Fig. 7.
Class I HDACs are histone decrotonylases. a Histone H3 decrotonylation and deacetylation in vitro by HDAC1, HDAC2, or HDAC3/Ncor1 complex; 5.65 μM histones were crotonylated or acetylated in vitro and then subjected to removal of the modification by the indicated HDACs. HDAC1 was 0.25, 0.12, 0.06, and 0.03 μM. HDAC2 was 0.18, 0.09, 0.05, and 0.02 μM. HDAC3/Ncor1 complex was 0.45, 0.23, 0.11, and 0.06 μM. b Comparative kinetics of HDAC1 decrotonylation and deacetylation; 5.65 µM histones were crotonylated or acetylated and then subjected to removal of the modification by 0.03 µM HDAC1 for different lengths of time. Samples were analyzed by dot blotting and initial rates of reaction were determined by plotting substrate removal over time. Kinetic parameters Vmax, Km, and Kcat, error bars are SEM, n = 3. c Effect of HDAC inhibitors TSA, crotonate, and butyrate on deacetylation and decrotonylation by HDAC1 in vitro. Representative blots of two repeat experiments are shown. d Histone crotonylation by HDAC1 using crotonate in vitro. Incubation of crotonate, acetate, or butyrate with or without HDAC1 followed by western blotting analysis with anti-H3K18ac/bt/cr. Western blot of HDAC1 and crotonate assay is representative of two western blots