesvirus LANABarbera, Andrew J.; Chodaparambil, Jayanth V.; Kelley-Clarke, Brenna; Joukov, Vladimir; Walter, Johannes C.; Luger, Karolin; Kaye, Kenneth M.Science (Washington, DC, United States) (2006), 311 (5762), 856-861CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Kaposi's sarcoma-assocd. herpesvirus (KSHV) latency-assocd. nuclear antigen (LANA) mediates viral genome attachment to mitotic chromosomes. We find that N-terminal LANA docks onto chromosomes by binding nucleosomes through the folded region of histones H2A-H2B. The same LANA residues were required for both H2A-H2B binding and chromosome assocn. Further, LANA did not bind Xenopus sperm chromatin, which is deficient in H2A-H2B; chromatin binding was rescued after assembly of nucleosomes contg. H2A-H2B. We also describe the 2.9-angstrom crystal structure of a nucleosome complexed with the first 23 LANA amino acids. The LANA peptide forms a hairpin that interacts exclusively with an acidic H2A-H2B region that is implicated in the formation of higher order chromatin structure. Our findings present a paradigm for how nucleosomes may serve as binding platforms for viral and cellular proteins and reveal a previously unknown mechanism for KSHV latency.
- 80Marmorstein, R.; Trievel, R. C. Biochim. Biophys. Acta, Gene Regul. Mech. 2009, 1789, 5880Histone modifying enzymes: Structures, mechanisms, and specificitiesMarmorstein, Ronen; Trievel, Raymond C.Biochimica et Biophysica Acta, Gene Regulatory Mechanisms (2009), 1789 (1), 58-68CODEN: BBAGC6; ISSN:1874-9399. (Elsevier B.V.)A review. Histone modifying enzymes catalyze the addn. or removal of an array of covalent modifications in histone and non-histone proteins. Within the context of chromatin, these modifications regulate gene expression as well as other genomic functions and have been implicated in establishing and maintaining a heritable epigenetic code that contributes to defining cell identity and fate. Biochem. and structural characterization of histone modifying enzymes has yielded important insights into their resp. catalytic mechanisms, substrate specificities, and regulation. In this review, we summarize recent advances in understanding these enzymes, highlighting studies of the histone acetyltransferases (HATs) p300 (also now known as KAT3B) and Rtt109 (KAT11) and the histone lysine demethylases (HDMs) LSD1 (KDM1) and JMJD2A (KDM4A), present overriding themes that derive from these studies, and pose remaining questions concerning their regulatory roles in mediating DNA transactions.
- 81Musselman, C. A.; Lalonde, M.-E.; Côté, J.; Kutateladze, T. G. Nat. Struct. Mol. Biol. 2012, 19, 121881Perceiving the epigenetic landscape through histone readersMusselman, Catherine A.; Lalonde, Marie-Eve; Cote, Jacques; Kutateladze, Tatiana G.Nature Structural & Molecular Biology (2012), 19 (12), 1218-1227CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)A review. Post-translational modifications (PTMs) of histones provide a fine-tuned mechanism for regulating chromatin structure and dynamics. PTMs can alter direct interactions between histones and DNA and serve as docking sites for protein effectors, or readers, of these PTMs. Binding of the readers recruits or stabilizes various components of the nuclear signaling machinery at specific genomic sites, mediating fundamental DNA-templated processes, including gene transcription and DNA recombination, replication and repair. In this review, we highlight the latest advances in characterizing histone-binding mechanisms and identifying new epigenetic readers and summarize the functional significance of PTM recognition.
- 82Ruthenburg, A. J.; Li, H.; Patel, D. J.; Allis, C. D. Nat. Rev. Mol. Cell Biol. 2007, 8, 98382Multivalent engagement of chromatin modifications by linked binding modulesRuthenburg, Alexander J.; Li, Haitao; Patel, Dinshaw J.; Allis, C. DavidNature Reviews Molecular Cell Biology (2007), 8 (12), 983-994CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. Histone post-translational modifications (PTMs) play crucial roles in genome management, in part by recruiting specific factors that alter the structural properties of chromatin. These so-called effector complexes often comprise multiple histone-binding modules that may act in concert to regulate chromatin structure and DNA-related activities. Various chem. modifications on histones and regions of assocd. DNA play crucial roles in genome management by binding specific factors that, in turn, serve to alter the structural properties of chromatin. These so-called effector proteins have typically been studied with the biochemist's paring knife; the capacity to recognize specific chromatin PTMs has been mapped to an increasing no. of domains that frequently appear in the nuclear subset of the proteome, often present in large, multisubunit complexes that bristle with modification-dependent binding potential. The authors propose that multivalent interactions on a single histone tail and beyond may have a significant, if not dominant, role in chromatin transactions.
- 83Zhou, B.-R.; Feng, H.; Ghirlando, R.; Kato, H.; Gruschus, J.; Bai, Y. J. Mol. Biol. 2012, 421, 3083Histone H4 K16Q Mutation, an Acetylation Mimic, Causes Structural Disorder of Its N-Terminal Basic Patch in the NucleosomeZhou, Bing-Rui; Feng, Hanqiao; Ghirlando, Rodolfo; Kato, Hidenori; Gruschus, James; Bai, YawenJournal of Molecular Biology (2012), 421 (1), 30-37CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)Histone tails and their posttranslational modifications play important roles in regulating the structure and dynamics of chromatin. For histone H4, the basic patch K16R17H18R19 in the N-terminal tail modulates chromatin compaction and nucleosome sliding catalyzed by ATP-dependent ISWI chromatin remodeling enzymes while acetylation of H4 K16 affects both functions. The structural basis for the effects of this acetylation is unknown. Here, we investigated the conformation of histone tails in the nucleosome by soln. NMR. We found that backbone amides of the N-terminal tails of histones H2A, H2B, and H3 are largely observable due to their conformational disorder. However, only residues 1-15 in H4 can be detected, indicating that residues 16-22 in the tails of both H4 histones fold onto the nucleosome core. Surprisingly, we found that K16Q mutation in H4, a mimic of K16 acetylation, leads to a structural disorder of the basic patch. Thus, our study suggests that the folded structure of the H4 basic patch in the nucleosome is important for chromatin compaction and nucleosome remodeling by ISWI enzymes while K16 acetylation affects both functions by causing structural disorder of the basic patch K16R17H18R19.
- 84Dorigo, B.; Schalch, T.; Bystricky, K.; Richmond, T. J. J. Mol. Biol. 2003, 327, 8584Chromatin Fiber Folding: Requirement for the Histone H4 N-terminal TailDorigo, Benedetta; Schalch, Thomas; Bystricky, Kerstin; Richmond, Timothy J.Journal of Molecular Biology (2003), 327 (1), 85-96CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Science Ltd.)We have developed a self-assembly system for nucleosome arrays in which recombinant, post-translationally unmodified histone proteins are combined with DNA of defined-sequence to form chromatin higher-order structure. The nucleosome arrays obtained are highly homogeneous and sediment at 53 S when maximally folded in 1 mM or 100 mM MgCl2. The folding properties are comparable to established systems. Anal. ultracentrifugation is used to det. the consequence of individual histone tail domain deletions on array folding. Fully compacted chromatin fibers are obtained with any one of the histone tails deleted with the exception of the H4 N terminus. The region of the H4 tail, which mediates compaction, resides in the stretch of amino acids 14-19.
- 85Robinson, P. J. J.; An, W.; Routh, A.; Martino, F.; Chapman, L.; Roeder, R. G.; Rhodes, D. J. Mol. Biol. 2008, 381, 8168530 nm Chromatin Fibre Decompaction Requires both H4-K16 Acetylation and Linker Histone EvictionRobinson, Philip J. J.; An, Woojin; Routh, Andrew; Martino, Fabrizio; Chapman, Lynda; Roeder, Robert G.; Rhodes, DanielaJournal of Molecular Biology (2008), 381 (4), 816-825CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)The mechanism by which chromatin is decondensed to permit access to DNA is largely unknown. Here, using a model nucleosome array reconstituted from recombinant histone octamers, we have defined the relative contribution of the individual histone octamer N-terminal tails as well as the effect of a targeted histone tail acetylation on the compaction state of the 30 nm chromatin fiber. This study goes beyond previous studies as it is based on a nucleosome array that is very long (61 nucleosomes) and contains a stoichiometric concn. of bound linker histone, which is essential for the formation of the 30 nm chromatin fiber. We find that compaction is regulated in two steps: Introduction of H4 acetylated to 30% on K16 inhibits compaction to a greater degree than deletion of the H4 N-terminal tail. Further decompaction is achieved by removal of the linker histone.
- 86Allahverdi, A.; Yang, R.; Korolev, N.; Fan, Y.; Davey, C. A.; Liu, C. F.; Nordenskiold, L. Nucleic Acids Res. 2011, 39, 168086The effects of histone H4 tail acetylations on cation-induced chromatin folding and self-associationAllahverdi, Abdollah; Yang, Renliang; Korolev, Nikolay; Fan, Yanping; Davey, Curt A.; Liu, Chuan-Fa; Nordenskioeld, LarsNucleic Acids Research (2011), 39 (5), 1680-1691CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Understanding the mol. mechanisms behind regulation of chromatin folding through covalent modifications of the histone N-terminal tails is hampered by a lack of accessible chromatin contg. precisely modified histones. We study the internal folding and intermol. self-assocn. of a chromatin system consisting of satd. 12-mer nucleosome arrays contg. various combinations of completely acetylated lysines at positions 5, 8, 12 and 16 of histone H4, induced by the cations Na+, K+, Mg2+, Ca2+, cobalt-hexammine3+, spermidine3+ and spermine4+. Histones were prepd. using a novel semi-synthetic approach with native chem. ligation. Acetylation of H4-K16, but not its glutamine mutation, drastically reduces cation-induced folding of the array. Neither acetylations nor mutations of all the sites K5, K8 and K12 can induce a similar degree of array unfolding. The ubiquitous K+, (as well as Rb+ and Cs+) showed an unfolding effect on unmodified arrays almost similar to that of H4-K16 acetylation. We propose that K+ (and Rb+/Cs+) binding to a site on the H2B histone (R96-L99) disrupts H4K16 ε-amino group binding to this specific site, thereby deranging H4 tail-mediated nucleosome-nucleosome stacking and that a similar mechanism operates in the case of H4-K16 acetylation. Inter-array self-assocn. follows electrostatic behavior and is largely insensitive to the position or nature of the H4 tail charge modification.
- 87Dorigo, B.; Schalch, T.; Kulangara, A.; Duda, S.; Schroeder, R. R.; Richmond, T. J. Science 2004, 306, 157187Nucleosome arrays reveal the two-start organization of the chromatin fiberDorigo, Benedetta; Schalch, Thomas; Kulangaral, Alexandra; Duda, Sylwia; Schroeder, Rasmus R.; Richmond, Timothy J.Science (Washington, DC, United States) (2004), 306 (5701), 1571-1573CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Chromatin folding dets. the accessibility of DNA constituting eukaryotic genomes and consequently is profoundly important in the mechanisms of nuclear processes such as gene regulation. Nucleosome arrays compact to form a 30-nm chromatin fiber of hitherto disputed structure. Two competing classes of models have been proposed in which nucleosomes are either arranged linearly in a one-start higher order helix or zigzag back and forth in a two-start helix. We analyzed compacted nucleosome arrays stabilized by introduction of disulfide cross-links and show that the chromatin fiber comprises two stacks of nucleosomes in accord with the two-start model.
- 88Sinha, D.; Shogren-Knaak, M. A. J. Biol. Chem. 2010, 285, 1657288Role of Direct Interactions between the Histone H4 Tail and the H2A Core in Long Range Nucleosome ContactsSinha, Divya; Shogren-Knaak, Michael A.Journal of Biological Chemistry (2010), 285 (22), 16572-16581CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)In eukaryotic nuclei the majority of genomic DNA is believed to exist in higher order chromatin structures. Nonetheless, the nature of direct, long range nucleosome interactions that contribute to these structures is poorly understood. To det. whether these interactions are directly mediated by contacts between the histone H4 amino-terminal tail and the acidic patch of the H2A/H2B interface, as previously demonstrated for short range nucleosomal interactions, we have characterized the extent and effect of disulfide crosslinking between residues in histones contained in different strands of nucleosomal arrays. We show that in 208-12 5 S rDNA and 601-177-12 nucleosomal array systems, direct interactions between histones H4-V21C and H2A-E64C can be captured. This interaction depends on the extent of initial cross-strand assocn. but does not require these specific residues, because interactions with residues flanking H4-V21C can also be captured. Addnl., we find that trapping H2A-H4 intra-array interactions antagonizes the ability of these arrays to undergo intermol. self-assocn.
- 89Yang, D.; Arya, G. Phys. Chem. Chem. Phys. 2011, 13, 291189Structure and binding of the H4 histone tail and the effects of lysine 16 acetylationYang, Darren; Arya, GauravPhysical Chemistry Chemical Physics (2011), 13 (7), 2911-2921CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)The H4 histone tail plays a crit. role in chromatin folding and regulation - it mediates strong interactions with the acidic patch of proximal nucleosomes and its acetylation at lysine 16 (K16) leads to partial unfolding of chromatin. The mol. mechanism assocd. with the H4 tail/acidic patch interactions and its modulation via K16 acetylation remains unknown. Here we employ a combination of mol. dynamics simulations, mol. docking calcns., and free energy computations to investigate the structure of the H4 tail in soln., the binding of the H4 tail with the acidic patch, and the effects of K16 acetylation. The H4 tail exhibits a disordered configuration except in the region Ala15-Lys20, where it exhibits a strong propensity for an α-helical structure. This α-helical region is found to dock very favorably into the acidic patch groove of a nucleosome with a binding free energy of approx. -7 kcal mol-1. We have identified the specific interactions that stabilize this binding as well as the assocd. energetics. The acetylation of K16 is found to reduce the α-helix-forming propensity of the H4 tail and K16's accessibility for mediating external interactions. More importantly, K16 acetylation destabilizes the binding of the H4 tail at the acidic patch by mitigating specific salt bridges and longer-range electrostatic interactions mediated by K16. Our study thus provides new microscopic insights into the compaction of chromatin and its regulation via post-translational modifications of histone tails, which could be of interest to chromatin biol., cancer, epigenetics, and drug design.
