Supplementary Figure 10: Confirmation of p300 positive feedback loop using antibody-independent experiments.

(a) 4.8 pmoles of the individual NUCs (unmodified; H4K12ac-modified; H4Kac₅-modified; or H3Kac₅-modified) were incubated with 120 fmoles of wt p300 in the presence of excess ³H-acetyl-CoA at 30 °C. The reactions were quenched after 0, 5, 15 and 45 min with 4x SDS sample buffer and analyzed by denaturing PAGE followed by CBB staining (top) or fluorography (bottom). Note that hyperacetylated H3 and H4 migrate faster than the respective unmodified versions, with hyperacetylated H3 comigrating with unmodified H2B. For analysis of the reaction products by native gel electrophoresis, see Fig. 3c. (b) The acetyltransferase assay was performed essentially the same as described for (a), using cold acetyl-CoA. The peptides were trypsinized, propionylated and subjected to ultra-high performance liquid chromatography-coupled high-resolution mass spectrometry. The resulting LC-MS/MS data were searched against a database consisting of human histones H2A, H2B, H3, and H4, and filtered to the 90 % peptide and 95 % protein probability levels. Ascore derived PTM site localization probabilities were calculated for each PTM, and fragmentation spectral assignments were subject to manual inspection and validation using the original tandem mass spectra acquired in profile mode. (c) Chromatin acetylation experiment employing ΔBD p300 mutant as shown in Fig. 3b (replicate 2).