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Article

Modifications of Human Histone H3 Variants during Mitosis^†

Supporting Info

Benjamin A. Garcia,^‡^§ Cynthia M. Barber,^§ Sandra B. Hake, Celeste Ptak,^‡ Fiona B. Turner, Scott A. Busby,^‡ Jeffrey Shabanowitz,^‡ Richard G. Moran,^# C. David Allis, and Donald F. Hunt*^‡

Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908, Laboratory of Chromatin Biology, The Rockefeller University, New York, New York 10021, Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298, and Department of Pathology, University of Virginia, Charlottesville, Virginia 22904

Biochemistry, 2005, 44 (39), pp 13202–13213

DOI: 10.1021/bi050906n

Publication Date (Web): September 3, 2005

†

This work was supported by grants from the National Institutes of Health to D.F.H. (GM37537) and C.D.A. (GM40922), The Rockefeller University's Women & Science Fellowship Program to S.B.H., and the Ford Foundation to B.A.G.

‡

Department of Chemistry, University of Virginia.

B.A.G. and C.M.B. contributed equally to this work.

Department of Biochemistry and Molecular Genetics, University of Virginia.

The Rockefeller University.

Virginia Commonwealth University.

To whom correspondence should be addressed. Tel: 434-924-3610. Fax: 434-982-2781. E-mail: dfh@virginia.edu.

Department of Pathology, University of Virginia.

Abstract

Phosphorylation of histone H3 is a hallmark event in mitosis and is associated with chromosome condensation. Here, we use a combination of immobilized metal affinity chromatography and tandem mass spectrometry to characterize post-translational modifications associated with phosphorylation on the N-terminal tails of histone H3 variants purified from mitotically arrested HeLa cells. Modifications observed in vivo on lysine residues adjacent to phosphorylated Ser and Thr provide support for the existence of the “methyl/phos”, binary-switch hypothesis [Fischle, W., Wang, Y., and Allis, C. D. (2003) Nature 425, 475−479]. ELISA with antibodies selective for H3 at Ser10, Ser28, and Thr3 show reduced activity when adjacent Lys residues are modified. When used together, mass spectrometry and immunoassay methods provide a powerful approach for elucidation of the histone code and identification of histone post-translational modifications that occur during mitosis and other specific cellular events.

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