Histone marks enable formation of immiscible phase-separated chromatin compartments

Eukaryotic chromatin is organized into compartments for gene expression regulation, but the underlying mechanisms remain unclear. Here, we demonstrate that multivalent H3K27me3 and its reader, CBX7-PRC1, regulate facultative heterochromatin via a phase separation mechanism, similar to constitutive heterochromatin. Facultative and constitutive heterochromatin represent distinct, coexisting repressive condensates in nuclei. H3K27me3- and H3K9me3-marked nucleosomal arrays and their reader complexes phase separate into immiscible condensates, analogous to the relationship between facultative and constitutive heterochromatin in vivo. Overexpression of CBX7-PRC1 causes aberrant chromatin compartmentalization in vivo and up-regulation of cancer-related genes. CBX7 inhibitor effectively inhibits cancer cell proliferation possibly through phase separation-mediated compartment reorganization. Our data suggest that histone marks mediate chromatin compartmentalization via phase separation, and offer potential epigenetic mechanistic insights into tumor development and cancer therapy. Overall design: By employing RNA-seq, ChIP-seq, CUT&Tag, and Hi-C, we systematically examined the change in chromatin features and transcriptome upon overexpression of CBX7 in NIH3T3 cells.