Figure 5

In vitro characterization of p300 I1395G and CBP I1432G mutant. (a) In vitro histone acetylation assay showing GST-p300-I1395G mutant is inactive in histone acetylation. Both wild-type and I1395G p300 histone acetyltransferase (HAT) domain were expressed and purified from bacteria and applied to HAT assay using core histones prepared from HeLa cells. The resulting histone acetylation was revealed by western blotting (WB) analysis using a pan-acetylated lysine antibody. Acetyl-coA, 100 μm. The components of in vitro acetylation reactions were also shown by Coomassie blue staining. (b) In vitro histone crotonylation assay showing that both wild-type GST-p300-HAT and GST-p300-I1395G mutant catalyzing histone crotonylation and that the mutant was more active. In vitro reactions were carried out using core histone substrates as above except crotonyl-coA (100 μm) was used. (c) The HAT activity of GST-p300-HAT and GST-p300-I1395G mutant was compared in a time course experiment. (d) The histone crotonyltransferase (HCT) activity of GST-p300-HAT and GST-p300-I1395G mutant was compared in a time course experiment. (e) The quantification of relative HAT activity of GST-p300-HAT and GST-p300-I1395G mutant according to results in c. (f) The quantification of relative HCT activity of GST-p300-HAT and GST-p300-I1395G mutant according to results in d. (g) In vitro histone acetylation assay using wild-type and I1432G mutant CBP immunoaffinity purified from 293T cells. (h) In vitro histone crotonylation assay using wild-type and I1432G mutant CBP immunoaffinity purified from 293T cells.