PRC2.1 and PRC2.2 Specific Accessory Proteins Drive Recruitment of Different Forms of Canonical PRC1 [4C-seq]

Polycomb repressive complex 2 (PRC2) mediates H3K27me3 deposition, which is thought to recruit canonical PRC1 (cPRC1) via chromodomain-containing CBX proteins to promote stable repression of developmental genes. PRC2 forms two major subcomplexes, PRC2.1 and PRC2.2, but their specific roles remain unclear. Through genetic knockout and replacement of PRC2 subcomplex-specific subunits in naïve and primed pluripotent cells, we uncover distinct roles for PRC2.1 and PRC2.2 in mediating the recruitment of different forms of cPRC1. PRC2.1 catalyses the majority of H3K27me3 at Polycomb target genes and is sufficient to promote recruitment of CBX2/4-cPRC1, but not CBX7-cPRC1. Conversely, while PRC2.2 is poor at catalysing H3K27me3, we find that its accessory protein JARID2 is essential for recruitment of CBX7-cPRC1 and the consequent 3D chromatin interactions at Polycomb target genes. We therefore define distinct contributions of PRC2.1 and PRC2.2 specific accessory proteins to Polycomb mediated repression and uncover a new mechanism for cPRC1 recruitment. Overall design: Circularized chromosome conformation capture (4C) assays were performed using a modified published approach(van de Werken et al., 2012; Wang et al., 2019). Briefly, 10 million detached ESCs or EpiLCs were fixed with 1% final formaldehyde for 10?minutes, and quenched with final 0.125 M glycine. The cells were lysed in the 30?ml lysis buffer (50?mM Tris pH 7.5, 150?mM NaCl, 0.5% NP-40, 1% Triton, 5?mM EDTA, and proteinase inhibitors: 2 µg/mL Aprotinin, 1 µg/mL Leupeptin, 1mM PMSF ) with rotation at 4?°C for 45? minutes. Isolated nuclei were then fully digested by restricted enzyme DpnII overnight digestion followed by overnight ligation with 50 U T4 DNA ligase at cool room temperature (18~20 ?°C) . Ligated circular DNA were purified after de-crosslinking incubation with proteinase K at 65?°C for overnight, followed by further incubation with Rnase A at 37 ?°C for 1 hour . The purified DNA was further digested with restricted enzyme NlaIII and then ligated again and purified. The 4C libraries were amplified with the viewpoint-specific primers with inverse PCR using Roche Expand Long Template PCR System. For each viewpoint, at least 8 PCR reaction products were pooled to enhance the library complexity. The 4C PCR products were purified using Qiagen Quick PCR purification Kit. Tbx3 promoter viewpoint primers are AAGGGAAGAAGCTGCAGATC (Reading primer) and TGAAGGGAGCCCCACATG.