A dually modified H2A.Z species bearing ubiquitination and acetylation. (a) Schematics of H2A.Z N-terminal and C-terminal amino acid sequences showing lysine residues that are can be acetylated (Ac) or monoubiquitinated (Ub1) according to MS analysis. (b) Western blots for H2A.Z (top panel), acH2A.Z (middle panel) and Ring1B (bottom panel) for control (left lane) and tamoxifen-induced Ring1B KO (right lane) mES cells. The data suggest that the PRC1 component Ring1B is upstream of H2A.Zub1 and acH2A.Zub1 in mES cells. (c) Left: SDS-PAGE shows separation of HPLC-purified H2A.Zub1 (top band) from other H2A.Z species (bottom band). Red boxes indicate bands excised for MS analysis. Right: Extracted ion chromatograms of the excised bands show H2A.Zub1 is present in the upper band and is absent in the lower band. m/z values indicate the residual of H2A.Zub1 after d
-propionylation and chymotryptic digestion (upper trace). (d) Quantification of the C-terminal H2A.Z monoubiquitination at K120, K121 and K125 residues in mES cells. (e) MS/MS spectra assign the positional isomers of ubiquitination (Ub1) in each peak from Figure 3c, right panel ((1), (2), (3) and (4) respectively). The three peaks in the upper trace in Figure 3c correspond to differential sites of H2A.Zub1. (-GGR) sites indicate the branched peptide that results from monoubiquitin ligation at a given residue followed by chymotryptic digestion. * indicates d5-propionylation and therefore an absence of ubiquitin at a given lysine. (#,&,%) marks indicate key ions that localize the site of ubiquitination on the peptide. (f) Abundance of C-terminal ubiquitination of H2A.Z in wild type and Ring1B KO mES cells. Signal corresponds to area under the peak(s) observed by MS corresponding to the H2A.Z 118-127 peptide with one ubiquityl adduct, as in Figure 3c. (g) Quantitative MS analysis shows the prevalence of one acetylation (1ac), two acetylation (2ac), three acetylation (3ac) and four acetylation (4ac) species on any N-terminal lysines within non-ubiquitinated and ubiquitinated H2A.Z populations separated by SDS-PAGE. Despite its apparently repressive function, H2A.Zub1 is more frequently acetylated. Ac: acetylated; HPLC: high performance liquid chromatography; KO: knockout; mES: mouse embryonic stem cells; MS: mass spectrometry; PRC1: Polycomb repressive complex 1; Ub0: non-ubiquitinated; Ub1: ubiquitinated; WT: wild type.