Polycomb-mediated 3D-genome organization controls replication timing.

Polycomb (PcG) bodies are nuclear foci formed by polycomb protein complexes that contain PcG-bound DNA and are implicated in gene regulation during development and differentiation, although their precise molecular function remains unclear. Using tyramide signal amplification sequencing, we provide a comprehensive view of genomic regions associated with PcG bodies, including specific centromeric and telomeric sites. These regions are enriched for the repressive marks H3K27me3 and H3K9me3, depleted of the active mark H3K4me3, and display low chromatin accessibility. We find a high density of replication origins around PcG bodies, consistent with the replication factor origin recognition complex-associated protein (ORCA)/LRWD1 localizing at these sites. ORCA interacts with the polycomb-repressive complex, stabilizes the H3K27 methyltransferase, and facilitates H3K27me3 deposition at specific chromatin sites. Loss of ORCA affects chromatin organization around PcG bodies, leading to decompaction of the repeat regions, and enhanced initiation from replication origins. Our results suggest that ORCA associates with PcG-bound chromatin to maintain a repressive environment that regulates replication origin firing timing.