Mapping early PRC2 nucleation sites upon Suz12 reintroduction reveals features of de novo Polycomb recruitment

Polycomb domains safeguard cell identity by maintaining lineage-specific chromatin states enriched in repressive histone modifications, preserving the epigenetic memory of cell lineages. While Polycomb repressive complex 2 (PRC2) can reestablish its occupancy after perturbation, the mechanisms that guide early de novo Polycomb recruitment remain unclear. To study the characteristics of de novo Polycomb recruitment sites, we engineered an auxin-inducible degradation system to reversibly deplete and reintroduce the endogenous PRC2 core subunit Suz12 in mouse embryonic stem cells (mESCs). Genome-wide profiling at an early recovery time point revealed ~ 1,100 PRC2 nucleation sites characterized by rapid Suz12 and H3K27me3 re-accumulation, with minimal impact on gene expression. These sites were significantly enriched at bivalent promoters, coinciding with unmethylated CpG islands and chromatin states associated with developmental regulation, and were largely conserved in differentiated cells. Motif analysis revealed G/C-rich DNA sequences associated with E2F and zinc-finger proteins, alongside strong co-occupancy with MTF2 and JARID2, two PRC2 cofactors previously implicated in Polycomb targeting. H2AK119ub1 persisted at nucleation sites in the absence of PRC2, and reanalysis of published data showed a preferential association of canonical PRC1 (Cbx7-containing) at these regions, while a subset of nucleation sites overlapped with long-range chromatin interaction anchors. These findings support that PRC2 nucleation sites are associated with a combination of chromatin states, DNA sequence features, cofactor co-occupancy, and spatial genome organization, embedded within a pre-existing canonical-PRC1 context, suggesting that epigenetic memory can be reestablished through defined genomic and chromatin features.