- 90Barbera, A. J.; Ballestas, M. E.; Kaye, K. M. J. Virol. 2004, 78, 29490The Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 1 N terminus is essential for chromosome association, DNA replication, and episome persistenceBarbera, Andrew J.; Ballestas, Mary E.; Kaye, Kenneth M.Journal of Virology (2004), 78 (1), 294-301CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)To persist in latently infected, proliferating cells, Kaposi's sarcoma-assocd. herpesvirus (KSHV) episomes must replicate and efficiently segregate to progeny nuclei. Episome persistence in uninfected cells requires latency-assocd. nuclear antigen 1 (LANA1) in trans and cis-acting KSHV terminal repeat (TR) DNA. The LANA1 C terminus binds TR DNA, and LANA1 mediates TR-assocd. DNA replication in transient assays. LANA1 also concs. at sites of KSHV TR DNA episomes along mitotic chromosomes, consistent with a tethering role to efficiently segregate episomes to progeny nuclei. LANA1 amino acids 5-22 constitute a chromosome assocn. region. We now investigate LANA1 residues 5-22 with scanning alanine substitutions. Mutations targeting LANA1 5GMR7, 8LRS10, and 11GRS13 eliminated chromosome assocn., DNA replication, and episome persistence. LANA1 mutated at 14TG15 retained the ability to assoc. with chromosomes but was partially deficient in DNA replication and episome persistence. These results provide genetic support for a key role of the LANA1 N terminus in chromosome assocn., LANA1-mediated DNA replication, and episome persistence.
- 91Clarke, P. R.; Zhang, C. Nat. Rev. Mol. Cell Biol. 2008, 9, 46491Spatial and temporal coordination of mitosis by Ran GTPaseClarke, Paul R.; Zhang, ChuanmaoNature Reviews Molecular Cell Biology (2008), 9 (6), 464-477CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. Small nuclear GTPase Ran controls the directionality of macromol. transport between the nucleus and the cytoplasm. Ran also plays important roles during mitosis, when the nucleus is dramatically reorganized to allow chromosome segregation. Ran directs the assembly of the mitotic spindle, nuclear envelope dynamics, and the timing of cell-cycle transitions. The mechanisms that underlie these functions provide insights into the spatial and temporal coordination of the changes that occur in intracellular organization during the cell-division cycle.
- 92Carazo-Salas, R. E.; Guarguaglini, G.; Gruss, O. J.; Segref, A.; Karsenti, E.; Mattaj, I. W. Nature 1999, 400, 17892Generation of GTP-bound Ran by RCC1 is required for chromatin-induced mitotic spindle formationCarazo-Salas, Rafael E.; Guarguaglini, Giulia; Gruss, Oliver J.; Segref, Alexandra; Karsenti, Eric; Mattaj, Iain W.Nature (London) (1999), 400 (6740), 178-181CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)Chromosomes are segregated by two antiparallel arrays of microtubules arranged to form the spindle app. During cell division, the nucleation of cytosolic microtubules is prevented and spindle microtubules nucleate from centrosomes (in mitotic animal cells) or around chromosomes (in plants and some meiotic cells). The mol. mechanism by which chromosomes induce local microtubule nucleation in the absence of centrosomes is unknown, but it can be studied by adding chromatin beads to Xenopus egg exts. The beads nucleate microtubules that eventually reorganize into a bipolar spindle. RCC1, the guanine-nucleotide-exchange factor for the GTPase protein Ran, is a component of chromatin. Using the chromatin bead assay, the authors show here that the activity of chromosome-assocd. RCC1 protein is required for spindle formation. Ran itself, when in the GTP-bound state (Ran-GTP), induces microtubule nucleation and spindle-like structures in M-phase ext. The authors propose that RCC1 generates a high local concn. of Ran-GTP around chromatin which in turn induces the local nucleation of microtubules.
- 93Kalab, P.; Heald, R. J. Cell Sci. 2008, 121, 157793The RanGTP gradient - a GPS for the mitotic spindleKalab, Petr; Heald, RebeccaJournal of Cell Science (2008), 121 (10), 1577-1586CODEN: JNCSAI; ISSN:0021-9533. (Company of Biologists Ltd.)A review. GTPase Ran plays a key role in nuclear import and export, mitotic spindle assembly, and nuclear envelope formation. The cycling of Ran between its GTP- and GDP-bound forms is catalyzed by the chromatin-bound guanine nucleotide exchange factor RCC1 and the cytoplasmic Ran GTPase-activating protein, RanGAP. The result is an intracellular concn. gradient of RanGTP that equips eukaryotic cells with a 'genome-positioning system' (GPS). The binding of RanGTP to nuclear transport receptors (NTRs) of the importin-β superfamily mediates the effects of the gradient and generates further downstream gradients, which have been elucidated by FRET imaging and computational modeling. The Ran-dependent GPS spatially directs many functions required for genome segregation by the mitotic spindle during mitosis. Through exportin 1, RanGTP recruits essential centrosome and kinetochore components, whereas the RanGTP-induced release of spindle assembly factors (SAFs) from importins activates SAFs to nucleate, bind, and organize nascent spindle microtubules. Although a considerable fraction of cytoplasmic SAFs is active and RanGTP induces only partial further activation near chromatin, bipolar spindle assembly is robustly induced by cooperativity and pos.-feedback mechanisms within the network of Ran-activated SAFs. The RanGTP gradient is conserved, although its roles vary among different cell types and species, and much remains to be learned regarding its functions.
- 94Nemergut, M. E.; Mizzen, C. A.; Stukenberg, T.; Allis, C. D.; Macara, I. G. Science 2001, 292, 154094Chromatin docking and exchange activity enhancement of RCC1 by histones H2A and H2BNemergut, Michael E.; Mizzen, Craig A.; Stukenberg, Todd; Allis, C. David; Macara, Ian G.Science (Washington, DC, United States) (2001), 292 (5521), 1540-1543CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The Ran guanosine triphosphatase (GTPase) controls nucleocytoplasmic transport, mitotic spindle formation, and nuclear envelope assembly. These functions rely on the assocn. of the Ran-specific exchange factor, RCC1 (regulator of chromosome condensation 1), with chromatin. We find that RCC1 binds directly to mononucleosomes and to histones H2A and. H2B. RCC1 utilizes these histones to bind Xenopus sperm chromatin, and the binding of RCC1 to nucleosomes or histones stimulates the catalytic activity of RCC1. We propose that the docking of RCC1 to H2A/H2B establishes the polarity of the Ran-GTP gradient that drives nuclear envelope assembly, nuclear transport, and other nuclear events.
- 95England, J. R.; Huang, J.; Jennings, M. J.; Makde, R. D.; Tan, S. J. Mol. Biol. 2010, 398, 51895RCC1 Uses a Conformationally Diverse Loop Region to Interact with the Nucleosome: A Model for the RCC1-Nucleosome ComplexEngland, Joseph R.; Huang, Jiehuan; Jennings, Matthew J.; Makde, Ravindra D.; Tan, SongJournal of Molecular Biology (2010), 398 (4), 518-529CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)The binding of RCC1 (regulator of chromosome condensation 1) to chromatin is crit. for cellular processes such as mitosis, nucleocytoplasmic transport, and nuclear envelope formation because RCC1 recruits the small GTPase Ran (Ras-related nuclear protein) to chromatin and sets up a Ran-GTP gradient around the chromosomes. However, the mol. mechanism by which RCC1 binds to nucleosomes, the repeating unit of chromatin, is not known. Biochem. approaches were used to test structural models for how the RCC1 β-propeller protein could bind to the nucleosome. In contrast to the prevailing model, RCC1 does not appear to use the β-propeller face opposite to its Ran-binding face to interact with nucleosomes. Instead, it is found that RCC1 uses a conformationally flexible loop region, termed the switchback loop, in addn. to its N-terminal tail to bind to the nucleosome. The juxtaposition of the RCC1 switchback loop to its Ran binding surface suggests a novel mechanism for how nucleosome-bound RCC1 recruits Ran to chromatin. Furthermore, this model accounts for previously unexplained observations for how Ran can interact with the nucleosome both dependent and independent of RCC1 and how binding of the nucleosome can enhance RCC1's Ran nucleotide exchange activity.
- 96Rusche, L. N.; Kirchmaier, A. L.; Rine, J. Annu. Rev. Biochem. 2003, 72, 48196The establishment, inheritance, and function of silenced chromatin in Saccharomyces cerevisiaeRusche, Laura N.; Kirchmaier, Ann L.; Rine, JasperAnnual Review of Biochemistry (2003), 72 (), 481-516CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)A review. Genomes are organized into active regions known as euchromatin and inactive regions known as heterochromatin, or silenced chromatin. This review describes contemporary knowledge and models for how silenced chromatin in Saccharomyces cerevisiae forms, functions, and is inherited. In S. cerevisiae, Sir proteins are the key structural components of silenced chromatin. Sir proteins interact first with silencers, which dictate which regions are silenced, and then with histone tails in nucleosomes as the Sir proteins spread from silencers along chromosomes. Importantly, the spreading of silenced chromatin requires the histone deacetylase activity of Sir2p. This requirement leads to a general model for the spreading and inheritance of silenced chromatin or other special chromatin states. Such chromatin domains are marked by modifications of the nucleosomes or DNA, and this mark is able to recruit an enzyme that makes further marks. Thus, among different organisms, multiple forms of repressive chromatin can be formed using similar strategies but completely different proteins. We also describe emerging evidence that mutations that cause global changes in the modification of histones can alter the balance between euchromatin and silenced chromatin within a cell.
- 97McBryant, S. J.; Krause, C.; Woodcock, C. L.; Hansen, J. C. Mol. Cell. Biol. 2008, 28, 356397The silent information regulator 3 protein, SIR3p, binds to chromatin fibers and assembles a hypercondensed chromatin architecture in the presence of saltMcBryant, Steven J.; Krause, Christine; Woodcock, Christopher L.; Hansen, Jeffrey C.Molecular and Cellular Biology (2008), 28 (11), 3563-3572CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)The telomeres and mating-type loci of budding yeast adopt a condensed, heterochromatin-like state through recruitment of the silent information regulator (SIR) proteins SIR2p, SIR3p, and SIR4p. In this study we characterize the chromatin binding determinants of recombinant SIR3p and identify how SIR3p mediates chromatin fiber condensation in vitro. Purified full-length SIR3p was incubated with naked DNA, nucleosome core particles, or defined nucleosomal arrays, and the resulting complexes were analyzed by electrophoretic shift assays, sedimentation velocity, and electron microscopy. SIR3p bound avidly to all three types of templates. SIR3p loading onto its nucleosomal sites in chromatin produced thickened condensed fibers that retained a beaded morphol. At higher SIR3p concns., individual nucleosomal arrays formed oligomeric suprastructures bridged by SIR3p oligomers. When condensed SIR3p-bound chromatin fibers were incubated in Mg2+, they folded and oligomerized even further to produce hypercondensed higher-order chromatin structures. Collectively, these results define how SIR3p may function as a chromatin architectural protein and provide new insight into the interplay between endogenous and protein-mediated chromatin fiber condensation pathways.
- 98Johnson, A.; Li, G.; Sikorski, T. W.; Buratowski, S.; Woodcock, C. L.; Moazed, D. Mol. Cell 2009, 35, 76998Reconstitution of heterochromatin-dependent transcriptional gene silencingJohnson, Aaron; Li, Geng; Sikorski, Timothy W.; Buratowski, Stephen; Woodcock, Christopher L.; Moazed, DaneshMolecular Cell (2009), 35 (6), 769-781CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)Heterochromatin assembly in budding yeast requires the SIR complex, which contains the NAD-dependent deacetylase Sir2 and the Sir3 and Sir4 proteins. Sir3 binds to nucleosomes contg. deacetylated histone H4 lysine 16 (H4K16) and, with Sir4, promotes spreading of Sir2 and deacetylation along the chromatin fiber. Combined action of histone modifying and binding activities is a conserved hallmark of heterochromatin, but the relative contribution of each activity to silencing has remained unclear. Here, we reconstitute SIR-chromatin complexes using purified components and show that the SIR complex efficiently deacetylates chromatin templates and promotes the assembly of altered structures that silence Gal4-VP16-activated transcription. Silencing requires all three Sir proteins, even with fully deacetylated chromatin, and involves the specific assocn. of Sir3 with deacetylated H4K16. These results define a minimal set of components that mediate heterochromatic gene silencing and demonstrate distinct contributions for histone deacetylation and nucleosome binding in the silencing mechanism.
- 99Martino, F.; Kueng, S.; Robinson, P.; Tsai-Pflugfelder, M.; van Leeuwen, F.; Ziegler, M.; Cubizolles, F.; Cockell, M. M.; Rhodes, D.; Gasser, S. M. Mol. Cell 2009, 33, 32399Reconstitution of yeast silent chromatin: multiple contact sites and O-AADPR binding load SIR complexes onto nucleosomes in vitroMartino, Fabrizio; Kueng, Stephanie; Robinson, Philip; Tsai-Pflugfelder, Monika; van Leeuwen, Fred; Ziegler, Mathias; Cubizolles, Fabien; Cockell, Moira M.; Rhodes, Daniela; Gasser, Susan M.Molecular Cell (2009), 33 (3), 323-334CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)At yeast telomeres and silent mating-type loci, chromatin assumes a higher-order structure that represses transcription by means of the histone deacetylase Sir2 and structural proteins Sir3 and Sir4. Here, we present a fully reconstituted system to analyze SIR holocomplex binding to nucleosomal arrays. Purified Sir2-3-4 heterotrimers bind chromatin, cooperatively yielding a stable complex of homogeneous mol. wt. Remarkably, Sir2-3-4 also binds naked DNA, reflecting the strong, albeit nonspecific, DNA-binding activity of Sir4. The binding of Sir3 to nucleosomes is sensitive to histone H4 N-terminal tail removal, while that of Sir2-4 is not. Dot1-mediated methylation of histone H3K79 reduces the binding of both Sir3 and Sir2-3-4. Addnl., a byproduct of Sir2-mediated NAD hydrolysis, O-acetyl-ADP-ribose, increases the efficiency with which Sir3 and Sir2-3-4 bind nucleosomes. Thus, in small cumulative steps, each Sir protein, unmodified histone domains, and contacts with DNA contribute to the stability of the silent chromatin complex.
- 100Norris, A.; Bianchet, M. A.; Boeke, J. D. PLoS Genet. 2008, 4, e1000301There is no corresponding record for this reference.
