Functional Characterization of Super-Enhancers in Regulating Cell Identity Genes
DOI:
https://doi.org/10.5281/zenodo.22760827Keywords:
super-enhancer, BRD4, MED1, cell identity, enhancer deletion, H3K27ac, CRISPRi, macrophage differentiationAbstract
Super-enhancers, large clusters of regulatory elements bound at exceptionally high density by lineage-specifying
transcription factors and the Mediator and BRD4 coactivators, have been proposed to disproportionately control the
transcription of genes that define cell identity, but whether individual constituent enhancers within a super-enhancer
contribute equally to this control or act in a more specialized, non-redundant fashion remains contested. We profiled
H3K27ac, MED1 and BRD4 occupancy genome-wide during monocyte-to-macrophage differentiation and ranked
the resulting super-enhancer landscape by signal density, then used CRISPR interference (CRISPRi) and individual
CRISPR-Cas9 deletion of each constituent element within four macrophage identity-gene super-enhancers (at
MAFB, MERTK, CD163 and MRC1) to test their relative functional contribution. H3K27ac and BRD4 density
identified 612 super-enhancers occupying 3.1% of called enhancer space but associated with 41% of total
enhancer signal, strongly enriched near genes induced during differentiation. CRISPRi silencing of any single
constituent within the four target super-enhancers reduced target gene expression, but the magnitude varied more
than eightfold across constituents at the same locus, and deletion of the single highest-ranked constituent at each
locus reduced target gene expression by 58-81%, closely approaching the effect of deleting the entire
super-enhancer, whereas deletion of any of the remaining constituents individually reduced expression by no more
than 15%. Hi-C and 4C-seq showed that the dominant constituent also made the strongest and most frequent
contact with the target promoter, and its deletion weakened contact frequency at the remaining constituents as well,
consistent with a hub architecture in which one element nucleates the looping of the others rather than each
constituent contacting the promoter independently. These results indicate that macrophage identity-gene
super-enhancers are functionally hierarchical rather than fully redundant clusters, with activity concentrated in a
single dominant constituent.
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