Gene repression and silencers are poorly understood. We reasoned that H3K27me3-rich regions (MRRs) of the genome defined from clusters of H3K27me3 peaks may be used to identify silencers that can regulate gene expression via proximity or looping. MRRs are associated with chromatin interactions and interact preferentially with each other. MRR-associated genes and long-range chromatin interactions are susceptible to H3K27me3 depletion. MRR component removal at interaction anchors by CRISPR leads to upregulation of interacting target genes, altered H3K27me3 and H3K27ac levels at interacting regions, and altered chromatin interactions. Chromatin interactions did not change at regions with high H3K27me3, but regions with lower H3K27me3 and higher H3K27ac levels showed losses in chromatin interactions, while new interactions emerged at high H3K27ac regions. The MRR knockout cells also showed changes in phenotype associated with cell identity, and altered xenograft tumor growth. Our results characterize H3K27me3-rich regions and their mechanisms of functioning.

H3K27me3-rich genomic regions can function as silencers to repress gene expression via chromatin interactions