- 101Singer, M. S.; Kahana, A.; Wolf, A. J.; Meisinger, L. L.; Peterson, S. E.; Goggin, C.; Mahowald, M.; Gottschling, D. E. Genetics 1998, 150, 613101Identification of high-copy disruptors of telomeric silencing in Saccharomyces cerevisiaeSinger, Miriam S.; Kahana, Alon; Wolf, Alexander J.; Meisinger, Lia L.; Peterson, Suzanne E.; Goggin, Colin; Mahowald, Maureen; Gottschling, Daniel E.Genetics (1998), 150 (2), 613-632CODEN: GENTAE; ISSN:0016-6731. (Genetics Society of America)The ends of chromosomes in Saccharomyces cerevisiae initiate a repressive chromatin structure that spreads internally and inhibits the transcription of nearby genes, a phenomenon termed telomeric silencing. To investigate the mol. basis of this process, we carried out a genetic screen to identify genes whose overexpression disrupts telomeric silencing. We thus isolated 10 DOT genes (disruptor of telomeric silencing). Among these were genes encoding chromatin component Sir4p, DNA helicase Dna2p, ribosomal protein L32, and two proteins of unknown function, Asf1p and Ifh1p. The collection also included genes that had not previously been identified: DOT1, DOT4, DOT5, DOT6, and TLC1, which encodes the RNA template component of telomerase. With the exception of TLC1, all these genes, particularly DOT1 and DOT4, also reduced silencing at other repressed loci (HM loci and rDNA) when overexpressed. Moreover, deletion of the latter two genes weakened silencing as well, suggesting that DOT1 and DOT4 normally play important roles in gene repression. DOT1 deletion also affected telomere tract length. The function of Dot1p is not known. The sequence of Dot4p suggests that it is a ubiquitin-processing protease. Taken together, the DOT genes include both components and regulators of silent chromatin.
- 102Ng, H. H.; Ciccone, D. N.; Morshead, K. B.; Oettinger, M. A.; Struhl, K. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 1820102Lysine-79 of histone H3 is hypomethylated at silenced loci in yeast and mammalian cells: A potential mechanism for position-effect variegationNg, Huck Hui; Ciccone, David N.; Morshead, Katrina B.; Oettinger, Marjorie A.; Struhl, KevinProceedings of the National Academy of Sciences of the United States of America (2003), 100 (4), 1820-1825CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Methylation of lysine-79 (K79) within the globular domain of histone H3 by Dot1 methylase is important for transcriptional silencing and for assocn. of the Sir silencing proteins in yeast. Here, we show that the level of H3-K79 methylation is low at all Sir-dependent silenced loci but not at other transcriptionally repressed regions. Hypomethylation of H3-K79 at the telomeric and silent mating-type loci, but not the ribosomal DNA, requires the Sir proteins. Overexpression of Sir3 concomitantly extends the domain of Sir protein assocn. and H3-K79 hypomethylation at telomeres. In mammalian cells, H3-K79 methylation is found at loci that are active for V(D)J recombination, but not at recombinationally inactive loci that are heterochromatic. These results suggest that H3-K79 methylation is an evolutionarily conserved marker of active chromatin regions, and that silencing proteins block the ability of Dot1 to methylate histone H3. Further, they suggest that Sir proteins preferentially bind chromatin with hypomethylated H3-K79 and then block H3-K79 methylation. This pos. feedback loop, and the reverse loop in which H3-K79 methylation weakens Sir protein assocn. and leads to further methylation, suggests a model for position-effect variegation.
- 103van Leeuwen, F.; Gafken, P. R.; Gottschling, D. E. Cell 2002, 109, 745103Dot1p modulates silencing in yeast by methylation of the nucleosome coreVan Leeuwen, Fred; Gafken, Philip R.; Gottschling, Daniel E.Cell (Cambridge, MA, United States) (2002), 109 (6), 745-756CODEN: CELLB5; ISSN:0092-8674. (Cell Press)DOT1 was originally identified as a gene affecting telomeric silencing in S. cerevisiae. We now find that Dot1p methylates histone H3 on lysine 79, which maps to the top and bottom of the nucleosome core. Methylation occurs only when histone H3 is assembled in chromatin. In vivo, Dot1p is solely responsible for this methylation and methylates ∼90% of histone H3. In dot1Δ cells, silencing is compromised and silencing proteins become redistributed at the expense of normally silenced loci. We suggest that methylation of histone H3 lysine 79 limits silencing to discrete loci by preventing the binding of Sir proteins elsewhere along the genome. Because Dot1p and histone H3 are conserved, similar mechanisms are likely at work in other eukaryotes.
- 104Arnaudo, N.; Fernández, I. S.; McLaughlin, S. H.; Peak-Chew, S. Y.; Rhodes, D.; Martino, F. Nat. Struct. Mol. Biol. 2013, 20, 1119104The N-terminal acetylation of Sir3 stabilizes its binding to the nucleosome core particleArnaudo, Nadia; Fernandez, Israel S.; McLaughlin, Stephen H.; Peak-Chew, Sew Y.; Rhodes, Daniela; Martino, FabrizioNature Structural & Molecular Biology (2013), 20 (9), 1119-1121CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)The N-terminal acetylation of Sir3 is essential for heterochromatin establishment and maintenance in yeast, but its mechanism of action is unknown. The crystal structure of the N-terminally acetylated BAH domain of Saccharomyces cerevisiae Sir3 bound to the nucleosome core particle reveals that the N-terminal acetylation stabilizes the interaction of Sir3 with the nucleosome. Addnl., we present a new method for the prodn. of protein-nucleosome complexes for structural anal.
- 105Yang, D.; Fang, Q.; Wang, M.; Ren, R.; Wang, H.; He, M.; Sun, Y.; Yang, N.; Xu, R.-M. Nat. Struct. Mol. Biol. 2013, 20, 1116105Nα-acetylated Sir3 stabilizes the conformation of a nucleosome-binding loop in the BAH domainYang, Dongxue; Fang, Qianglin; Wang, Mingzhu; Ren, Ren; Wang, Hong; He, Meng; Sun, Youwei; Yang, Na; Xu, Rui-MingNature Structural & Molecular Biology (2013), 20 (9), 1116-1118CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)In Saccharomyces cerevisiae, acetylation of the Sir3 N terminus is important for transcriptional silencing. This covalent modification promotes the binding of the Sir3 BAH domain to the nucleosome, but a mechanistic understanding of this phenomenon is lacking. By X-ray crystallog., we show here that the acetylated N terminus of Sir3 does not interact with the nucleosome directly. Instead, it stabilizes a nucleosome-binding loop in the BAH domain.
- 106Connelly, J. J.; Yuan, P.; Hsu, H.-C.; Li, Z.; Xu, R.-M.; Sternglanz, R. Mol. Cell. Biol. 2006, 26, 3256106Structure and function of the Saccharomyces cerevisiae Sir3 BAH domainConnelly, Jessica J.; Yuan, Peihua; Hsu, Hao-Chi; Li, Zhizhong; Xu, Rui-Ming; Sternglanz, RolfMolecular and Cellular Biology (2006), 26 (8), 3256-3265CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)Previous work has shown that the N terminus of the Saccharomyces cerevisiae Sir3 protein is crucial for the function of Sir3 in transcriptional silencing. Here, we show that overexpression of N-terminal fragments of Sir3 in strains lacking the full-length protein can lead to some silencing of HML and HMR. Sir3 contains a BAH (bromo-adjacent homol.) domain at its N terminus. Overexpression of this domain alone can lead to silencing as long as Sir1 is overexpressed and Sir2 and Sir4 are present. Overexpression of the closely related Orc1 BAH domain can also silence in the absence of any Sir3 protein. A previously characterized hypermorphic sir3 mutation, D205N, greatly improves silencing by the Sir3 BAH domain and allows it to bind to DNA and oligonucleosomes in vitro. A previously uncharacterized region in the Sir1 N terminus is required for silencing by both the Sir3 and Orc1 BAH domains. The structure of the Sir3 BAH domain has been detd. In the crystal, the mol. multimerizes in the form of a left-handed superhelix. This superhelix may be relevant to the function of the BAH domain of Sir3 in silencing.
- 107Kato, H.; Jiang, J.; Zhou, B.-R.; Rozendaal, M.; Feng, H.; Ghirlando, R.; Xiao, T. S.; Straight, A. F.; Bai, Y. Science 2013, 340, 1110107A conserved mechanism for centromeric nucleosome recognition by centromere protein CENP-CKato, Hidenori; Jiang, Jiansheng; Zhou, Bing-Rui; Rozendaal, Marieke; Feng, Hanqiao; Ghirlando, Rodolfo; Xiao, T. Sam; Straight, Aaron F.; Bai, YawenScience (Washington, DC, United States) (2013), 340 (6136), 1110-1113CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Chromosome segregation during mitosis requires assembly of the kinetochore complex at the centromere. Kinetochore assembly depends on specific recognition of the histone variant CENP-A in the centromeric nucleosome by centromere protein C (CENP-C). Here, the authors defined the determinants of this recognition mechanism and discovered that CENP-C binds a hydrophobic region in the CENP-A tail and docks onto the acidic patch of histone H2A and H2B. The authors further found that the more broadly conserved CENP-C motif used the same mechanism for CENP-A nucleosome recognition. These findings revealed a conserved mechanism for protein recruitment to centromeres and a histone recognition mode whereby a disordered peptide binds the histone tail through hydrophobic interactions facilitated by nucleosome docking.
- 108Henikoff, S.; Ahmad, K.; Malik, H. S. Science 2001, 293, 1098108The centromere paradox: Stable inheritance with rapidly evolving DNAHenikoff, Steven; Ahmad, Kami; Malik, Harmit S.Science (Washington, DC, United States) (2001), 293 (5532), 1098-1102CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The review. Every eukaryotic chromosome has a centromere, the locus responsible for poleward movement at mitosis and meiosis. Although conventional loci are specified by their DNA sequences, current evidence favors a chromatin-based inheritance mechanism for centromeres. The chromosome segregation machinery is highly conserved across all eukaryotes, but the DNA and protein components specific to centromeric chromatin are evolving rapidly. Incompatibilities between rapidly evolving centromeric components may be responsible for both the organization of centromeric regions and the reproductive isolation of emerging species.
- 109Verdaasdonk, J. S.; Bloom, K. Nat. Rev. Mol. Cell Biol. 2011, 12, 320109Centromeres: unique chromatin structures that drive chromosome segregationVerdaasdonk, Jolien S.; Bloom, KerryNature Reviews Molecular Cell Biology (2011), 12 (5), 320-332CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. Fidelity during chromosome segregation is essential to prevent aneuploidy. The proteins and chromatin at the centromere form a unique site for kinetochore attachment and allow the cell to sense and correct errors during chromosome segregation. Centromeric chromatin is characterized by distinct chromatin organization, epigenetics, centromere-assocd. proteins, and histone variants. These include histone H3 variant centromeric protein A (CENP-A), the compn. and deposition of which have been widely investigated. Studies have examd. the structural and biophys. properties of the centromere and have suggested that the centromere is not simply a 'landing pad' for kinetochore formation, but plays an essential role in mitosis by assembling and directing the organization of the kinetochore.
- 110Hori, T.; Amano, M.; Suzuki, A.; Backer, C. B.; Welburn, J. P.; Dong, Y.; McEwen, B. F.; Shang, W.-H.; Suzuki, E.; Okawa, K.; Cheeseman, I. M.; Fukagawa, T. Cell 2008, 135, 1039110CCAN makes multiple contacts with centromeric DNA to provide distinct pathways to the outer kinetochoreHori, Tetsuya; Amano, Miho; Suzuki, Aussie; Backer, Chelsea B.; Welburn, Julie P.; Dong, Yimin; McEwen, Bruce F.; Shang, Wei-Hao; Suzuki, Emiko; Okawa, Katsuya; Cheeseman, Iain M.; Fukagawa, TatsuoCell (Cambridge, MA, United States) (2008), 135 (6), 1039-1052CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Kinetochore specification and assembly requires the targeted deposition of specialized nucleosomes contg. the histone H3 variant CENP-A at centromeres. However, CENP-A is not sufficient to drive full-kinetochore assembly, and it is not clear how centromeric chromatin is established. Here, we identify CENP-W as a component of the DNA-proximal constitutive centromere-assocd. network (CCAN) of proteins. We demonstrate that CENP-W forms a DNA-binding complex together with the CCAN component CENP-T. This complex directly assocs. with nucleosomal DNA and with canonical histone H3, but not with CENP-A, in centromeric regions. CENP-T/CENP-W functions upstream of other CCAN components with the exception of CENP-C, an addnl. putative DNA-binding protein. Our anal. indicates that CENP-T/CENP-W and CENP-C provide distinct pathways to connect the centromere with outer kinetochore assembly. In total, our results suggest that the CENP-T/CENP-W complex is directly involved in establishment of centromere chromatin structure coordinately with CENP-A.
- 111McGinty, R. K.; Henrici, R. C.; Tan, S. Nature 2014, 514, 591111Crystal structure of the PRC1 ubiquitylation module bound to the nucleosomeMcGinty, Robert K.; Henrici, Ryan C.; Tan, SongNature (London, United Kingdom) (2014), 514 (7524), 591-596CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)The Polycomb group of epigenetic enzymes represses expression of developmentally regulated genes in many eukaryotes. This group includes the Polycomb repressive complex 1 (PRC1), which ubiquitylates nucleosomal histone H2A Lys 119 using its E3 ubiquitin ligase subunits, Ring1B and Bmi1, together with an E2 ubiquitin-conjugating enzyme, UbcH5c. However, the mol. mechanism of nucleosome substrate recognition by PRC1 or other chromatin enzymes is unclear. Here we present the crystal structure of the human Ring1B-Bmi1-UbcH5c E3-E2 complex (the PRC1 ubiquitylation module) bound to its nucleosome core particle substrate. The structure shows how a chromatin enzyme achieves substrate specificity by interacting with several nucleosome surfaces spatially distinct from the site of catalysis. Our structure further reveals an unexpected role for the ubiquitin E2 enzyme in substrate recognition, and provides insight into how the related histone H2A E3 ligase, BRCA1, interacts with and ubiquitylates the nucleosome.
