Histone variant macroH2A1.2 is mono-ubiquitinated at its histone domain

Ogawa, Yasushi; Ono, Taketoshi; Wakata, Yuka; Okawa, Katsuya; Tagami, Hideaki; Ki, Shibahara

doi:10.1016/j.bbrc.2005.08.046

Cited by 27 publications

(19 citation statements)

References 29 publications

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“…A slower-migrating, crossreacting band above the mH2A1 signal was repeatedly observed by immunoblotting (IB) and is consistent with the monoubiquitylated form of mH2A given its size (an Ϸ8-kDa shift; Fig. 1b, asterisk) (28,29). Multiple HPLC runs were carried out, and peak fractions from all runs were pooled for MS; a small portion of the total pool was examined by SDS/PAGE to determine the major constituents of this sample (Fig.…”

Section: Resultssupporting

confidence: 56%

“…Nevertheless, PTMs of other H2A variants, such as mH2A, are just beginning to be uncovered. For example, MS approaches have recently uncovered monoubiquitylation of K115 on overexpressed mH2A1.2, a site analogous to K119ub of histone H2A (28,29). Methylation of K17, K122, and K238 has been reported, and so has phosphorylation of T128, a site verified by us in this work (29).…”

Section: Discussionsupporting

confidence: 61%

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A phosphorylated subpopulation of the histone variant macroH2A1 is excluded from the inactive X chromosome and enriched during mitosis

Bernstein

Muratore-Schroeder²,

Diaz

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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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 damageassociated H2A.X, macroH2A (mH2A), and H2ABbd (Barr bodydeficient). 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 Ϸ30-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.macroH2A ͉ histone modifications ͉ phosphorylation

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Section: Resultssupporting

confidence: 56%

Section: Discussionsupporting

confidence: 61%

A phosphorylated subpopulation of the histone variant macroH2A1 is excluded from the inactive X chromosome and enriched during mitosis

Bernstein

Muratore-Schroeder²,

Diaz

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…The low abundance of ubiquitination, approximately 10% for H2A and 1-2% for H2B, could explain why ubiquitination was not detected (4). Recently ubiquitination on macroH2A1.2 was identified by mass spectrometry but only when upfront enrichment of ubiquitinated protein was performed before analyzing the protein (30). Also acetylation of Lys-9 for histone H2A and Lys-5 for histone H2B was only identified in cells treated with a histone deacetylase inhibitor (which induces histone hyperacetylation) indicating the low abundance of these modification in nontreated cells (16).…”

Section: Discussionmentioning

confidence: 99%

Characterization of Histone H2A and H2B Variants and Their Post-translational Modifications by Mass Spectrometry

Bonenfant

Coulot

Towbin

et al. 2006

Molecular & Cellular Proteomics

125

111

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The nucleosome, the fundamental structural unit of chromatin, contains an octamer of core histones H3, H4, H2A, and H2B. Incorporation of histone variants alters the functional properties of chromatin. To understand the global dynamics of chromatin structure and function, analysis of histone variants incorporated into the nucleosome and their covalent modifications is required. Here we report the first global mass spectrometric analysis of histone H2A and H2B variants derived from Jurkat cells. A combination of mass spectrometric techniques, HPLC separations, and enzymatic digestions using endoproteinase Glu-C, endoproteinase Arg-C, and trypsin were used to identify histone H2A and H2B subtypes and their modifications. We identified nine histone H2A and 11 histone H2B subtypes, among them proteins that only had been postulated at the gene level. The two main H2A variants, H2AO and H2AC, as well as H2AL were either acetylated at Lys-5 or phosphorylated at Ser-1. For the replacement histone H2AZ, acetylation at Lys-4 and Lys-7 was found. Within the eukaryotic cell nucleus the genetic information is organized in a highly conserved structural polymer, termed chromatin, that supports and controls crucial functions of the genome. The fundamental unit of eukaryotic chromatin, the nucleosome, consists of 146 base pairs of genomic DNA wrapped around an octamer of the core histone proteins H2A, H2B, H3, and H4. The amino-terminal tails of each of the four core histones are subject to several types of covalent modifications, including acetylation, methylation, and phosphorylation. These modifications affect lysines (acetylation, mono-, di-, and trimethylation), serines and threonines (phosphoryla-

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“…The histone tails protrude from the nucleosome and contain sites of posttranslational modification, 22,23 suggesting that modification of macroH2A1 may also play a role in its correct localization. The decrease in percentage of cells with Xi-enrichment of chimeras containing mH2A1-HD tail sequences suggests that these chimeras may have altered affinities for factors that mediate macroH2A1 enrichment on the Xi.…”

Section: Many Short Stretches Of Residues Within the Histone Domain Pmentioning

confidence: 98%

The Histone Domain of macroH2A1 Contains Several Dispersed Elements that Are Each Sufficient to Direct Enrichment on the Inactive X Chromosome

Nusinow

Sharp

Morris

et al. 2007

Journal of Molecular Biology

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Histone variants replace the core histones in a substantial fraction of nucleosomes, affecting chromatin structure and impacting chromatin-templated processes. In many instances incorporation of histone variants results in formation of specialized regions of chromatin. Proper localization of histone variants to distinct regions of the genome is critical for their function, yet how this specific localization is achieved remains unclear. macroH2A1 is enriched on the inactive X chromosome in female mammalian cells, where it functions to maintain gene silencing. macroH2A1 consists of a histone H2A-like histone domain and a large, globular C-terminal macro domain that is not present in other histone proteins. The histone domain of macroH2A1 is alone sufficient to direct enrichment on the inactive X chromosome when expressed in female cells, indicating that sequences important for correct localization lie in this domain. Here we investigate whether divergent sequences of the H2A variant macroH2A1 contribute to its correct localization. We mapped the regions of the macroH2A1 histone domain that are sufficient for localization to the inactive X chromosome using chimeras between H2A and the histone domain of macroH2A1. Multiple short sequences dispersed along the macroH2A1 histone domain individually supported enrichment on the inactive X chromosome when introduced into H2A. These sequences map to the surface of the macroH2A1/H2B dimer, but are buried in the crystal structure of the macroH2A1 containing nucleosome, suggesting that they may contribute to recognition by macroH2A1/H2B deposition factors.

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Histone variant macroH2A1.2 is mono-ubiquitinated at its histone domain

Cited by 27 publications

References 29 publications

A phosphorylated subpopulation of the histone variant macroH2A1 is excluded from the inactive X chromosome and enriched during mitosis

A phosphorylated subpopulation of the histone variant macroH2A1 is excluded from the inactive X chromosome and enriched during mitosis

Characterization of Histone H2A and H2B Variants and Their Post-translational Modifications by Mass Spectrometry

The Histone Domain of macroH2A1 Contains Several Dispersed Elements that Are Each Sufficient to Direct Enrichment on the Inactive X Chromosome

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