UNLABELLED: The dynamic regulation of epigenetic states relies on complex macromolecular interactions. PRC2, the methyltransferase complex responsible for depositing H3K27me3, interacts with distinct accessory proteins to form the mutually exclusive subcomplexes PHF1-PRC2.1, MTF2-PRC2.1, PHF19-PRC2.1, and PRC2.2. The functions of these subcomplexes are unclear and thought to be highly redundant. Here we show that PRC2 subcomplexes have distinct roles in epigenetic repression of lineage-specific genes and stem cell differentiation. Using a human pluripotent stem cell model, we engineered a comprehensive set of separation-of-function mutants to dissect the roles of individual protein-protein and DNA-protein interactions. Our results show that PRC2.1 and PRC2.2 deposit H3K27me3 locus-specifically, resulting in opposing outcomes in cardiomyocyte differentiation. We find that MTF2 stimulates PRC2.1-mediated repression in stem cells and cardiac differentiation through its interaction with DNA and H3K36me3, while PHF19 antagonizes it. Furthermore, MTF2-PRC2.1 maintains normal cardiomyocyte function. Together, these results reveal the importance and specificity of individual macromolecular interactions in Polycomb-mediated epigenetic repression in human stem cells and differentiation.

HIGHLIGHTS: The PRC2.1 and PRC2.2 subcomplexes have distinct specificities for H3K27me3 depositionThe three PCL accessory proteins have distinct functions in regulating the PRC2 core complex, with MTF2 and PHF19 antagonizing each otherInteractions between the PCL proteins and the PRC2 core, DNA, and H3K36me3 dictate PRC2 occupancy and activity at developmental genesPRC2.1 and PRC2.2 play opposing roles in stem cell cardiomyocyte differentiationMTF2 plays key functions in regulating differentiation timing and action potential rhythm in cardiomyocytes.