- 112Cao, R.; Tsukada, Y.-I.; Zhang, Y. Mol. Cell 2005, 20, 845112Role of Bmi-1 and Ring1A in H2A ubiquitylation and Hox gene silencingCao, Ru; Tsukada, Yu-ichi; Zhang, YiMolecular Cell (2005), 20 (6), 845-854CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)Polycomb group (PcG) proteins exist in at least two biochem. distinct protein complexes, the EED-EZH2 complex and the PRC1 complex, that resp. possess H3-K27 methyltransferase and H2A-K119 ubiquitin E3 ligase activities. How the enzymic activities are regulated and what their role is in Hox gene silencing are not clear. Here, the authors demonstrate that Bmi-1 and Ring1A, two components of the PRC1 complex, play important roles in H2A ubiquitylation and Hox gene silencing. Both proteins pos. regulate H2A ubiquitylation. Chromatin immunopptn. (ChIP) assays demonstrate that Bmi-1 and other components of the two PcG complexes bind to the promoter of HoxC13. Knockout of Bmi-1 results in significant loss of H2A ubiquitylation and upregulation of Hoxc13 expression, whereas EZH2-mediated H3-K27 methylation is not affected. These results suggest that EZH2-mediated H3-K27 methylation functions upstream of PRC1 and establishes a crit. role for Bmi-1 and Ring1A in H2A ubiquitylation and Hox gene silencing.
- 113Grau, D. J.; Chapman, B. A.; Garlick, J. D.; Borowsky, M.; Francis, N. J.; Kingston, R. E. Genes Dev. 2011, 25, 2210113Compaction of chromatin by diverse polycomb group proteins requires localized regions of high chargeGrau, Daniel J.; Chapman, Brad A.; Garlick, Joe D.; Borowsky, Mark; Francis, Nicole J.; Kingston, Robert E.Genes & Development (2011), 25 (20), 2210-2221CODEN: GEDEEP; ISSN:0890-9369. (Cold Spring Harbor Laboratory Press)Polycomb group (PcG) proteins are required for the epigenetic maintenance of developmental genes in a silent state. Proteins in the Polycomb-repressive complex 1 (PRC1) class of the PcG are conserved from flies to humans and inhibit transcription. One hypothesis for PRC1 mechanism is that it compacts chromatin, based in part on electron microscopy expts. demonstrating that Drosophila PRC1 compacts nucleosomal arrays. We show that this function is conserved between Drosophila and mouse PRC1 complexes and requires a region with an overrepresentation of basic amino acids. While the active region is found in the Posterior Sex Combs (PSC) subunit in Drosophila, it is unexpectedly found in a different PRC1 subunit, a Polycomb homolog called M33, in mice. We provide exptl. support for the general importance of a charged region by predicting the compacting capability of PcG proteins from species other than Drosophila and mice and by testing several of these proteins using soln. assays and microscopy. We infer that the ability of PcG proteins to compact chromatin in vitro can be predicted by the presence of domains of high pos. charge and that PRC1 components from a variety of species conserve this highly charged region. This supports the hypothesis that compaction is a key aspect of PcG function.
- 114Simon, J. A.; Kingston, R. E. Nat. Rev. Mol. Cell Biol. 2009, 10, 697114Mechanisms of Polycomb gene silencing: Knowns and unknownsSimon, Jeffrey A.; Kingston, Robert E.Nature Reviews Molecular Cell Biology (2009), 10 (10), 697-708CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. Polycomb proteins form chromatin-modifying complexes that implement transcriptional silencing in higher eukaryotes. Hundreds of genes are silenced by Polycomb proteins, including dozens of genes that encode crucial developmental regulators in organisms ranging from plants to humans. Two main families of complexes, called Polycomb repressive complex 1 (PRC1) and PRC2, are targeted to repressed regions. Recent studies have advanced our understanding of these complexes, including their potential mechanisms of gene silencing, the roles of chromatin modifications, their means of delivery to target genes and the functional distinctions among variant complexes. Emerging concepts include the existence of a Polycomb barrier to transcription elongation and the involvement of non-coding RNAs in the targeting of Polycomb complexes. These findings have an impact on the epigenetic programming of gene expression in many biol. systems.
- 115Buchwald, G.; van der Stoop, P.; Weichenrieder, O.; Perrakis, A.; van Lohuizen, M.; Sixma, T. K. EMBO J. 2006, 25, 2465115Structure and E3-ligase activity of the Ring-Ring complex of Polycomb proteins Bmi1 and Ring1bBuchwald, Gretel; van der Stoop, Petra; Weichenrieder, Oliver; Perrakis, Anastassis; van Lohuizen, Maarten; Sixma, Titia K.EMBO Journal (2006), 25 (11), 2465-2474CODEN: EMJODG; ISSN:0261-4189. (Nature Publishing Group)Polycomb group (PcG) proteins Ring1b and Bmi1 (B-cell-specific Moloney murine leukemia virus integration site 1) are crit. components of the chromatin-modulating PRC1 complex. Histone H2A ubiquitination by the PRC1 complex strongly depends on the Ring1b protein. Here we show that the E3-ligase activity of Ring1b on histone H2A is enhanced by Bmi1 in vitro. The N-terminal Ring-domains are sufficient for this activity and Ring1a can replace Ring1b. E2 enzymes UbcH5a, b, c or UbcH6 support this activity with varying processivity and selectivity. All four E2s promote autoubiquitination of Ring1b without affecting E3-ligase activity. We solved the crystal structure of the Ring-Ring heterodimeric complex of Ring1b and Bmi1. In the structure the arrangement of the Ring-domains is similar to another H2A E3 ligase, the BRCA1/BARD1 complex, but complex formation depends on an N-terminal arm of Ring1b that embraces the Bmi1 Ring-domain. Mutation of a crit. residue in the E2/E3 interface shows that catalytic activity resides in Ring1b and not in Bmi1. These data provide a foundation for understanding the crit. enzymic activity at the core of the PRC1 polycomb complex, which is implicated in stem cell maintenance and cancer.
- 116Bentley, M. L.; Corn, J. E.; Dong, K. C.; Phung, Q.; Cheung, T. K.; Cochran, A. G. EMBO J. 2011, 30, 3285116Recognition of UbcH5c and the nucleosome by the Bmi1/Ring1b ubiquitin ligase complexBentley, Matthew L.; Corn, Jacob E.; Dong, Ken C.; Phung, Qui; Cheung, Tommy K.; Cochran, Andrea G.EMBO Journal (2011), 30 (16), 3285-3297CODEN: EMJODG; ISSN:0261-4189. (Nature Publishing Group)The Polycomb repressive complex 1 (PRC1) mediates gene silencing, in part by monoubiquitination of histone H2A on lysine 119 (uH2A). Bmi1 and Ring1b are crit. components of PRC1 that heterodimerize via their N-terminal RING domains to form an active E3 ubiquitin ligase. We have detd. the crystal structure of a complex between the Bmi1/Ring1b RING-RING heterodimer and the E2 enzyme UbcH5c and find that UbcH5c interacts with Ring1b only, in a manner fairly typical of E2-E3 interactions. However, we further show that the Bmi1/Ring1b RING domains bind directly to duplex DNA through a basic surface patch unique to the Bmi1/Ring1b RING-RING dimer. Mutation of residues on this interaction surface leads to a loss of H2A ubiquitination activity. Computational modeling of the interface between Bmi1/Ring1b-UbcH5c and the nucleosome suggests that Bmi1/Ring1b interacts with both nucleosomal DNA and an acidic patch on histone H4 to achieve specific monoubiquitination of H2A. Our results point to a novel mechanism of substrate recognition, and control of product formation, by Bmi1/Ring1b.
- 117Leung, J. W.; Agarwal, P.; Canny, M. D.; Gong, F.; Robison, A. D.; Finkelstein, I. J.; Durocher, D.; Miller, K. M. PLoS Genet. 2014, 10, e1004178117Nucleosome acidic patch promotes RNF168- and RING1B/BMI1-dependent H2AX and H2A ubiquitination and DNA damage signalingLeung, Justin W.; Agarwal, Poonam; Canny, Marella D.; Gong, Fade; Robison, Aaron D.; Finkelstein, Ilya J.; Durocher, Daniel; Miller, Kyle M.PLoS Genetics (2014), 10 (3), e1004178/1-e1004178/14, 14 pp.CODEN: PGLEB5; ISSN:1553-7404. (Public Library of Science)Histone ubiquitinations are crit. for the activation of the DNA damage response (DDR). In particular, RNF168 and RING1B/BMI1 function in the DDR by ubiquitinating H2A/H2AX on Lys-13/15 and Lys-118/119, resp. However, it remains to be defined how the ubiquitin pathway engages chromatin to provide regulation of ubiquitin targeting of specific histone residues. Here we identify the nucleosome acid patch as a crit. chromatin mediator of H2A/H2AX ubiquitination (ub). The acidic patch is required for RNF168- and RING1B/BMI1-dependent H2A/H2AXub in vivo. The acidic patch functions within the nucleosome as nucleosomes contg. a mutated acidic patch exhibit defective H2A/H2AXub by RNF168 and RING1B/BMI1 in vitro. Furthermore, direct perturbation of the nucleosome acidic patch in vivo by the expression of an engineered acidic patch interacting viral peptide, LANA, results in defective H2AXub and RNF168-dependent DNA damage responses including 53BP1 and BRCA1 recruitment to DNA damage. The acidic patch therefore is a crit. nucleosome feature that may serve as a scaffold to integrate multiple ubiquitin signals on chromatin to compose selective ubiquitinations on histones for DNA damage signaling.
- 118Kato, H.; van Ingen, H.; Zhou, B.-R.; Feng, H.; Bustin, M.; Kay, L. E.; Bai, Y. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 12283118Architecture of the high mobility group nucleosomal protein 2-nucleosome complex as revealed by methyl-based NMRKato, Hidenori; van Ingen, Hugo; Zhou, Bing-Rui; Feng, Hanqiao; Bustin, Michael; Kay, Lewis E.; Bai, YawenProceedings of the National Academy of Sciences of the United States of America (2011), 108 (30), 12283-12288, S12283/1-S12283/15CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Chromatin structure and function are regulated by numerous proteins through specific binding to nucleosomes. The structural basis of many of these interactions is unknown, as in the case of the high mobility group nucleosomal (HMGN) protein family that regulates various chromatin functions, including transcription. Here, we report the architecture of the HMGN2-nucleosome complex detd. by a combination of methyl-transverse relaxation optimized NMR spectroscopy (methyl-TROSY) and mutational anal. We found that HMGN2 binds to both the acidic patch in the H2A-H2B dimer and to nucleosomal DNA near the entry/exit point, "stapling" the histone core and the DNA. These results provide insight into how HMGNs regulate chromatin structure through interfering with the binding of linker histone H1 to the nucleosome as well as a structural basis of how phosphorylation induces dissocn. of HMGNs from chromatin during mitosis. Importantly, our approach is generally applicable to the study of nucleosome-binding interactions in chromatin.
- 119Zhou, B.-R.; Feng, H.; Kato, H.; Dai, L.; Yang, Y.; Zhou, Y.; Bai, Y. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 19390119Structural insights into the histone H1-nucleosome complexZhou, Bing-Rui; Feng, Hanqiao; Kato, Hidenori; Dai, Liang; Yang, Yuedong; Zhou, Yaoqi; Bai, YawenProceedings of the National Academy of Sciences of the United States of America (2013), 110 (48), 19390-19395,S19390/1-S19390/27CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Linker H1 histones facilitate formation of higher-order chromatin structures and play important roles in various cell functions. Despite several decades of effort, the structural basis of how H1 interacts with the nucleosome remains elusive. Here, we investigated Drosophila H1 in complex with the nucleosome, using soln. NMR spectroscopy and other biophys. methods. We found that the globular domain of H1 bridges the nucleosome core and one 10-base pair linker DNA asym., with its α3 helix facing the nucleosomal DNA near the dyad axis. Two short regions in the C-terminal tail of H1 and the C-terminal tail of one of the two H2A histones are also involved in the formation of the H1-nucleosome complex. Our results lead to a residue-specific structural model for the globular domain of the Drosophila H1 in complex with the nucleosome, which is different from all previous expt.-based models and has implications for chromatin dynamics in vivo.
- 120Rattner, B. P.; Yusufzai, T.; Kadonaga, J. T. Mol. Cell 2009, 34, 620120HMGN proteins act in opposition to ATP-dependent chromatin remodeling factors to restrict nucleosome mobilityRattner, Barbara P.; Yusufzai, Timur; Kadonaga, James T.Molecular Cell (2009), 34 (5), 620-626CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)The high-mobility group N (HMGN) proteins are abundant nonhistone chromosomal proteins that bind specifically to nucleosomes at two high-affinity sites. Here we report that purified recombinant human HMGN1 (HMG14) and HMGN2 (HMG17) potently repress ATP-dependent chromatin remodeling by four different mol. motor proteins. In contrast, mutant HMGN proteins with double Ser-to-Glu mutations in their nucleosome-binding domains are unable to inhibit chromatin remodeling. The HMGN-mediated repression of chromatin remodeling is reversible and dynamic. With the ACF chromatin remodeling factor, HMGN2 does not directly inhibit the ATPase activity but rather appears to reduce the affinity of the factor to chromatin. These findings suggest that HMGN proteins serve as a counterbalance to the action of the many ATP-dependent chromatin remodeling activities in the nucleus.
- 121Lim, J.-H.; West, K. L.; Rubinstein, Y.; Bergel, M.; Postnikov, Y. V.; Bustin, M. EMBO J. 2005, 24, 3038121Chromosomal protein HMGN1 enhances the acetylation of lysine 14 in histone H3Lim, Jae-Hwan; West, Katherine L.; Rubinstein, Yaffa; Bergel, Michael; Postnikov, Yuri V.; Bustin, MichaelEMBO Journal (2005), 24 (17), 3038-3048CODEN: EMJODG; ISSN:0261-4189. (Nature Publishing Group)The acetylation levels of lysine residues in nucleosomes, which are detd. by the opposing activities of histone acetyltransferases (HATs) and deacetylases, play an important role in regulating chromatin-related processes, including transcription. We report that HMGN1, a nucleosomal binding protein that reduces the compaction of the chromatin fiber, increases the levels of acetylation of K14 in H3. The levels of H3K14ac in Hmgn1-/- cells are lower than in Hmgn1+/+ cells. Induced expression of wild-type HMGN1, but not of a mutant that does not bind to chromatin, in Hmgn1-/- cells elevates the levels of H3K14ac. In vivo, HMGN1 elevates the levels of H3K14ac by enhancing the action of HAT. In vitro, HMGN1 enhances the ability of PCAF to acetylate nucleosomal, but not free, H3. Thus, HMGN1 modulates the levels of H3K14ac by binding to chromatin. We suggest that HMGN1, and perhaps similar architectural proteins, modulates the levels of acetylation in chromatin by altering the equil. generated by the opposing enzymic activities that continuously modify and de-modify the histone tails in nucleosomes.
