Fig. 4: H3K27Ac ChIP–seq and RNA–seq analyses identify SAT1 as a critical regulator of acetate-mediated effects.
a, Distribution of H3K27 acetylation (H3K27Ac) ChIP–seq reads from vehicle-treated (blue) and acetate- treated (red) S2-013 cells within ±10 kb of the TSS. b, Density of ChIP–seq reads for H3K27ac ±10 kb from the TSS in vehicle- and acetate-treated S2-013 cells. c, DAVID-based pathway enrichment analysis of differentially H3K27-acetylated gene promoters in vehicle and acetate-treated S2-013 cell line (statistical analysis using Fisher’s exact test). d, Volcano plot depicting differentially regulated genes (1.5-fold change cutoff) in S2-013 cells upon acetate treatment. e, Venn diagram of ChIP–seq and RNA–seq data showing 282 genes that are upregulated in RNA–seq data with differentially acetylated H3K27 in their gene promoters. f, Heatmap showing 48 differentially regulated genes upregulated by acetate in an ACSS2-dependent manner. The colour bar shows the ratio of fragments per kilobase of transcript per million mapped reads (FPKM) values of siScr, siScr + acetate, siACSS2, or siACSS2 + acetate to that of siScr. g, Volcano plot showing the Cox coefficient and −log(P value) of the 48 regulated genes extracted from the OncoLnc database for survival of patients with PDAC. Prospective oncogenes and tumour suppressors are shown in red and green, respectively. h, Principal component analysis (PCA) plot of S2-013 cells treated with acetate under acidosis relative to untreated cells as determined by LC–MS/MS-based metabolomics (n = 5 biological replicates per group). i, Heatmap of top 25 altered metabolites from S2-013 cells treated with acetate under acidosis and displayed with row Z-score normalization, as determined by LC–MS/MS-based metabolomics (n = 5 biological replicates per group). j, H3K27ac at putative enhancer regions proximal to the SAT1 gene. The y axis shows reads per bin per million. k, Relative mRNA levels of the SAT1 gene in siScr and siACSS2 S2-013 cells in the presence or absence of acetate for 24 h under acidosis (n = 4 in each group, from independent biological replicates; one-way ANOVA with Bonferroni’s post-hoc test, mean ± s.e.m.). l, Relative levels of SAT1 and ACSS2 proteins in scrambled control (siScr) and ACSS2 knockdown (siACSS2) S2-013 cells in the presence and absence of 5 mM acetate for 24 h under acidosis. Immunoblots are representative of two independent experiments.
