EPOP and MTF2 Activate PRC2 Activity through DNA-sequence specificity

The Polycomb repressive complex 2 (PRC2) establishes transcriptionally silent chromatin by catalyzing histone H3 lysine 27 trimethylation (H3K27me3). Although cofactors such as MTF2 and JARID2 are known to direct PRC2 to nucleation sites and stimulate its catalytic activity, the role of the PRC2-associated factor EPOP has remained unclear and even controversial. Here, we combine biochemical reconstitution and a cellular EED-rescue system to define EPOP's contribution to initial PRC2 recruitment and de novo H3K27me3 deposition. We show that EPOP, like MTF2, directly stimulates PRC2 histone methyltransferase activity in vitro. In vivo, EPOP is dispensable for initial PRC2 recruitment but promotes de novo H3K27me3 deposition in cooperation with MTF2 and JARID2. Mechanistically, electrophoretic mobility shift assays and reconstituted chromatin substrates reveal that EPOP and MTF2 enhance PRC2–nucleosome binding in a sequence- and chromatin-dependent manner: EPOP preferentially potentiates activity on GA-rich dinucleosomes, whereas MTF2 favors GCN-rich substrates. Together, these findings position EPOP as a positive regulator of PRC2 function during the establishment of H3K27me3 domains, and reveal a paradigm in which distinct cofactors confer DNA-sequence-specific control of de novo H3K27me3 deposition. Overall design: To dissect the contribution of PRC2 accessory factors to initial PRC2 recruitment and de novo H3K27me3 deposition, we used a mouse embryonic stem cell (mESC) system in which the core PRC2 subunit EED is knocked out, eliminating PRC2 chromatin binding and H3K27me3 after passaging. Re-expression of HA-tagged EED (EED-rescue) was induced and profiled at 0 h (pre-rescue baseline) and 24 h (early de novo phase). We performed ChIP-seq for HA-EED (proxy for PRC2 chromatin recruitment) and ChIP-seq for H3K27me3 (histone mark deposition) across a factorial panel of genotypes: wild-type accessory factor background and targeted knockouts of MTF2, JARID2, EPOP, as single KOs and double KOs (MTF2/JARID2, EPOP/MTF2, EPOP/JARID2).This design enables direct comparison of initial PRC2 recruitment (HA-EED ChIP-seq) and de novo H3K27me3 deposition (H3K27me3 ChIP-seq) kinetics in the presence or absence of individual cofactors during the onset of Polycomb domain formation.