Figure 6. H3S10A affects histone crosstalk in cis.

(A) Mass spectrometric analysis of H3X/H3Y heterodimers expressing one biotin-labeled subunit, with a S10A point mutation either located on the same H3 molecule (in cis), or in trans. Left: Schematic of asymmetric nucleosomes. Right: Robust avidin-affinity purification of biotin-labeled H3 molecules. Mass spec quantitation of peptides (described on top of the graphs) from the strains with genotypes indicated on the x-axes. For each peptide, raw peak area scores were normalized by dividing by the peak areas of an internal control, the unmodified H3 peptide containing K42. Average and std. dev. of triplicate measures of these normalized values are graphed on the y-axes. As expected, S10A-containing peptides were only detected when present in cis on the biotin-labeled histone, but not when expressed in trans on the heterodimeric partner. (B) H3K9 acetylation levels are diminished in cis in H3S10A mutants. Data are shown for mass spec analysis of the indicated mutant strains. Left two bars show data for wild-type and double H3S10A mutants on the background of a wild-type H3-H3 interface. Right four panels show H3K9Ac levels on an avidin-purified biotin-tagged H3 molecule, showing data for H3X acetylation in the context of the pseudo-wild type H3X/Y background, H3X acetylation on the same (cis) or opposite tail from an H3S10A mutation, and H3X acetylation in a double H3XS10A/H3YS10A mutant. (C) Western blots confirming effects of symmetric S10A on K9 acetylation both on the WT H3-H3 background, and on the X-Y background, as indicated. For the right panel, the epitope tag present on H3X allows separate probing of the H3X and H3Y molecules. Total protein on blots was visualized by Ponceau S staining. Note that although the S10A mutation in principle might affect anti-H3K9ac antibody (Abcam ab10812) binding, these results are highly concordant with the mass spectrometry measurements in panel (B), where the ability to detect lysine acetylation (based on peptide mass) is unaffected by the difference between S10 and S10A. (D–F) Quantitation of blots from panel (C). K9Ac signals were normalized to total protein detected by Ponceau S. Strains analyzed were: WT wt-C (PKY4610), S10A wt-C (PKY5003), pWT-XY (PKY4983), S10A-XY-cis (PKY5005), S10A-XY trans (PKY4986) and S10A-XY double (PKY5042).