Fig 1: ACAA2 is associated with promoters and required for H3K9 butyrylation. Twelve-week-old C57BL/6J mice were maintained on a low-fat (10 Kcal %) or high-fat (60 Kcal %) diet for 4 days before subjecting them to transverse aortic constriction (TAC) or sham surgeries. They were then maintained on the same diets for 7 days before they were assessed by echocardiography, sacrificed, and the hearts isolated and pooled (n = 3 each). Chromatin was extracted from the heart tissue and subjected to anti-ACAA2 ChIP–Seq. A–B. The average signal (Avg Signal) of the chromatin-bound ACAA2 sequence reads (Y axis) assembled at gene promotors (−2 Kb to +2 Kb from the transcription start sites [TSS], X axis) were graphed for each condition (see color keycode at top) or C–D. all reads were represented by heatmaps aligned across the same region. Each input was generated from a pool of Sham and TAC heart chromatin. E. ACAA2-bound sequence fragments (Y axis) aligned to chromosome coordinates (X axis) are shown for Clasp1, Arpc4, and Usp1 genes. Each ChIP–Seq reaction was conducted with a pool of 3 hearts (LV). The anti-ACAA2 ChIP–Seq was 2x for the low-fat diet with similar results (1 set, sham and TAC, in this figure and 1 set previously published, GEO accession # GSE119391) and 1x for the high-fat diet. F. A diagram of the β-oxidation pathway and the enzymes that catalyze it depicted in red. Deletion of ACADS (ΔACADS), HADHA (ΔHADHA), or ACAA2 (ΔACAA2) and its consequences on the intermediate metabolites are shown, where the upward arrow signifies an increase and the downward arrow signifies a decrease in the indicated intermediates. Accumulation of acetoacetyl-CoA upon deletion of ACAA2 inhibited the trifunctional enzyme activity. Short-chain acyl-CoA was salvaged by ketogenesis as depicted in light blue. G. Recombinant H3K9Ac, H3K9Bu, and H3K18Bu nucleosomes (25, 50, and 75 ng of protein) were analyzed by Western blotting using the corresponding antibodies. H. Protein from human Hap1, Hap1ΔACAA2, J. Hap1ΔHADHA, or L. ΔHap1ΔACADS cells was extracted, fractionated into cytosol (Cyto), membrane/mitochondria (Mem/mito), nucleoplasm (Nuc), and analyzed by Western blotting for the molecules listed on the right of each panel (n = 3–6 each). I., K., and M. H3K9Bu or H3K9Cr signals were normalized to H3 or POL II, averaged, and the relative values calculated and graphed after adjusting one of the Hap1 sample levels to 1. Error bars represent S.E.M. The brackets encompass the values that were statistically compared for significance, with the p values listed above each.