In vitro histone acetyltransferase (HAT) assays

For HAT assays on recombinant nucleosomes containing full-length histone H3.1, purified wild type or mutated p300_BRPHZ (300 nM) was incubated with recombinant mononucleosome (100 nM) in HAT reaction buffer (50 mM Tris pH 8.0, 0.1 mM EDTA, 10% glycerol, 1 mM PMSF and 1mM DTT) in a total volume of 50 μL. After pre-warming at 37 °C for 5 minutes, reactions were initiated with the addition of acetyl-CoA (Sigma) to a final concentration of 0.1 mM and incubated for 10~80 minutes at 37 °C. For assays comparing the HAT activities on recombinant nucleosomes containing full-length histone H3.1 and N-terminally truncated H3.1, wild type p300_BRPHZ fragment (50 nM) and monobucleosome (500 nM) were incubated under the same condition for 1 to 6 hours. Reactions were quenched by flash-freezing in liquid nitrogen and then analyzed by SDS-PAGE and Western blot analysis. Western blot results were quantified by LI-COR Odyssey and normalized to a common standard sample.