Macromolecular interactions dictate Polycomb-mediated epigenetic repression [ChIP-seq]

Dynamic regulation of epigenetic states relies on complex macromolecular interactions such as at the protein-protein and protein-DNA level. It remains an unsolved question how H3K27me3, the hallmark histone modification for facultative heterochromatin, and its writer Polycomb repressive complex 2 (PRC2) are spatiotemporally regulated during development. Here we engineered separation-of-function mutants to surgically dissect the roles of individual macromolecular interactions required for PRC2 function. We show that Polycomb-mediated silencing is precisely regulated by the dynamic interactions among the PRC2 core complex, its accessory proteins, DNA sequences, and other histone modifications. Combining CRISPR-mediated engineering of separation-of-function mutants, human stem cell differentiation models, next-generation sequencing approaches, and reconstituted biochemical assays, we identified distinct regulatory functions of these macromolecular interactions on epigenetic repression in human pluripotent stem cells and cardiac differentiation. Disruption of key interactions led to distinct and opposing effects on cardiomyocyte differentiation, suggesting their highly specified roles in cell fate determination. Together, these results reveal the importance of individual macromolecular interactions at the center of PRC2 controlling epigenetic repression.