Fig 2: KAT2A primarily catalyzes acetylation on recombinant histone substrates. Western blots of (A) acetyl-, (B) succinyl-, and (C) malonyl-lysine following in vitro activity assay with 0, 5, or 50 μM acyl-CoA, KAT2A-catalytic domain (top) or full-length KAT2A (bottom), and histone H3.1 or calf thymus (CT) histone. Membranes were stained with Coomassie Blue for determining protein loading. All experiments were performed in triplicate with reproducible results each time.

Fig 3: Transient knockdown of KAT2A leads to a decrease in histone acetylation. (A) Western blot of HEK293T lysate supernatant confirming that KAT2A is knocked down relative to the nontargeting control. (B) Western blot showing that transient shRNA knockdown leads to expression of a compensatory isoform in both the supernatant (spnt) and pellet fractions with sh2. (C) Western blot showing that siRNA knockdown of KAT2A leads to expression of a higher molecular weight compensatory isoform. (D) Western blot showing that global lysine acetylation, succinylation, and malonylation of 293T supernatant does not change with KAT2A knockdown. (E) Western blot probing lysine acetylation, succinylation, and malonylation of the histone portion of lysed 293T cell pellet. Quantification of lysine modification relative to the control for three independent replicates is shown in the bar graph below. P value for lysine acetylation decrease is 0.0002. All experiments were performed in triplicate with similar results each time. Coomassie Blue (CB) staining of the membranes shows equal loading for all experiments.

Fig 4: KAT2A robustly catalyzes histone acetylation as determined by label-free quantification mass spectrometry. (A) Histone coverage in the LFQ MS experiment. Coverage region is indicated in gray with the percent of overall histone shown above. (B) Table summarizing LFQ MS results for KAT2A modified histone peptides with modified lysines bolded. Ratios represent fold enrichment in samples containing KAT2A vs control samples without KAT2A. All peptides with ratios > 1 are shown for reactions with succinyl- and malonyl-CoA. Peptides with ratios > 3 are shown for reactions with acetyl-CoA. Additional nonsignificant (ns) acetyl-lysine peptides with ratios > 1 were found and are available in the Supporting Information. XAll modifications are found on lysine if the amino acid is not indicated. For peptides where modified lysine is not bolded, corresponding masses were found, but the modification site could not be identified specifically. YP values were determined using unpaired t test analysis in Prism. P values of >0.05 (ns), ≤0.05 (*), and ≤0.01 (**). All peptides with significant abundance are shown in the table.

Fig 5: Possible explanation for the preference of malonyl-CoA over succinyl-CoA by KAT2A. The structure of KAT2A in complex with propionyl-CoA (PDB 5h84) is used for this analysis. KAT2A’s surface contact potential map (generated using PyMOL) is shown, with the blue color indicating positive potential, the red color indicating negative potential, and the white color indicating a neutral surface. The propionyl-CoA molecule is shown in stick representation. The propionyl group is next to a negative surface of KAT2A, to which malonyl and succinyl-CoA would also be adjacent if they are bound to KAT2A. The longer succinyl-CoA would present the negatively charged carboxylate closer to the negative surface of KAT2A, causing an unfavorable interaction and explaining why succinyl-CoA would be a worse substrate than malonyl-CoA.