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, the CBX7-PRC1 complex, regulate facultative heterochromatin via a phase separation mechanism. Facultative and constitutive heterochromatin represent distinct, coexisting condensates in nuclei. In vitro, H3K27me3- and H3K9me3-marked nucleosomal arrays and their reader complexes can phase separate into immiscible condensates that are analogous to the relationship between facultative and constitutive heterochromatin in vivo. Moreover, overexpression of CBX7-PRC1 causes aberrant chromatin compartmentalization as demonstrated by H3K9me3 CUT&Tag and up-regulation of genes related to cancer, such as acute myeloblastic leukemia (AML). Chromobox 7 (CBX7) inhibitor effectively inhibits cancer cell proliferation, possibly through phase-separation-mediated compartment reorganization. Our data demonstrate how the specificity of compartmentalization is achieved based on the formation of immiscible phase-separated condensates and offer potential epigenetic mechanistic insights into tumor development.