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 re-establish its occupancy after perturbation, the mechanisms that guide de novo Polycomb recruitment remain unclear. To address this, 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 histone H3K27me3 re-accumulation with strong signal, 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 identified 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. Notably, a subset of nucleation sites overlapped with long-range chromatin interaction anchors in histone H3K27me3 HiChIP datasets. These findings reveal that PRC2 de novo nucleation sites are associated with a combination of chromatin states, DNA sequence features, cofactor co-occupancy and spatial genome organization, suggesting that epigenetic memory can be re-established through defined genomic and chromatin features. Author summaryPolycomb group proteins are key epigenetic regulators that silence gene expression by establishing and dispersing repressive chromatin domains marked by histone modifications such as histone H3K27me3 and H2AK119ub1 and are critical for defining cell identity. During differentiation, Polycomb domains are dynamically redistributed, implying a mechanism for de novo targeting to specific loci. How the Polycomb Repressive Complex 2 (PRC2) is initially recruited to these nucleation sites and which features stabilize its binding remain poorly understood. To explore this, we studied the characteristics of the de novo recruitment sites of PRC2 in mouse embryonic stem cells (mESCs) using an auxin-inducible degradation (AID) system targeting PRC2 to deplete and reintroduce the core subunit Suz12. We identified recruitment sites after complete clearance of the histone H3K27me3 through ChIP-seq at a very early time point after Suz12 reintroduction. Most nucleation sites were located at bivalent promoters of developmental genes and correlated with unmethylated CpG islands. Motif analysis revealed over-represented sequences and accessory partners such as MTF2 and JARID2, along with long-range chromatin interactions. These nucleation sites were also conserved in differentiated cells, highlighting their potential role in developmental regulation. Our study provides insight into how Polycomb domains are established and how epigenetic memory is maintained in stem cells.