Figure 3. Histone Crotonylation is Regulated Metabolically by the Concentration of Crotonyl-CoA.

(A) In vitro p300 HAT/HCT reactions were performed with indicated concentrations of crotonyl-CoA and acetyl-CoA. Reaction products were immunoblotted with the indicated antibodies.

(B) LC-MS analysis of cellular crotonyl-CoA levels extracted from HeLa S3 cells cultured with the indicated concentration of sodium crotonate (pH 7.4) or sodium acetate (pH 7.4) added to full media for 12 hours. The data represent mean peak area ± standard deviation of four independent experiments. Summary of p-value is as follows: ns (p>0.05), * (p≤0.05), ** (p≤0.01), *** (p≤0.001), **** (p≤0.0001).

(C) Histones were acid extracted from HeLa S3 cells treated as in (B) and immunoblotted by the indicated antibodies.

(D) HeLa S3 cells were transfected with control or ACSS2-specific siRNA (pool of 5). 12 hours prior to harvest 10 mM crotonate was added to the media as indicated. 72 hours post-transfection cells were harvested and subject to LC-MS analysis as in (B). The data represent mean peak area ± standard deviation of four independent experiments. p-value summary as in (B).

(E) Same as (D), except 72 hours post-transfection, histones and whole cell lysates were prepared, and immunoblotted with the indicated antibodies. Immunoblots of acid extracts are shown above the black line, while immunoblots of whole cell lysates are shown bellow the black line.

(F) HeLa S3 cells were transfected with control or specific siRNAs to ACSS2. ACSS2 #1-3 are unique single RNAs, and ACSS2 #4 is the pool of 5 used in (D–E). Analysis and labeling as in (C) and (E). See also Figures S3 and S4.