title: A phosphorylated subpopulation of the histone variant macroH2A1 is excluded from the inactive X chromosome and enriched during mitosis.
abstract: Histone variants play an important role in numerous biological processes through changes in nucleosome structure and stability and possibly through mechanisms influenced by posttranslational modifications unique to a histone variant. The family of histone H2A variants includes members such as H2A.Z, the DNA damage-associated H2A.X, macroH2A (mH2A), and H2ABbd (Barr body-deficient). Here, we have undertaken the challenge to decipher the posttranslational modification-mediated "histone code" of mH2A, a variant generally associated with certain forms of condensed chromatin such as the inactive X chromosome in female mammals. By using female human cells as a source of mH2A, endogenous mH2A was purified and analyzed by mass spectrometry. Although mH2A is in low abundance compared with conventional histones, we identified a phosphorylation site, S137ph, which resides within the "hinge" region of mH2A. This lysine-rich hinge is an approximately 30-aa stretch between the H2A and macro domains, proposed to bind nucleic acids. A specific antibody to S137ph was raised; by using this reagent, S137 phosphorylation was found to be present in both male and female cells and on both splice variants of the mH2A1 gene. Although mH2A is generally enriched on the inactive X chromosome in female cells, mH2AS137ph is excluded from this heterochromatic structure. Thus, a phosphorylated subpopulation of mH2A appears to play a unique role in chromatin regulation beyond X inactivation. We provide evidence that S137ph is enriched in mitosis, suggestive of a role in the regulation of mH2A posttranslational modifications throughout the cell cycle.
authors.affiliation: Laboratory of Chromatin Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA. bernste@rockefeller.edu
authors.forename: Emily
authors.initials: E
authors.lastname: Bernstein
authors.forename: Tara L
authors.initials: TL
authors.lastname: Muratore-Schroeder
authors.forename: Robert L
authors.initials: RL
authors.lastname: Diaz
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Chow
authors.forename: Lakshmi N
authors.initials: LN
authors.lastname: Changolkar
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Shabanowitz
authors.forename: Edith
authors.initials: E
authors.lastname: Heard
authors.forename: John R
authors.initials: JR
authors.lastname: Pehrson
authors.forename: Donald F
authors.initials: DF
authors.lastname: Hunt
authors.forename: C David
authors.initials: CD
authors.lastname: Allis
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D002460:Cell Line; D041321:Chromosomes, Human, X / Q000235:genetics*; D005260:Female; D006657:Histones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008297:Male; D008938:Mitosis / Q000235:genetics*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D049951:X Chromosome Inactivation
chemical_list: D006657:Histones; C120756:macroH2A histone