ORCA-mediated Polycomb Repressive Complex stabilization maintains 3D-genome architecture and controls replication timing

Polycomb bodies, also called Polycomb group (PcG) protein complexes, are implicated in gene regulation, especially during development, differentiation, and during the maintenance of tissue-specific functions in multicellular organisms. Predominantly, in transformed cells, PcG bodies  form distinct nuclear foci, within which PcG-bound DNA is localized. These structures likely represent misregulated gene expression due to the loss of normal regulatory mechanisms, yet the molecular function of these bodies in cellular homeostasis remains unknown. Using TSA-Seq (Tyramide signal amplification), we provide the first-of-a-kind comprehensive view of the genomic regions, including specific centromeric and telomeric sites that are in proximity to the PcG bodies. Further, these regions are enriched for the repressive histone marks, H3K27me3, H3K9me3 and depleted for active marks, H3K4me3 consistent with a lack of accessible chromatin. We find high density of replication origins around PcG bodies, consistent with the replication factor, ORCA/LRWD1 localizing at these sites. ORCA interacts with the PRC complex, stabilizes the H3K27 methyltransferase, and facilitates the deposition of this histone mark at specific chromatin sites. Loss of ORCA impacts the chromatin organization around the PcG bodies, leading to decompaction of the repeat regions, and enhanced initiation from replication origins. Our results indicate that ORCA interacts with the PcG complex-bound chromatin and maintains a repressed heterochromatin environment to control the timing of replication origin firing.