Serine 31 phosphorylation of histone variant H3.3 is specific to regions bordering centromeres in metaphase chromosomes

doi:10.1073/pnas.0502413102

Serine 31 phosphorylation of histone variant H3.3 is specific to regions bordering centromeres in metaphase chromosomes

^*Laboratory of Chromatin Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021; ^†Department of Chemistry, University of Virginia, Charlottesville, VA 22901; and Departments of ^§Biochemistry and Molecular Genetics and ^¶Pathology, University of Virginia, Charlottesville, VA 22908

Communicated by Robert G. Roeder, The Rockefeller University, New York, NY, March 23, 2005 (received for review March 1, 2005)

Abstract

Histones are the fundamental components of the nucleosome. Physiologically relevant variation is introduced into this structure through chromatin remodeling, addition of covalent modifications, or replacement with specialized histone variants. The histone H3 family contains an evolutionary conserved variant, H3.3, which differs in sequence in only five amino acids from the canonical H3, H3.1, and was shown to play a role in the transcriptional activation of genes. Histone H3.3 contains a serine (S) to alanine (A) replacement at amino acid position 31 (S31). Here, we demonstrate by both MS and biochemical methods that this serine is phosphorylated (S31P) during mitosis in mammalian cells. In contrast to H3 S10 and H3 S28, which first become phosphorylated in prophase, H3.3 S31 phosphorylation is observed only in late prometaphase and metaphase and is absent in anaphase. Additionally, H3.3 S31P forms a speckled staining pattern on the metaphase plate, whereas H3 S10 and H3 S28 phosphorylation localizes to the outer regions of condensed DNA. Furthermore, in contrast to phosphorylated general H3, H3.3 S31P is localized in distinct chromosomal regions immediately adjacent to centromeres. These findings argue for a unique function for the phosphorylated isoform of H3.3 that is distinct from its suspected role in gene activation.

Footnotes

↵ ∥ To whom correspondence should be addressed. E-mail: alliscd{at}rockefeller.edu.
↵ ‡ B.A.G. and M.K. contributed equally to this work.
Author contributions: S.B.H. and C.D.A. designed research; S.B.H., B.A.G., M.K., S.P.B., and J.S. performed research; S.B.H., B.A.G., M.K., S.P.B., J.S., and D.F.H. analyzed data; S.B.H. and B.A.G. wrote the paper; and D.F.H. and C.D.A. corrected the manuscript and provided discussion.
Abbreviations: IB, immunoblotting; IF, immunofluorescence; CS, chromosome spreads.