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

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 were associated with chromatin interactions and interact preferentially with each other. MRR component removal at interaction anchors by CRISPR led 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 low H3K27me3 and high H3K27ac levels showed changes in chromatin interactions. The MRR knockout cells also showed changes in phenotype associated with cell identity, and altered xenograft tumor growth. MRR-associated genes and long-range chromatin interactions were susceptible to H3K27me3 depletion. Our results characterized H3K27me3-rich regions and their mechanisms of functioning via looping. Overall design: K562 DMSO/5µM GSK343 ChIP-seq have 2 biological replicates for each type of antibody. HAP1 Wild-type (WT)/EZH2 Knockout (EZH2KO) ChIP-seq have 2 biological replicates for each type of antibody. K562 DMSO/5µM GSK343 RNA-seq have 2 biological replicates for each condition. K562 DMSO/1µM GSK343 RNA-seq have 3 biological and 3 technical replicates for each condition. HAP1 WT/EZH2KO RNA-seq have 2 biological replicates for each condition. K562 CRISPR Empty Vector (EV)/Knockout (KO) RNA-seq have 2 biological replicates for each CRISPR clone. K562 EV/MRR2-A1 knockout (IGF1KO)ChIP-seq have 2 biological replicates for each type of antibody. 4C experiments have 2 biological replicates except TMCO4 4C, which only has 1 biological replicate.