To gain a more quantitative picture of H3K27Ac dynamics upon butyrate treatment, we performed differential enrichment analyses with DESeq2 on reproducible peaks, which showed that most regions acetylated at baseline (>68%) were not significantly (fold change > 2 and adjusted P value < 0.05) affected by 3-24 h butyrate treatment (Supplementary Fig. 3). After 3 h exposure, butyrate mostly triggered hyper-acetylation (6.3% hyper-acetylated vs. 2.2% hypo-acetylated peaks), whereas 12 h butyrate exposure showed similar proportions of hyper- and hypo-acetylation at baseline H3K27Ac+ regions (13.3% vs. 12.8%). Finally, 24 h of treatment mostly induced hypo-acetylation of existing peaks (13.1% vs. 18.6%, Supplementary Fig. 3).
To better annotate at which regulatory sites butyrate-induced histone acetylation dynamics occur, we intersected regions of dynamic acetylation with TSS locations and putative typical enhancer or super-enhancer regions as defined by the ROSE algorithm 28,35. At untreated conditions (indicated as 0 h), histone acetylation was primarily located distant from TSS regions, with the bulk of H3K27Ac+ sites located 50-500 kb away from the TSS (Fig. 3A, bar graph). Furthermore, ~52% and ~19% of acetylated peaks were associated with typical enhancers and super-enhancers, respectively, whereas 17% of acetylated peaks were associated with TSS regions (Fig. 3A, donut graph).