EZH2 Serine 21 Phosphorylation Restrains Compact-State PRC2 Activation and H3K27me3 Propagation

Polycomb Repressive Complex 2 (PRC2) propagates H3K27me3 through EED-dependent allosteric activation, yet how cells modulate the magnitude of this positive-feedback response remains poorly understood. Here, we identify phosphorylation of EZH2 serine 21 as a post-translational mechanism that attenuates PRC2 allosteric responsiveness. Prior structural studies have established that activator-bound PRC2 adopts both compact and extended active conformations. Using cryo-EM classification of wild-type and phospho-null EZH2 S21A PRC2 complexes, we find that the phospho-null EZH2 S21A substitution changes the distribution of particles across these pre-existing states, shifting PRC2 from a predominantly extended conformation to one enriched for the compact, allosterically activated conformation. Consistent with this structural transition, EZH2 S21A increases basal PRC2 activity, lowers the EC ₅₀ for H3K27me3-dependent stimulation, and accelerates H3K27me3 accumulation on peptide and nucleosome substrates. Disruption of the EED-EZH2 interface suppresses the S21A gain-of-activity phenotype, indicating that S21 phosphorylation constrains PRC2 by limiting productive EED-EZH2 allosteric coupling. In mesenchymal stem cells, loss of this phosphorylation-dependent restraint broadens H3K27me3 domains, reduces canonical PRC1 enrichment at high-occupancy Polycomb target loci, misregulates lineage-associated transcriptional programs, and impairs differentiation. These findings identify EZH2 S21 phosphorylation as a molecular rheostat that limits compact-state PRC2 activation, constrains H3K27me3 spreading, and preserves Polycomb-dependent developmental competence.