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.