- 122Lim, J.-H.; Catez, F.; Birger, Y.; West, K. L.; Prymakowska-Bosak, M.; Postnikov, Y. V.; Bustin, M. Mol. Cell 2004, 15, 573122Chromosomal protein HMGN1 modulates histone H3 phosphorylationLim, Jae-Hwan; Catez, Frederic; Birger, Yehudit; West, Katherine L.; Prymakowska-Bosak, Marta; Postnikov, Yuri V.; Bustin, MichaelMolecular Cell (2004), 15 (4), 573-584CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)Here we demonstrate that HMGN1, a nuclear protein that binds to nucleosomes and reduces the compaction of the chromatin fiber, modulates histone posttranslational modifications. In Hmgn1-/- cells, loss of HMGN1 elevates the steady-state levels of phospho-S10-H3 and enhances the rate of stress-induced phosphorylation of S10-H3. In vitro, HMGN1 reduces the rate of phospho-S10-H3 by hindering the ability of kinases to modify nucleosomal, but not free, H3. During anisomycin treatment, the phosphorylation of HMGN1 precedes that of H3 and leads to a transient weakening of the binding of HMGN1 to chromatin. We propose that the reduced binding of HMGN1 to nucleosomes, or the absence of the protein, improves access of anisomysin-induced kinases to H3. Thus, the levels of posttranslational modifications in chromatin are modulated by nucleosome binding proteins that alter the ability of enzymic complexes to access and modify their nucleosomal targets.
- 123Postnikov, Y.; Bustin, M. Biochim. Biophys. Acta, Gene Regul. Mech. 2010, 1799, 62123Regulation of chromatin structure and function by HMGN proteinsPostnikov, Yuri; Bustin, MichaelBiochimica et Biophysica Acta, Gene Regulatory Mechanisms (2010), 1799 (1-2), 62-68CODEN: BBAGC6; ISSN:1874-9399. (Elsevier B.V.)A review. High mobility group nucleosome-binding (HMGN) proteins are architectural non-histone chromosomal proteins that bind to nucleosomes and modulate the structure and function of chromatin. The interaction of HMGN proteins with nucleosomes is dynamic and the proteins compete with the linker histone H1 chromatin-binding sites. HMGNs reduce the H1-mediated compaction of the chromatin fiber and facilitate the targeting of regulatory factors to chromatin. They modulate the cellular epigenetic profile, affect gene expression, and impact biol. processes such as development and the cellular response to environmental and hormonal signals. Here, the authors review the role of HMGN in chromatin structure, the link between HMGN proteins and histone modifications, and discuss the consequence of this link on nuclear processes and cellular phenotype.
- 124Prymakowska-Bosak, M.; Misteli, T.; Herrera, J. E.; Shirakawa, H.; Birger, Y.; Garfield, S.; Bustin, M. Mol. Cell. Biol. 2001, 21, 5169124Mitotic phosphorylation prevents the binding of HMGN proteins to chromatinPrymakowska-Bosak, Marta; Misteli, Tom; Herrera, Julio E.; Shirakawa, Hitoshi; Birger, Yehudit; Garfield, Susan; Bustin, MichaelMolecular and Cellular Biology (2001), 21 (15), 5169-5178CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)Condensation of the chromatin fiber and transcriptional inhibition during mitosis is assocd. with the redistribution of many DNA- and chromatin-binding proteins, including members of the high-mobility-group N (HMGN) family. Here we study the mechanism governing the organization of HMGN proteins in mitosis. Using site-specific antibodies and quant. gel anal. with proteins extd. from synchronized HeLa cells, we demonstrate that, during mitosis, the conserved serine residues in the nucleosomal binding domain (NBD) of this protein family are highly and specifically phosphorylated. Nucleosome mobility shift assays with both in vitro-phosphorylated proteins and with point mutants bearing neg. charges in the NBD demonstrate that the neg. charge abolishes the ability of the proteins to bind to nucleosomes. Fluorescence loss of photobleaching demonstrates that, in living cells, the neg. charge in the NBD increases the intranuclear mobility of the protein and significantly decreases the relative time that it is bound to chromatin. Expression of wild-type and mutant proteins in HmgN1-/- cells indicates that the neg. charged protein is not bound to chromosomes. We conclude that during mitosis the NBD of HMGN proteins is highly phosphorylated and that this modification regulates the interaction of the proteins with chromatin.
- 125Mattiroli, F.; Uckelmann, M.; Sahtoe, D. D.; van Dijk, W. J.; Sixma, T. K. Nat. Commun. 2014, 5, 3291125The nucleosome acidic patch plays a critical role in RNF168-dependent ubiquitination of histone H2AMattiroli Francesca; Uckelmann Michael; Sahtoe Danny D; van Dijk Willem J; Sixma Titia KNature communications (2014), 5 (), 3291 ISSN:.During DNA damage response, the RING E3 ligase RNF168 ubiquitinates nucleosomal H2A at K13-15. Here we show that the ubiquitination reaction is regulated by its substrate. We define a region on the RING domain important for target recognition and identify the H2A/H2B dimer as the minimal substrate to confer lysine specificity to the RNF168 reaction. Importantly, we find an active role for the substrate in the reaction. H2A/H2B dimers and nucleosomes enhance the E3-mediated discharge of ubiquitin from the E2 and redirect the reaction towards the relevant target, in a process that depends on an intact acidic patch. This active contribution of a region distal from the target lysine provides regulation of the specific K13-15 ubiquitination reaction during the complex signalling process at DNA damage sites.
- 126Carriere, V.; Roussel, L.; Ortega, N.; Lacorre, D.-A.; Americh, L.; Aguilar, L.; Bouche, G.; Girard, J.-P. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 282126IL-33, the IL-1-like cytokine ligand for ST2 receptor, is a chromatin-associated nucleus factor in vivoCarriere, Virginie; Roussel, Lucie; Ortega, Nathalie; Lacorre, Delphine-Armelle; Americh, Laure; Aguilar, Luc; Bouche, Gerard; Girard, Jean-PhilippeProceedings of the National Academy of Sciences of the United States of America (2007), 104 (1), 282-287CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Recent studies indicate that IL-1α functions intracellularly in path ways independent of its cell surface receptors by translocating to the nucleus and regulating transcription. Similarly, the chromatin-assocd. protein HMGB1 acts as both a nuclear factor and a secreted proinflammatory cytokine. Here, we show that IL-33, an IL-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-assocd. cytokines, is an endothelium-derived, chromatin-assocd. nuclear factor with transcriptional repressor properties. We found that IL-33 is identical to NF-HEV, a nuclear factor preferentially expressed in high endothelial venules (HEV), that we previously characterized. Accordingly, in situ hybridization demonstrated that endothelial cells constitute a major source of IL-33 mRNA in chronically inflamed tissues from patients with rheumatoid arthritis and Crohn's disease. Immunostaining with three distinct antisera, directed against the N-terminal part and IL-1-like C-terminal domain, revealed that IL-33 is a heterochromatin-assocd. nuclear factor in HEV endothelial cells in vivo. Assocn. of IL-33 with heterochromatin was also obsd. in human and mouse cells under living conditions. In addn., colocalization of IL-33 with mitotic chromatin was noted. Nuclear localization, heterochromatin-assocn., and targeting to mitotic chromosomes were all found to be mediated by an evolutionarily conserved homeodomain-like helix-turn-helix motif within the IL-33 N-terminal part. Finally, IL-33 was found to possess transcriptional repressor properties, assocd. with the homeodomain-like helix-turn-helix motif. Together, these data suggest that, similarly to IL1α and HMGB1, IL-33 is a dual function protein that may function as both a proinflammatory cytokine and an intracellular nuclear factor with transcriptional regulatory properties.
- 127Kalashnikova, A. A.; Porter-Goff, M. E.; Muthurajan, U. M.; Luger, K.; Hansen, J. C. J. R. Soc., Interface 2013, 10, 20121022127The role of the nucleosome acidic patch in modulating higher order chromatin structureKalashnikova, Anna A.; Porter-Goff, Mary E.; Muthurajan, Uma M.; Luger, Karolin; Hansen, Jeffrey C.Journal of the Royal Society, Interface (2013), 10 (82), 20121022/1-20121022/9CODEN: JRSICU; ISSN:1742-5689. (Royal Society)A review. Higher order folding of chromatin fiber is mediated by interactions of the histone H4 N-terminal tail domains with neighboring nucleosomes. Mechanistically, the H4 tails of one nucleosome bind to the acidic patch region on the surface of adjacent nucleosomes, causing fiber compaction. The functionality of the chromatin fiber can be modified by proteins that interact with the nucleosome. The co-structures of five different proteins with the nucleosome (LANA, IL-33, RCC1, Sir3 and HMGN2) recently have been examd. by exptl. and computational studies. Interestingly, each of these proteins displays steric, ionic and hydrogen bond complementarity with the acidic patch, and therefore will compete with each other for binding to the nucleosome. We first review the mol. details of each interface, focusing on the key non-covalent interactions that stabilize the protein-acidic patch interactions. We then propose a model in which binding of proteins to the nucleosome disrupts interaction of the H4 tail domains with the acidic patch, preventing the intrinsic chromatin folding pathway and leading to assembly of alternative higher order chromatin structures with unique biol. functions.
- 128Clapier, C. R.; Cairns, B. R. Annu. Rev. Biochem. 2009, 78, 273128The biology of chromatin remodeling complexesClapier, Cedric R.; Cairns, Bradley R.Annual Review of Biochemistry (2009), 78 (), 273-304CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)A review. The packaging of chromosomal DNA by nucleosomes condenses and organizes the genome, but occludes many regulatory DNA elements. However, this constraint also allows nucleosomes and other chromatin components to actively participate in the regulation of transcription, chromosome segregation, DNA replication, and DNA repair. To enable dynamic access to packaged DNA and to tailor nucleosome compn. in chromosomal regions, cells have evolved a set of specialized chromatin remodeling complexes (remodelers). Remodelers use the energy of ATP hydrolysis to move, destabilize, eject, or restructure nucleosomes. Here, we address many aspects of remodeler biol.: their targeting, mechanism, regulation, shared and unique properties, and specialization for particular biol. processes. We also address roles for remodelers in development, cancer, and human syndromes.
- 129Chaban, Y.; Ezeokonkwo, C.; Chung, W.-H.; Zhang, F.; Kornberg, R. D.; Maier-Davis, B.; Lorch, Y.; Asturias, F. J. Nat. Struct. Mol. Biol. 2008, 15, 1272129Structure of a RSC-nucleosome complex and insights into chromatin remodelingChaban, Yuriy; Ezeokonkwo, Chukwudi; Chung, Wen-Hsiang; Zhang, Fan; Kornberg, Roger D.; Maier-Davis, Barbara; Lorch, Yahli; Asturias, Francisco J.Nature Structural & Molecular Biology (2008), 15 (12), 1272-1277CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)ATP-dependent chromatin-remodeling complexes, such as RSC, can reposition, evict, or restructure nucleosomes. A structure of a RSC-nucleosome complex with a nucleosome detd. by cryo-EM shows the nucleosome bound in a central RSC cavity. Extensive interaction of RSC with histones and DNA seems to destabilize the nucleosome and lead to an overall ATP-independent rearrangement of its structure. Nucleosomal DNA appears disordered and largely free to bulge out into soln. as required for remodeling, but the structure of the RSC-nucleosome complex indicates that RSC is unlikely to displace the octamer from the nucleosome to which it is bound. Consideration of the RSC-nucleosome structure and published biochem. information suggests that ATP-dependent DNA translocation by RSC may result in the eviction of histone octamers from adjacent nucleosomes.
- 130Asturias, F. J.; Chung, W.-H.; Kornberg, R. D.; Lorch, Y. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 13477130Structural analysis of the RSC chromatin-remodeling complexAsturias, Francisco J.; Chung, Wen-Hsiang; Kornberg, Roger D.; Lorch, YahliProceedings of the National Academy of Sciences of the United States of America (2002), 99 (21), 13477-13480CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Electron microscopy of the RSC chromatin-remodeling complex reveals a ring of protein densities around a central cavity. The size and shape of the cavity correspond closely to those of a nucleosome. Results of nuclease protection anal. are consistent with nucleosome binding in the cavity. Such binding could explain the ability of RSC to expose nucleosomal DNA in the presence of ATP without loss of assocd. histones.
- 131Saha, A.; Wittmeyer, J.; Cairns, B. R. Nat. Struct. Mol. Biol. 2005, 12, 747131Chromatin remodeling through directional DNA translocation from an internal nucleosomal siteSaha, Anjanabha; Wittmeyer, Jacqueline; Cairns, Bradley R.Nature Structural & Molecular Biology (2005), 12 (9), 747-755CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)The RSC chromatin remodeler contains Sth1, an ATP-dependent DNA translocase. On DNA substrates, RSC/Sth1 tracks along one strand of the duplex with a 3' → 5' polarity and a tracking requirement of one base, properties that may enable directional DNA translocation on nucleosomes. The binding of RSC or Sth1 elicits a DNase I-hypersensitive site approx. two DNA turns from the nucleosomal dyad, and the binding of Sth1 requires intact DNA at this location. Results with various nucleosome substrates suggest that RSC/Sth1 remains at a fixed position on the histone octamer and that Sth1 conducts directional DNA translocation from a location about two turns from the nucleosomal dyad, drawing in DNA from one side of the nucleosome and pumping it toward the other. These studies suggest that nucleosome mobilization involves directional DNA translocation initiating from a fixed internal site on the nucleosome.
