We also tested whether increased histone acetylation upon butyrate treatment was linked to elevated gene expression levels. Several strongly upregulated genes (Log2 fold change > 4) displayed hyper-acetylation at their TSS regions (representative examples are F2RL1 and CLGN, which show 1500 and 675 fold increased expression respectively; Fig. 4D). Indeed, acetylation at the TSS of the 50 most upregulated genes was increased at all measured timepoints (Fig. 4E, left histogram). Acetylation at the TSS of a broader set of the 100 most upregulated genes was also increased at 3 h after butyrate exposure, although H3K27Ac levels returned to near basal levels after 12 and 24 h (Fig. 4E, middle histogram). However, the 451 remaining upregulated genes, which showed weaker induction and higher baseline TSS acetylation, showed reduced TSS H3K27Ac signals in response to prolonged (>3 h) butyrate treatment (Fig. 4E, right histogram). Area under the curve quantification validated that acetylation at the TSS of strongly upregulated genes rapidly increased, whereas acetylation at the TSS of moderately or weakly upregulated genes actually decreased (Fig. 4F). Notably, the most strongly upregulated genes (i.e. top 50/100) displayed lower baseline acetylation (Fig. 4E) and RNA expression (Supplementary Fig. 4), whereas less strongly upregulated genes had established TSS acetylation and higher expression prior to butyrate treatment (Supplementary Fig. 4). These results suggests that transient hyper-acetylation at the TSS of genes may be sufficient to induce gene activation at poorly expressed or silent genes, whereas the butyrate-mediated induction of already robustly expressed genes is regulated by TSS acetylation-independent mechanisms.