cPRC1.2 and CTCF-Mediated Transition from Poised to Active Chromatin Loops Drives Bivalent Gene Activation

Polycomb Repressive Complex 1 (PRC1) and CCCTC-binding factor (CTCF) are critical regulators of 3D chromatin architecture that influence cellular transcriptional programs. Spatial chromatin structures comprise conserved compartments, topologically associating domains (TADs), and dynamic, cell-type-specific chromatin loops. Although the role of CTCF in chromatin organization is well-known, the involvement of PRC1 is less understood. In this study, we identified an unexpected, essential role for the canonical Pcgf2-containing PRC1 complex (cPRC1.2), a known transcriptional repressor, in activating bivalent genes during differentiation. Our Hi-C analysis revealed that cPRC1.2 forms chromatin loops at bivalent promoters, rendering them silent yet poised for activation. Using mouse embryonic stem cells (ESCs) with CRISPR/Cas9-mediated gene editing, we found that the loss of Pcgf2, though not affecting the global level of H2AK119ub1, disrupts these cPRC1.2 loops in ESCs and impairs the transcriptional induction of crucial target genes necessary for neuronal differentiation. Furthermore, we identified CTCF enrichment at cPRC1.2 loop anchors and at Polycomb group (PcG) bodies, nuclear foci with concentrated PRC1 and its tethered chromatin domains, suggesting that PRC1 and CTCF cooperatively shape chromatin loop structures. Through virtual 4C and other genomic analyses, we discovered that establishing neuronal progenitor cell (NPC) identity involves a switch from cPRC1.2-mediated chromatin loops to CTCF-mediated active loops, enabling the expression of critical lineage-specific factors. This study uncovers a novel mechanism by which pre-formed PRC1 and CTCF loops at lineage-specific genes maintain a poised state for subsequent gene activation, advancing our understanding of the role of chromatin architecture in controlling cell fate transitions. We aim to investigate how PRC1 regulates chromatin organization during neuronal differentiation by targeting Pcgf2, an important PRC1 component. We generated Pcgf2 knock-out (KO), Pcgf4 KO, Phc1 KO, Phc1-SAM-domain KO, and Irx3-anchor KO mESC lines via the CRISPR/Cas9 system. Triplicate WT and knock-out mESC lines were differentiated first into embryonic bodies (EBs) and then into neuro-progenitor cells (NPCs). Duplicated samples were subjected to RNA-seq. WT, Pcgf2 KO, Irx3-anchor KO ESC and NPC; and U2OS samples were subjected to ChIP-seq. WT and Pcgf2 KO ESC and Pcgf2 KO NPC samples were subjected to Hi-C.