- 132Leschziner, A. E.; Saha, A.; Wittmeyer, J.; Zhang, Y.; Bustamante, C.; Cairns, B. R.; Nogales, E. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 4913132Conformational flexibility in the chromatin remodeler RSC observed by electron microscopy and the orthogonal tilt reconstruction methodLeschziner, Andres E.; Saha, Anjanabha; Wittmeyer, Jacqueline; Zhang, Yongli; Bustamante, Carlos; Cairns, Bradley R.; Nogales, EvaProceedings of the National Academy of Sciences of the United States of America (2007), 104 (12), 4913-4918CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Chromatin remodeling complexes (remodelers) are large, multi-subunit macromol. assemblies that use ATP hydrolysis to alter the structure and positioning of nucleosomes. The mechanisms proposed for remodeler action on nucleosomes are diverse, and require structural evaluation and insights. Previous reconstructions of remodelers using electron microscopy revealed interesting features, but also significant discrepancies, prompting new approaches. Here, we use the orthogonal tilt reconstruction method, which is well suited for heterogeneous samples, to provide a reconstruction of the yeast RSC (_remodel the _structure of _chromatin) complex. Two interesting features are revealed: first, we observe a deep central cavity within RSC, displaying a remarkable surface complementarity for the nucleosome. Second, we are able to visualize two distinct RSC conformers, revealing a major conformational change in a large protein "arm," which may shift to further envelop the nucleosome. We present a model of the RSC-nucleosome complex that rationalizes the single mol. results obtained by using optical tweezers and also discuss the mechanistic implications of our structures.
- 133Racki, L. R.; Yang, J. G.; Naber, N.; Partensky, P. D.; Acevedo, A.; Purcell, T. J.; Cooke, R.; Cheng, Y.; Narlikar, G. J. Nature 2009, 462, 1016133The chromatin remodeller ACF acts as a dimeric motor to space nucleosomesRacki, Lisa R.; Yang, Janet G.; Naber, Nariman; Partensky, Peretz D.; Acevedo, Ashley; Purcell, Thomas J.; Cooke, Roger; Cheng, Yifan; Narlikar, Geeta J.Nature (London, United Kingdom) (2009), 462 (7276), 1016-1021CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Evenly spaced nucleosomes directly correlate with condensed chromatin and gene silencing. The ATP-dependent chromatin assembly factor (ACF) forms such structures in vitro and is required for silencing in vivo. ACF generates and maintains nucleosome spacing by constantly moving a nucleosome towards the longer flanking DNA faster than the shorter flanking DNA. How the enzyme rapidly moves back and forth between both sides of a nucleosome to accomplish bidirectional movement is unknown. Here, we show that nucleosome movement depends cooperatively on two ACF mols., indicating that ACF functions as a dimer of ATPases. Further, the nucleotide state dets. whether the dimer closely engages one or both sides of the nucleosome. Three-dimensional reconstruction by single-particle electron microscopy of the ATPase-nucleosome complex in an activated ATP state reveals a dimer architecture in which the two ATPases face each other. Our results indicate a model in which the two ATPases work in a coordinated manner, taking turns to engage either side of a nucleosome, thereby allowing processive bidirectional movement. This novel dimeric motor mechanism differs from that of dimeric motors such as kinesin and dimeric helicases that processively translocate unidirectionally and reflects the unique challenges faced by motors that move nucleosomes.
- 134Yamada, K.; Frouws, T. D.; Angst, B.; Fitzgerald, D. J.; DeLuca, C.; Schimmele, K.; Sargent, D. F.; Richmond, T. J. Nature 2011, 472, 448134Structure and mechanism of the chromatin remodelling factor ISW1aYamada, Kazuhiro; Frouws, Timothy D.; Angst, Brigitte; Fitzgerald, Daniel J.; DeLuca, Carl; Schimmele, Kyoko; Sargent, David F.; Richmond, Timothy J.Nature (London, United Kingdom) (2011), 472 (7344), 448-453CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Site-specific recognition of DNA in eukaryotic organisms depends on the arrangement of nucleosomes in chromatin. In the yeast Saccharomyces cerevisiae, ISW1a and related chromatin remodeling factors are implicated in establishing the nucleosome repeat during replication and altering nucleosome position to affect gene activity. Here we have solved the crystal structures of S. cerevisiae ISW1a lacking its ATPase domain both alone and with DNA bound at resolns. of 3.25 Å and 3.60 Å, resp., and we have visualized two different nucleosome-contg. remodeling complexes using cryo-electron microscopy. The composite x-ray and electron microscopy structures combined with site-directed photocrosslinking analyses of these complexes suggest that ISW1a uses a dinucleosome substrate for chromatin remodeling. Results from a remodeling assay corroborate the dinucleosome model. We show how a chromatin remodeling factor could set the spacing between two adjacent nucleosomes acting as a ' protein ruler'.
- 135Saravanan, M.; Wuerges, J.; Bose, D.; McCormack, E. A.; Cook, N. J.; Zhang, X.; Wigley, D. B. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 20883135Interactions between the nucleosome histone core and Arp8 in the INO80 chromatin remodeling complexSaravanan, Matheshwaran; Wuerges, Jochen; Bose, Daniel; McCormack, Elizabeth A.; Cook, Nicola J.; Zhang, Xiaodong; Wigley, Dale B.Proceedings of the National Academy of Sciences of the United States of America (2012), 109 (51), 20883-20888, S20883/1-S20883/29CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Actin-related protein Arp8 is a component of the INO80 chromatin remodeling complex. Yeast Arp8 (yArp8) comprises two domains: a 25-KDa N-terminal domain, found only in yeast, and a 75-KDa C-terminal domain (yArp8CTD) that contains the actin fold and is conserved across other species. The crystal structure shows that yArp8CTD contains three insertions within the actin core. Using a combination of biochem. and EM, we show that Arp8 forms a complex with nucleosomes, and that the principal interactions are via the H3 and H4 histones, mediated through one of the yArp8 insertions. We show that recombinant yArp8 exists in monomeric and dimeric states, but the dimer is the biol. relevant form required for stable interactions with histones that exploits the twofold symmetry of the nucleosome core. Taken together, these data provide unique insight into the stoichiometry, architecture, and mol. interactions between components of the INO80 remodeling complex and nucleosomes, providing a first step toward building up the structure of the complex.
- 136Tosi, A.; Haas, C.; Herzog, F.; Gilmozzi, A.; Berninghausen, O.; Ungewickell, C.; Gerhold, C. B.; Lakomek, K.; Aebersold, R.; Beckmann, R.; Hopfner, K.-P. Cell 2013, 154, 1207136Structure and Subunit Topology of the INO80 Chromatin Remodeler and Its Nucleosome ComplexTosi, Alessandro; Haas, Caroline; Herzog, Franz; Gilmozzi, Andrea; Berninghausen, Otto; Ungewickell, Charlotte; Gerhold, Christian B.; Lakomek, Kristina; Aebersold, Ruedi; Beckmann, Roland; Hopfner, Karl-PeterCell (Cambridge, MA, United States) (2013), 154 (6), 1207-1219CODEN: CELLB5; ISSN:0092-8674. (Cell Press)INO80/SWR1 family chromatin remodelers are complexes composed of >15 subunits and mol. masses exceeding 1 MDa. Their important role in transcription and genome maintenance is exchanging the histone variants H2A and H2A. Z. We report the architecture of S. cerevisiae INO80 using an integrative approach of electron microscopy, crosslinking and mass spectrometry. INO80 has an embryo-shaped head-neck-body-foot architecture and shows dynamic open and closed conformations. We can assign an Rvb1/Rvb2 heterododecamer to the head in close contact with the Ino80 Snf2 domain, Ies2, and the Arp5 module at the neck. The high-affinity nucleosome-binding Nhp10 module localizes to the body, whereas the module that contains actin, Arp4, and Arp8 maps to the foot. Structural and biochem. analyses indicate that the nucleosome is bound at the concave surface near the neck, flanked by the Rvb1/2 and Arp8 modules. Our anal. establishes a structural and functional framework for this family of large remodelers.
- 137Chittuluru, J. R.; Chaban, Y.; Monnet-Saksouk, J.; Carrozza, M. J.; Sapountzi, V.; Selleck, W.; Huang, J.; Utley, R. T.; Cramet, M.; Allard, S.; Cai, G.; Workman, J. L.; Fried, M. G.; Tan, S.; Côté, J.; Asturias, F. J. Nat. Struct. Mol. Biol. 2011, 18, 1196137Structure and nucleosome interaction of the yeast NuA4 and Piccolo-NuA4 histone acetyltransferase complexesChittuluru, Johnathan R.; Chaban, Yuriy; Monnet-Saksouk, Julie; Carrozza, Michael J.; Sapountzi, Vasileia; Selleck, William; Huang, Jiehuan; Utley, Rhea T.; Cramet, Myriam; Allard, Stephane; Cai, Gang; Workman, Jerry L.; Fried, Michael G.; Tan, Song; Cote, Jacques; Asturias, Francisco J.Nature Structural & Molecular Biology (2011), 18 (11), 1196-1203CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)We have used EM and biochem. to characterize the structure of NuA4, an essential yeast histone acetyltransferase (HAT) complex conserved throughout eukaryotes, and we have detd. the interaction of NuA4 with the nucleosome core particle (NCP). The ATM-related Tra1 subunit, which is shared with the SAGA coactivator complex, forms a large domain joined to a second region that accommodates the catalytic subcomplex Piccolo and other NuA4 subunits. EM anal. of a NuA4-NCP complex shows the NCP bound at the periphery of NuA4. EM characterization of Piccolo and Piccolo-NCP provided further information about subunit organization and confirmed that histone acetylation requires minimal contact with the NCP. A small conserved region at the N terminus of Piccolo subunit enhancer of Polycomb-like 1 (Epl1) is essential for NCP interaction, whereas the subunit yeast homolog of mammalian Ing1 2 (Yng2) apparently positions Piccolo for efficient acetylation of histone H4 or histone H2A tails. Taken together, these results provide an understanding of the NuA4 subunit organization and the NuA4-NCP interactions.
- 138Boudreault, A. A.; Cronier, D.; Selleck, W.; Lacoste, N.; Utley, R. T.; Allard, S.; Savard, J.; Lane, W. S.; Tan, S.; Côté, J. Genes Dev. 2003, 17, 1415138Yeast Enhancer of Polycomb defines global Esa1-dependent acetylation of chromatinBoudreault, Alexandre A.; Cronier, Dominique; Selleck, William; Lacoste, Nicolas; Utley, Rhea T.; Allard, Stephane; Savard, Julie; Lane, William S.; Tan, Song; Cote, JacquesGenes & Development (2003), 17 (11), 1415-1428CODEN: GEDEEP; ISSN:0890-9369. (Cold Spring Harbor Laboratory Press)Drosophila Enhancer of Polycomb, E(Pc), is a suppressor of position-effect variegation and an enhancer of both Polycomb and trithorax mutations. A homologous yeast protein, Epl1, is a subunit of the NuA4 histone acetyltransferase complex. Epl1 depletion causes cells to accumulate in G2/M and global loss of acetylated histones H4 and H2A. In relation to the Drosophila protein, mutation of Epl1 suppresses gene silencing by telomere position effect. Epl1 protein is found in the NuA4 complex and a novel highly active smaller complex named Piccolo NuA4 (picNuA4). The picNuA4 complex contains Esa1, Epl1, and Yng2 as subunits and strongly prefers chromatin over free histones as substrate. Epl1 conserved N-terminal domain bridges Esa1 and Yng2 together, stimulating Esa1 catalytic activity and enabling acetylation of chromatin substrates. A recombinant picNuA4 complex shows characteristics similar to the native complex, including strong chromatin preference. Cells expressing only the N-terminal half of Epl1 lack NuA4 HAT activity, but possess picNuA4 complex and activity. These results indicate that the essential aspect of Esa1 and Epl1 resides in picNuA4 function. We propose that picNuA4 represents a nontargeted histone H4/H2A acetyltransferase activity responsible for global acetylation, whereas the NuA4 complex is recruited to specific genomic loci to perturb locally the dynamic acetylation/deacetylation equil.
- 139Huang, J.; Tan, S. Mol. Cell. Biol. 2013, 33, 159139Piccolo NuA4-catalyzed acetylation of nucleosomal histones: critical roles of an Esa1 tudor/chromo barrel loop and an Epl1 enhancer of polycomb A (EPcA) basic regionHuang, Jiehuan; Tan, SongMolecular and Cellular Biology (2013), 33 (1), 159-169CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)Piccolo NuA4 is an essential yeast histone acetyltransferase (HAT) complex that targets histones H4 and H2A in nucleosome substrates. While Piccolo NuA4's catalytic subunit Esa1 alone is unable to acetylate nucleosomal histones, its accessory subunits, Yng2 and Epl1, enable Esa1 to bind to and to act on nucleosomes. We previously detd. that the Tudor domain of Esa1 and the EPcA homol. domain of Epl1 play crit. roles in Piccolo NuA4's ability to act on the nucleosome. In this work, we pinpoint a loop within the Esa1 Tudor domain and a short basic region at the N terminus of the Epl1 EPcA domain as necessary for this nucleosomal HAT activity. We also show that this Esa1 Tudor domain loop region is positioned close to nucleosomal DNA and that the Epl1 EPcA basic region is in proximity to the N-terminal histone H2A tail, the globular region of histone H4, and also to nucleosomal DNA when Piccolo NuA4 interacts with the nucleosome. Since neither region identified is required for Piccolo NuA4 to bind to nucleosomes and yet both are needed to acetylate nucleosomes, these regions may function after the enzyme binds nucleosomes to disengage substrate histone tails from nucleosomal DNA.
- 140Harshman, S. W.; Young, N. L.; Parthun, M. R.; Freitas, M. A. Nucleic Acids Res. 2013, 41, 9593140H1 histones: current perspectives and challengesHarshman, Sean W.; Young, Nicolas L.; Parthun, Mark R.; Freitas, Michael A.Nucleic Acids Research (2013), 41 (21), 9593-9609CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)A review. H1 and related linker histones are important both for maintenance of higher-order chromatin structure and for the regulation of gene expression. The biol. of the linker histones is complex, as they are evolutionarily variable, exist in multiple isoforms and undergo a large variety of posttranslational modifications in their long, unstructured, NH2- and C-terminal tails. We review recent progress in understanding the structure, genetics and posttranslational modifications of linker histones, with an emphasis on the dynamic interactions of these proteins with DNA and transcriptional regulators. We also discuss various exptl. challenges to the study of H1 and related proteins, including limitations of immunol. reagents and practical difficulties in the anal. of posttranslational modifications by mass spectrometry.
