javascript:void(0)Discussion
Although we and others have demonstrated that butyrate and other HDAC inhibitors can regulate the activity of mast cells and other immune cells, it remains incompletely understood how such broadly-acting molecules can exert rather precise and cell-type specific transcriptional changes. By integrating RNA-Seq and ChIP-Seq datasets obtained from butyrate-treated primary human mast cells, we uncovered that butyrate controls mast cell function through complex yet highly specific changes in histone acetylation – including a loss of acetylation at super-enhancers that control key mast cell degranulation genes. Pharmacological inhibition of super-enhancers indeed suppressed mast cell degranulation, similar to what was observed for butyrate.
We found that gene repression following butyrate treatment is primarily associated with H3K27Ac depletion around the TSS of highly expressed genes. Although seemingly counterintuitive, many studies have reported gene repression triggered by HDAC inhibitors44. Highly expressed genes were previously shown to be prime targets of HDAC inhibitors in (cancer) cell lines45,46, including KIT in transformed human mast cells47, in line with our own findings. This may be explained by preferential binding of HDACs to highly expressed genes, where they are considered to be critical for maintaining the precise acetylation-deacetylation balance required for productive gene transcription46,48. As histone acetylation attracts many proteins involved in transcriptional control, a global redistribution of histone acetylation, as caused by butyrate, is likely to redirect these chromatin readers away from their target regulatory regions46. While our findings agree with these notions, we also show that many highly-expressed and strongly acetylated genes are completely impervious to HDAC inhibition by butyrate, indicating that additional mechanisms determine the remarkable selectivity by which butyrate affects the epigenome and transcriptome of mast cells.
In agreement with findings by Rada-Iglesias et al. 49, our analysis ofH3K27Ac dynamics in mast cells revealed that butyrate-induced hyper-acetylation is mostly an early and transient event, since hypo-acetylation became the dominant effect at 24 hours. This suggests that histone deacetylation might be a secondary effect of HDAC inhibition, as HATs gain a competitive advantage for acetyl groups and are able to deposit these at new locations. In primary human mast cells, loss of H3K27Ac at gene regulatory regions upon butyrate treatment was not caused by notable reductions in HAT expression (data not shown). Important to note here is that a previous study of the HDAC inhibitor largazole revealed that exposure to low concentrations solely induced gene activation, whereas higher concentrations shifted the balance towards gene repression 50. Whether a similar dose-dependent response exists for butyrate will need to be addressed in subsequent studies.
To our best knowledge, we are the first to define super-enhancers in primary human mast cells, and describe their potential relevance for mast cell biology. Super-enhancer-associated genes in human mast cells were strongly linked to immune effector cell processes, such as cell activation, exocytosis and secretion. Likely due to their high HDAC and HAT occupancy, ~84% of super-enhancers contained hypo-acetylated regions after butyrate treatment, which correlated with reduced transcriptional output of many nearby genes. Among these affected super-enhancer-associated genes are many core regulators of mast cell identity and function, including the KIT receptor, FceRI signaling components and degranulation-associated factors. Interestingly, perturbation of super-enhancer activity by JQ-1 inhibited degranulation of primary human mast cells, most likelyvia downregulation of various key mast cell identity genes that are also targeted for repression by butyrate. Indeed, specific repression of core cell identity gene expression by HDAC inhibitors has been previously reported50,51. Thus, it appears plausible that at least part of the inhibitory effect of butyrate is a direct consequence of super-enhancer destabilization. How mast cell activation subsequently reorganizes the chromatin landscape, a phenomenon recently described by Cildir et al 52, and which specific (combination of) repressed genes form the foundation of butyrate’s inhibitory effects, are important topics for future studies.
In summary, our data indicate that butyrate inhibits mast cell activation via a surprisingly selective suppression of the core mast cell transcriptional program, in part by targeting super-enhancers. Acquiring a deeper understanding of the mechanisms of action of butyrate, and other HDAC inhibitors, may in the future offer improved ways to combat mast cell-mediated diseases such as allergies and asthma.