In vitro histone acylation assay

Based on substrate specificity, histone H3 was assayed with HATs p300, CBP, PCAF and Gcn5, and histone H4 with HATs MOF, NatA and Tip60. In vitro enzymatic assays were carried by incubating 0.5 µg of each HAT with 10 µg of recombinant histones H3 or H4 in the presence of 0.5 mM of short-chain acyl-CoAs (acetyl-, crotonyl-, malonyl-, succinyl-, propionyl-, butyryl-, glutaryl- and β-hydroxybutyryl CoA;—Sigma-Aldrich) in 1X HAT buffer (25 mM Tris-HCl pH = 8, 25 mM KCl, 1 mM DTT, 0.1 mM AEBSF and 5 mM sodium butyrate) for 60 min at 30 °C; the final volume was 50 µL. For competition assays, reactions were carried out in the presence of 10 µM of acetyl-CoA and 10 µM of other acyl-CoAs. Background control reactions were performed in the absence of HATs. Reactions were stopped by freezing and samples were dried in a SpeedVac and resuspended in 20 µL of 100 mM ammonium bicarbonate (pH 8.0). Histones were derivatized with propionic anhydride (or d₁₀-propionic anhydride when analyzing lysine propionylation). For this procedure, fresh propionylation reagent was prepared by mixing propionic anhydride with acetonitrile in the ratio 1:3 (v/v). Propionylation reagent was added to each sample in 1:4 (v/v). Ammonium bicarbonate was quickly added to the solution to re-establish pH 8.0. Samples were dried down to 10–20 µL in a SpeedVac, reconstituted with 20 µL of ammonium bicarbonate and the propionylation procedure was repeated one more time. Samples were then digested with trypsin at a 1:10 ratio (wt/wt) for 6 h at 37 °C. Samples were desalted by C18 stage-tip. For this procedure, a small piece of a C18 solid phase extraction disk was deposited into a pipette tip to create a stage-tip. The C18 resin was flushed with 100% acetonitrile by slow centrifugation using a centrifuge adaptor to hold the stage-tips in place in a 1.5 ml Eppendorf tube. The resin was then equilibrated by flushing 80 µl of 0.1% TFA. Samples were acidified to pH 4.0 or lower with acetic acid and loaded onto the disk by slow centrifugation. Samples were then washed by flushing 70–80 µL of 0.1% TFA and eluted into a clean Eppendorf tube by flushing 70 µL of 75% acetonitrile and 0.5% acetic acid by slow centrifugation. Samples were dried in a SpeedVac and resuspended at 0.5 μg per μL in 0.1 M acetic acid for nano LC-MS/MS analysis⁶⁶.