- 141Allan, J.; Hartman, P. G.; Crane-Robinson, C.; Aviles, F. X. Nature 1980, 288, 675141The structure of histone H1 and its location in chromatinAllan, J.; Hartman, P. G.; Crane-Robinson, C.; Aviles, F. X.Nature (London, United Kingdom) (1980), 288 (5792), 675-9CODEN: NATUAS; ISSN:0028-0836.The lysine-rich histones were shown to be a unified family of proteins on the basis of their primary structure. Each histone had an amino acid chain which fell into 3 distinct domains. Only the central domain (∼80 residues) was in a folded conformation. It was protected from trypsin digestion in chromatin and corresponded to the segment of highest sequence conservation. Without the flanking domains, the central domain was able to close 2 full turns of DNA in the nucleosome and could thus locate the H1 mol. The central domain alone could protect the extra ∼20 base pairs of DNA present in the chromatosome above that in the core particle. Full compaction of chromatin by H1 may require the intact mol.
- 142Syed, S. H.; Goutte-Gattat, D.; Becker, N.; Meyer, S.; Shukla, M. S.; Hayes, J. J.; Everaers, R.; Angelov, D.; Bednar, J.; Dimitrov, S. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 9620142Single-base resolution mapping of H1-nucleosome interactions and 3D organization of the nucleosomeSyed, Sajad Hussain; Goutte-Gattat, Damien; Becker, Nils; Meyer, Sam; Shukla, Manu Shubhdarshan; Hayes, Jeffrey J.; Everaers, Ralf; Angelov, Dimitar; Bednar, Jan; Dimitrov, StefanProceedings of the National Academy of Sciences of the United States of America (2010), 107 (21), 9620-9625, S9620/1-S9620/10CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Despite the key role of the linker histone H1 in chromatin structure and dynamics, its location and interactions with nucleosomal DNA have not been elucidated. The authors used a combination of electron cryomicroscopy, hydroxyl radical footprinting, and nanoscale modeling to analyze the structure of precisely positioned mono-, di-, and trinucleosomes contg. physiol. assembled full-length histone H1 or truncated mutants of this protein. Single-base resoln. ·OH footprinting shows that the globular domain of histone H1 (GH1) interacts with the DNA minor groove located at the center of the nucleosome and contacts a 10-bp region of DNA localized sym. with respect to the nucleosomal dyad. In addn., GH1 interacts with and organizes about one helical turn of DNA in each linker region of the nucleosome. Also a seven amino acid residue region (121-127) in the C-terminus of histone H1 was required for the formation of the stem structure of the linker DNA. A mol. model on the basis of these data and coarse-grain DNA mechanics provides novel insights on how the different domains of H1 interact with the nucleosome and predicts a specific H1-mediated stem structure within linker DNA.
- 143Fan, L.; Roberts, V. A. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 8384143Complex of linker histone H5 with the nucleosome and its implications for chromatin packingFan, Li; Roberts, Victoria A.Proceedings of the National Academy of Sciences of the United States of America (2006), 103 (22), 8384-8389CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Linker histones are essential for chromatin filament formation, and they play key roles in the regulation of gene expression. Despite the detn. of structures of the nucleosome and linker histones, the location of the linker histone on the nucleosome is still a matter of debate. Here we show by computational docking that the globular domain of linker histone variant H5 (GH5) has three distinct DNA-binding sites, through which GH5 contacts the DNA at the nucleosome dyad and the linker DNA strands entering and exiting the nucleosome. Our results explain the extensive mutagenesis and crosslinking data showing that side chains spread throughout the GH5 surface interact with nucleosomal DNA. The nucleosome DNA contacts pos. charged side chains that are conserved within the linker histone family, indicating that our model extends to linker histone-nucleosome interactions in general. Furthermore, our model provides a structural mechanism for formation of a dinucleosome complex specific to the linker histone H5, explaining its efficiency in chromatin compaction and transcription regulation. Thus, this work provides a basis for understanding how structural differences within the linker histone family result in functional differences, which in turn are important for gene regulation.
- 144Meyer, S.; Becker, N. B.; Syed, S. H.; Goutte-Gattat, D.; Shukla, M. S.; Hayes, J. J.; Angelov, D.; Bednar, J.; Dimitrov, S.; Everaers, R. Nucleic Acids Res. 2011, 39, 9139144From crystal and NMR structures, footprints and cryo-electron-micrographs to large and soft structures: nanoscale modeling of the nucleosomal stemMeyer, Sam; Becker, Nils B.; Syed, Sajad Hussain; Goutte-Gattat, Damien; Shukla, Manu Shubhdarshan; Hayes, Jeffrey J.; Angelov, Dimitar; Bednar, Jan; Dimitrov, Stefan; Everaers, RalfNucleic Acids Research (2011), 39 (21), 9139-9154CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)The interaction of histone H1 with linker DNA results in the formation of the nucleosomal stem structure, with considerable influence on chromatin organization. In a recent paper, we published results of biochem. footprinting and cryo-electron-micrographs of reconstituted mono-, di- and tri-nucleosomes, for H1 variants with different lengths of the cationic C-terminus. Here, we present a detailed account of the anal. of the exptl. data on the stem structure including thermal fluctuations of the stem structure. By combining (i) crystal and NMR structures of the nucleosome core particle and H1, (ii) the known nano-scale structure and elasticity of DNA, (iii) footprinting information on the location of protected sites on the DNA backbone and (iv) cryo-electron micrographs of reconstituted tri-nucleosomes, we arrive at a description of a polymorphic, hierarchically organized stem with a typical length of 20±2 base pairs. A comparison to linker conformations inferred for poly-601 fibers with different linker lengths suggests that intra-stem interactions stabilize and facilitate the formation of dense chromatin fibers.
- 145Zhou, Y. B.; Gerchman, S. E.; Ramakrishnan, V.; Travers, A.; Muyldermans, S. Nature 1998, 395, 402145Position and orientation of the globular domain of linker histone H5 on the nucleosomeZhou, Y.-B.; Gerchman, Sue Ellen; Ramakrishnan, V.; Travers, Andrew; Muyldermans, SergeNature (London) (1998), 395 (6700), 402-405CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)It is essential to identify the exact location of the linker histone within nucleosomes, the fundamental packing units of chromatin, in order to understand how condensed, transcriptionally inactive chromatin forms. Here, using a site-specific protein-DNA photocrosslinking method1, we map the binding site and the orientation of the globular domain of linker histone H5 on mixed-sequence chicken nucleosomes. We show, in contrast to an earlier model2, that the globular domain forms a bridge between one terminus of chromatosomal DNA and the DNA in the vicinity of the dyad axis of sym. of the core particle. Helix III of the globular domain binds in the major groove of the first helical turn of the chromatosomal DNA, whereas the secondary DNA-binding site on the opposite face of the globular domain of histone H5 makes contact with the nucleosomal DNA close to its midpoint. We also infer that helix I and helix II of the globular domain of histone H5 probably face, resp., the solvent and the nucleosome. This location places the basic carboxy-terminal region of the globular domain in a position from which it could simultaneously bind the nucleosome-linking DNA strands that exit and enter the nucleosome.
- 146Bharath, M. M. S.; Chandra, N. R.; Rao, M. R. S. Nucleic Acids Res. 2003, 31, 4264146Molecular modeling of the chromatosome particleBharath, M. M. Srinivas; Chandra, Nagasuma R.; Rao, M. R. S.Nucleic Acids Research (2003), 31 (14), 4264-4274CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)In an effort to understand the role of the linker histone in chromatin folding, its structure and location in the nucleosome has been studied by mol. modeling methods. The structure of the globular domain of the rat histone H1d, a highly conserved part of the linker histone, built by homol. modeling methods, revealed a three-helical bundle fold that could be described as a helix-turn-helix variant with its characteristic properties of binding to DNA at the major groove. Using the information of its preferential binding to four-way Holliday junction (HJ) DNA, a model of the domain complexed to HJ was built, which was subsequently used to position the globular domain onto the nucleosome. The model revealed that the primary binding site of the domain interacts with the extra 20 bp of DNA of the entering duplex at the major groove while the secondary binding site interacts with the minor groove of the central gyre of the DNA superhelix of the nucleosomal core. The positioning of the globular domain served as an anchor to locate the C-terminal domain onto the nucleosome to obtain the structure of the chromatosome particle. The resulting structure had a stem-like appearance, resembling that obsd. by electron microscopic studies. The C-terminal domain which adopts a high mobility group (HMG)-box-like fold, has the ability to bend DNA, causing DNA condensation or compaction. It was obsd. that the three S/TPKK motifs in the C-terminal domain interact with the exiting duplex, thus defining the path of linker DNA in the chromatin fiber. This study has provided an insight into the probable individual roles of globular and the C-terminal domains of histone H1 in chromatin organization.
- 147Wong, J.; Li, Q.; Levi, B. Z.; Shi, Y. B.; Wolffe, A. P. EMBO J. 1997, 16, 7130147Structural and functional features of a specific nucleosome containing a recognition element for the thyroid hormone receptorWong, Jiemin; Li, Qiao; Levi, Ben-Zion; Shi, Yun-Bo; Wolffe, Alan P.EMBO Journal (1997), 16 (23), 7130-7145CODEN: EMJODG; ISSN:0261-4189. (Oxford University Press)The Xenopus thyroid hormone receptor βA (TRβA) gene contains an important thyroid hormone response element (TRE) that is assembled into a positioned nucleosome. We det. the translational position of the nucleosome contg. the TRE and the rotational positioning of the double helix with respect to the histone surface. Histone H1 is incorporated into the nucleosome leading to an asym. protection to micrococcal nuclease cleavage of linker DNA relative to the nucleosome core. Histone H1 assocn. is without significant consequence for the binding of the heterodimer of thyroid hormone receptor and 9-cis retinoic acid receptor (TR/RXR) to nucleosomal DNA in vitro, or for the regulation of TRβA gene transcription following microinjection into the oocyte nucleus. Small alterations of 3 and 6 bp in the translational positioning of the TRE in chromatin are also without effect on the transcriptional activity of the TRβA gene, whereas a small change in the rotational position of the TRE (3 bp) relative to the histone surface significantly reduces the binding of TR/RXR to the nucleosome and decreases transcriptional activation directed by TR/RXR. Our results indicate that the specific architecture of the nucleosome contg. the TRE may have regulatory significance for expression of the TRβA gene.
- 148Pachov, G. V.; Gabdoulline, R. R.; Wade, R. C. Nucleic Acids Res. 2011, 39, 5255148On the structure and dynamics of the complex of the nucleosome and the linker histonePachov, Georgi V.; Gabdoulline, Razif R.; Wade, Rebecca C.Nucleic Acids Research (2011), 39 (12), 5255-5263CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Several different models of the linker histone (LH)-nucleosome complex have been proposed, but none of them has unambiguously revealed the position and binding sites of the LH on the nucleosome. Using Brownian dynamics-based docking together with normal mode anal. of the nucleosome to account for the flexibility of two flanking 10 bp long linker DNAs (L-DNA), we identified binding modes of the H5-LH globular domain (GH5) to the nucleosome. For a wide range of nucleosomal conformations with the L-DNA ends less than 65 Å apart, one dominant binding mode was identified for GH5 and found to be consistent with fluorescence recovery after photobleaching (FRAP) expts. GH5 binds asym. with respect to the nucleosomal dyad axis, fitting between the nucleosomal DNA and one of the L-DNAs. For greater distances between L-DNA ends, docking of GH5 to the L-DNA that is more restrained and less open becomes favored. These results suggest a selection mechanism by which GH5 preferentially binds one of the L-DNAs and thereby affects DNA dynamics and accessibility and contributes to formation of a particular chromatin fiber structure. The two binding modes identified would, resp., favor a tight zigzag chromatin structure or a loose solenoid chromatin fiber.
- 149An, W.; Leuba, S. H.; van Holde, K.; Zlatanova, J. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3396149Linker histone protects linker DNA on only one side of the core particle and in a sequence-dependent mannerAn, Woojin; Leuba, Sanford H.; Van Holde, Kensal; Zlatanova, JordankaProceedings of the National Academy of Sciences of the United States of America (1998), 95 (7), 3396-3401CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The protection against micrococcal nuclease digestion afforded to chromatosomal DNA by the presence of a linker histone (H1°) has been quant. measured in two reconstituted systems. We have used chromatosomes reconstituted at two distinct positions on a DNA fragment contg. the 5S rRNA gene from Lytechinus variegatus and at a specific position on a sequence contg. Gal4- and USF-binding sites. In all cases, we find asym. protection, with ≈20 bp protected on one side of the core particle and no protection on the other. We demonstrated through crosslinking expts. that the result is not due to any sliding of the histone core caused by either linker histone addn. or micrococcal nuclease cleavage. Because the core particle is itself a sym. object, the preferred asym. location of a linker histone must be dictated by unknown elements in the DNA sequence.
- 150Vogler, C.; Huber, C.; Waldmann, T.; Ettig, R.; Braun, L.; Izzo, A.; Daujat, S.; Chassignet, I.; Lopez-Contreras, A. J.; Fernandez-Capetillo, O.; Dundr, M.; Rippe, K.; Längst, G.; Schneider, R. PLoS Genet. 2010, 6, e1001234There is no corresponding record for this reference.
- 151Thakar, A.; Gupta, P.; Ishibashi, T.; Finn, R.; Silva-Moreno, B.; Uchiyama, S.; Fukui, K.; Tomschik, M.; Ausio, J.; Zlatanova, J. Biochemistry 2009, 48, 10852151H2A.Z and H3.3 Histone Variants Affect Nucleosome Structure: Biochemical and Biophysical StudiesThakar, Amit; Gupta, Pooja; Ishibashi, Toyotaka; Finn, Ron; Silva-Moreno, Begonia; Uchiyama, Susumu; Fukui, Kiichi; Tomschik, Miroslav; Ausio, Juan; Zlatanova, JordankaBiochemistry (2009), 48 (46), 10852-10857CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Histone variants play important roles in regulation of chromatin structure and function. To understand the structural role played by histone variants H2A.Z and H3.3, both of which are implicated in transcription regulation, we conducted extensive biochem. and biophys. anal. on mononucleosomes reconstituted from either random-sequence DNA derived from native nucleosomes or a defined DNA nucleosome positioning sequence and recombinant human histones. Using established electrophoretic and sedimentation anal. methods, we compared the properties of nucleosomes contg. canonical histones and histone variants H2A.Z and H3.3 (in isolation or in combination). We find only subtle differences in the compaction and stability of the particles. Interestingly, both H2A.Z and H3.3 affect nucleosome positioning, either creating new positions or altering the relative occupancy of the existing nucleosome position space. On the other hand, only H2A.Z-contg. nucleosomes exhibit altered linker histone binding. These properties could be physiol. significant as nucleosome positions and linker histone binding partly det. factor binding accessibility.
