Mapping Post-translational Modifications of the Histone Variant MacroH2A1 Using Tandem Mass Spectrometry

• Published in: Molecular & cellular proteomics Vol. 5; no. 1; pp. 194 - 203
• Main Authors: Chu, Feixia, Nusinow, Dmitri A., Chalkley, Robert J., Plath, Kathrin, Panning, Barbara, Burlingame, Alma L.
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 01.01.2006
• Subjects: Histones - chemistry; Histones - metabolism; Immunoprecipitation; Lysine - chemistry; Methylation; Phosphorylation; Protein Processing, Post-Translational; Spectrometry, Mass, Electrospray Ionization; Ubiquitin - metabolism
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1074/mcp.M500285-MCP200

Post-translational histone modifications modulate chromatin-templated processes and therefore affect cellular proliferation, growth, and development. Although post-translational modifications on the core histones have been under intense investigation for several years, the modifications on variant histones are poorly understood. We used tandem mass spectrometry to identify covalent modifications on a histone H2A variant, macroH2A1.2. MacroH2A1.2 can be monoubiquitinated; however, the site of monoubiquitination has not been documented. In this study we used green fluorescent protein-tagged macroH2A1.2 to determine that Lys 115 is a site of ubiquitination. In addition, we found that this variant H2A is methylated on the ε amino group of lysine residues Lys 17 , Lys 122 , and Lys 238 and phosphorylated on Thr 128 . Three of these modifications were also found to be present in the endogenous protein by mass spectrometric analysis. These results provide the first direct evidence that multiple post-translational modifications are imposed on macroH2A1.2, suggesting that, like canonical H2A, this variant H2A is subject to regulation by combinatorial use of covalent modifications.