Dually modified H2A.Z is Ring1B dependent and enriched in bivalent chromatin. (a) Extracted ion chromatograms of upper (H2A.Zub1) and lower (H2A.Zub0) PAGE bands showing presence of various acetyl forms of the N-terminus (1-19) of H2A.Z. (b) Site-specific distribution of monoacetyl (top panel) or diacetyl (bottom) moieties on the N-terminal lysines in H2A.Zub0 and H2A.Zub1 fractions. Monoacetylation is preferred on K7 in H2A.Zub0, while K11 is preferred in H2A.Zub1 fractions. Diacetylation often occurs on lysines proximal to each other. Remarkably, diacetylated K4/K11 is observed and specific for H2A.Zub1 fractions. (c) Left: western blot image shows the relative levels of ubiquitinated (Ub1) and non-ubiquitinated (Ub0) species in H2A.Z and acH2A.Z in mononucleosomal fractions enriched by immunoprecipitation with antibodies against H3K27me3, H3K4me3 or phosphorylated histone 3 serine 10 residue (H3S10P). Both H2A.Zub1 and acH2A.Zub1 species are more enriched in the H3K27me3 fraction, relative to H2A.Zub0 and acH2A.Zub0 species. The phosphorylation of H3S10 is a hallmark of mitosis, and it anti-correlates with H2Aub1 and H2A.Zub1 levels, thus serving as a negative control [22, 52]. Right: Bar plot shows a quantification of the ratio between ubiquitinated and non-ubiquitinated species in H2A.Z (black) and acH2A.Z (white) for each lane. The data are an average of triplicate experiments, normalized by input. Error bars indicate standard deviation. Ac: acetylated; H3S10P: phosphorylated histone 3 serine 10 residue; IP: immunoprecipitation; me3: trimethylation; Ub0: non-ubiquitinated; Ub1: ubiquitinated; WB: western blot.