- 152McGhee, J. D.; Nickol, J. M.; Felsenfeld, G.; Rau, D. C. Cell 1983, 33, 831152Higher order structure of chromatin: orientation of nucleosomes within the 30 nm chromatin solenoid is independent of species and spacer lengthMcGhee, James D.; Nickol, Joanne M.; Felsenfeld, Gary; Rau, Donald C.Cell (Cambridge, MA, United States) (1983), 33 (3), 831-41CODEN: CELLB5; ISSN:0092-8674.Elec. dichroism was used to study the arrangement of nucleosomes in 30-nm chromatin solenoidal fibers prepd. from a variety of sources (CHO cells, HeLa cells, rat liver, chicken erythrocytes, and sea urchin sperm) in which the nucleosome spacer length varies in the ∼10-∼80-base-pair range. Field-free relaxation times are consistent only with structures contg. 6 ± 1 nucleosomes for every 11 nm of solenoidal length. With very few assumptions about the arrangement of the spacer DNA, the dichroism data are consistent with the same orientation of the chromatosomes for every chromatin sample examd. The orientation, which maintains the faces of the radially arranged chromatosomes inclined at an angle of 20-33° to the solenoid axis, thus appears to be a general structural feature of the higher-order chromatin fiber.
- 153Widom, J.; Klug, A. Cell 1985, 43, 207153Structure of the 300Å chromatin filament: x-ray diffraction from oriented samplesWidom, J.; Klug, A.Cell (Cambridge, MA, United States) (1985), 43 (1), 207-13CODEN: CELLB5; ISSN:0092-8674.X-ray diffraction patterns have been obtained from partially oriented samples of 300-Å chromatin filaments. The chromatin was prepd. by methods that preserve its structure, and conditions were found in which the 300-Å filaments spontaneously form ordered aggregates, so that it was not necessary to pull fibers. The diffraction patterns show a meridional band at 110 Å, and equatorial bands at 340, 57, 37, and 27 Å. These patterns, together with patterns calcd. from the known 7-Å electron d. map of the nucleosome core particle, imply side-to-side packing of nucleosomes in the direction of the 300-Å filament and radial packing around it. These results are consistent with the solenoid model of J. R. Finch and A. Klug (1976) and are inconsistent with many other proposed models.
- 154Thoma, F.; Koller, T.; Klug, A. J. Cell Biol. 2003, 83, 403There is no corresponding record for this reference.
- 155Worcel, A.; Strogatz, S.; Riley, D. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 1461155Structure of chromatin and the linking number of DNAWorcel, Abraham; Strogatz, Steven; Riley, DonaldProceedings of the National Academy of Sciences of the United States of America (1981), 78 (3), 1461-5CODEN: PNASA6; ISSN:0027-8424.Recent observations suggest that the basic supranucleosomal structure of chromatin is a zigzag helical ribbon with a repeat unit made of 2 nucleosomes connected by a relaxed spacer DNA. A remarkable feature of 1 particular ribbon is that it solves the apparent paradox between the no. of DNA turns/nucleosome and the total linking no. of a nucleosome-contg. closed circular DNA mol. The repeat unit of the proposed structure, which contains 2 nucleosomes with -1.75 DNA turns/nucleosome and 1 spacer crossover/repeat, contributes -2 to the linking no. of closed circular DNA. Space-filling models show that the cylindrical 250-Å chromatin fiber can be generated by twisting the ribbon.
- 156Woodcock, C. L.; Frado, L. L.; Rattner, J. B. J. Cell Biol. 2002, 99, 42There is no corresponding record for this reference.
- 157Williams, S. P.; Athey, B. D.; Muglia, L. J.; Schappe, R. S.; Gough, A. H.; Langmore, J. P. Biophys. J. 1986, 49, 233157Chromatin fibers are left-handed double helixes with diameter and mass per unit length that depend on linker lengthWilliams, Shawn P.; Athey, Brian D.; Muglia, Louis J.; Schappe, R. Scott; Gough, Albert H.; Langmore, John P.Biophysical Journal (1986), 49 (1), 233-48, 373CODEN: BIOJAU; ISSN:0006-3495.Four classes of models were proposed for the internal structure of eukaryotic chromosome fibers: the solenoid, twisted-ribbon, crossed-linker, and superbead models. Electron image and x-ray scattering data were collected from nuclei and isolated chromatin fibers of 7 different tissues to distinguish between these models. The fiber diams. were related to the linker lengths by the equation: D(N) = 19.3 + 0.23 N, where D(N) is the external diam. (nm) and N is the linker length (base pairs). The no. of nucleosomes per unit length of the fibers was also related to linker length. Detailed studies were done on the highly regular chromatin from erythrocytes of Necturus (mud puppy) and sperm of Thyone (sea cucumber). Necturus Chromatin fibers [N = 48 base pairs (bp)] had diams. of 31 nm and had 7.5 nucleosomes per 10 nm along the axis. Thyone Chromatin fibers (N = 87 bp) had diams. of 39 nm and had 12 nucleosomes per 10 nm along the axis. Fourier transforms of electron micrographs of Necturus fibers showed left-handed helical symmetry with a pitch of 25.8 nm and pitch angle of 32°, consistent with a double helix. Comparable conclusions were drawn from the Thyone data. The data did not support the solenoid, twisted-ribbon, or supranucleosomal particle models. The data did support 2 crossed-linker models having left-handed double-helical symmetry and conserved nucleosome interactions.
- 158Wong, H.; Victor, J.-M.; Mozziconacci, J. PLoS One 2007, 2, e877158An all-atom model of the chromatin fiber containing linker histones reveals a versatile structure tuned by the nucleosomal repeat lengthWong Hua; Victor Jean-Marc; Mozziconacci JulienPloS one (2007), 2 (9), e877 ISSN:.In the nucleus of eukaryotic cells, histone proteins organize the linear genome into a functional and hierarchical architecture. In this paper, we use the crystal structures of the nucleosome core particle, B-DNA and the globular domain of H5 linker histone to build the first all-atom model of compact chromatin fibers. In this 3D jigsaw puzzle, DNA bending is achieved by solving an inverse kinematics problem. Our model is based on recent electron microscopy measurements of reconstituted fiber dimensions. Strikingly, we find that the chromatin fiber containing linker histones is a polymorphic structure. We show that different fiber conformations are obtained by tuning the linker histone orientation at the nucleosomes entry/exit according to the nucleosomal repeat length. We propose that the observed in vivo quantization of nucleosomal repeat length could reflect nature's ability to use the DNA molecule's helical geometry in order to give chromatin versatile topological and mechanical properties.
- 159Eltsov, M.; MacLellan, K. M.; Maeshima, K.; Frangakis, A. S.; Dubochet, J. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 19732159Analysis of cryo-electron microscopy images does not support the existence of 30-nm chromatin fibers in mitotic chromosomes in situEltsov, Mikhail; MacLellan, Kirsty M.; Maeshima, Kazuhiro; Frangakis, Achilleas S.; Dubochet, JacquesProceedings of the National Academy of Sciences of the United States of America (2008), 105 (50), 19732-19737CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Although the formation of 30-nm chromatin fibers is thought to be the most basic event of chromatin compaction, it remains controversial because high-resoln. imaging of chromatin in living eukaryotic cells had not been possible until now. Cryo-electron microscopy of vitreous sections is a relatively new technique, which enables direct high-resoln. observation of cell structures in a close-to-native state. We used cryo-electron microscopy and image processing to further investigate the presence of 30-nm chromatin fibers in human mitotic chromosomes. HeLa S3 cells were vitrified by high-pressure freezing, thin-sectioned, and then imaged under the cryo-electron microscope without any further chem. treatment or staining. For an unambiguous interpretation of the images, the effects of the contrast transfer function were computationally cor. The mitotic chromosomes of the HeLa S3 cells appeared as compact structures with a homogeneous grainy texture, in which there were no visible 30-nm fibers. Power spectra of the chromosome images also gave no indication of 30-nm chromatin folding. These results, together with our observations of the effects of chromosome swelling, strongly suggest that, within the bulk of compact metaphase chromosomes, the nucleosomal fiber does not undergo 30-nm folding, but exists in a highly disordered and interdigitated state, which is, on the local scale, comparable with a polymer melt.
- 160Maeshima, K.; Hihara, S.; Eltsov, M. Curr. Opin. Cell Biol. 2010, 22, 291160Chromatin structure: does the 30-nm fiber exist in vivo?Maeshima, Kazuhiro; Hihara, Saera; Eltsov, MikhailCurrent Opinion in Cell Biology (2010), 22 (3), 291-297CODEN: COCBE3; ISSN:0955-0674. (Elsevier B.V.)A review. A long strand of DNA is wrapped around the core histone and forms a nucleosome. Although the nucleosome has long been assumed to be folded into 30-nm chromatin fibers, their structural details and how such fibers are organized into a nucleus or mitotic chromosome remain unclear. When the authors obsd. frozen hydrated (vitrified) human mitotic cells using cryo-electron microscopy, which enables direct high-resoln. imaging of the cellular structures in a close-to-native state, they found no higher-order structures including 30-nm chromatin fibers in the chromosome. Therefore, the authors have proposed that the nucleosome fibers exist in a highly disordered, interdigitated state like a 'polymer melt' that undergoes dynamic movement. The authors have postulated that a similar state exists in active interphase nuclei, resulting in several advantages in the transcription and DNA replication processes.
- 161Joti, Y.; Hikima, T.; Nishino, Y.; Kamada, F.; Hihara, S.; Takata, H.; Ishikawa, T.; Maeshima, K. Nucleus 2012, 3, 404161Chromosomes without a 30-nm chromatin fiberJoti Yasumasa; Hikima Takaaki; Nishino Yoshinori; Kamada Fukumi; Hihara Saera; Takata Hideaki; Ishikawa Tetsuya; Maeshima KazuhiroNucleus (Austin, Tex.) (2012), 3 (5), 404-10 ISSN:.How is a long strand of genomic DNA packaged into a mitotic chromosome or nucleus? The nucleosome fiber (beads-on-a-string), in which DNA is wrapped around core histones, has long been assumed to be folded into a 30-nm chromatin fiber, and a further helically folded larger fiber. However, when frozen hydrated human mitotic cells were observed using cryoelectron microscopy, no higher-order structures that included 30-nm chromatin fibers were found. To investigate the bulk structure of mitotic chromosomes further, we performed small-angle X-ray scattering (SAXS), which can detect periodic structures in noncrystalline materials in solution. The results were striking: no structural feature larger than 11 nm was detected, even at a chromosome-diameter scale (~1 μm). We also found a similar scattering pattern in interphase nuclei of HeLa cells in the range up to ~275 nm. Our findings suggest a common structural feature in interphase and mitotic chromatins: compact and irregular folding of nucleosome fibers occurs without a 30-nm chromatin structure.
- 162Nishino, Y.; Eltsov, M.; Joti, Y.; Ito, K.; Takata, H.; Takahashi, Y.; Hihara, S.; Frangakis, A. S.; Imamoto, N.; Ishikawa, T.; Maeshima, K. EMBO J. 2012, 31, 1644162Human mitotic chromosomes consist predominantly of irregularly folded nucleosome fibers without a 30-nm chromatin structureNishino, Yoshinori; Eltsov, Mikhail; Joti, Yasumasa; Ito, Kazuki; Takata, Hideaki; Takahashi, Yukio; Hihara, Saera; Frangakis, Achilleas S.; Imamoto, Naoko; Ishikawa, Tetsuya; Maeshima, KazuhiroEMBO Journal (2012), 31 (7), 1644-1653CODEN: EMJODG; ISSN:0261-4189. (Nature Publishing Group)How a long strand of genomic DNA is compacted into a mitotic chromosome remains one of the basic questions in biol. The nucleosome fiber, in which DNA is wrapped around core histones, has long been assumed to be folded into a 30-nm chromatin fiber and further hierarchical regular structures to form mitotic chromosomes, although the actual existence of these regular structures is controversial. Here, the authors show that human mitotic HeLa chromosomes are mainly composed of irregularly folded nucleosome fibers rather than 30-nm chromatin fibers. The authors' comprehensive and quant. study using cryo-electron microscopy and synchrotron x-ray scattering resolved the long-standing contradictions regarding the existence of 30-nm chromatin structures and detected no regular structure >11 nm. This finding suggests that the mitotic chromosome consists of irregularly arranged nucleosome fibers, with a fractal nature, which permits a more dynamic and flexible genome organization than would be allowed by static regular structures.
- 163Hansen, J. C. EMBO J. 2012, 31, 1621163Human mitotic chromosome structure: what happened to the 30-nm fiber?Hansen, Jeffrey C.EMBO Journal (2012), 31 (7), 1621-1623CODEN: EMJODG; ISSN:0261-4189. (Nature Publishing Group)A review. The long-standing view of chromosome packaging is that 10-nm beads-on-a-string chromatin fibers fold into higher-order 30-nm fibers, which further twist and coil to form highly condensed chromosomes. After 4 decades of intense pursuit of the structure and properties of 30-nm chromatin fibers, the work of Y. Nishino et al. (2012) demonstrates that regular 30-nm fibers are absent from human mitotic chromosomes. The emerging view is that chromosome-level condensation can be achieved through packaging of 10-nm fibers in a fractal manner.
- 164Baker, N. A.; Sept, D.; Joseph, S.; Holst, M. J.; McCammon, J. A. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 10037164Electrostatics of nanosystems: application to microtubules and the ribosomeBaker, Nathan A.; Sept, David; Joseph, Simpson; Holst, Michael J.; McCammon, J. AndrewProceedings of the National Academy of Sciences of the United States of America (2001), 98 (18), 10037-10041CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Evaluation of the electrostatic properties of biomols. has become a std. practice in mol. biophysics. Foremost among the models used to elucidate the electrostatic potential is the Poisson-Boltzmann equation; however, existing methods for solving this equation have limited the scope of accurate electrostatic calcns. to relatively small biomol. systems. Here we present the application of numerical methods to enable the trivially parallel soln. of the Poisson-Boltzmann equation for supramol. structures that are orders of magnitude larger in size. As a demonstration of this methodol., electrostatic potentials have been calcd. for large microtubule and ribosome structures. The results point to the likely role of electrostatics in a variety of activities of these structures.
