PRC2.1 and PRC2.2 Specific Accessory Proteins Drive Recruitment of Different Forms of Canonical PRC1 [ChIP]

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: Cells were collected and washed with PBS 2x, then suspended in 10 ml PBS. Next, cells were fixed with 1% formaldehyde (Sigma) with rotation for 10?minutes, followed by quenching with glycine. Fixed cells were washed with cold PBS (with proteinase inhibitors cocktails, PIC) then lysed in SDS lysis buffer (0.5% SDS, 100mM NaCl, 50mM Tris pH 8.0, 5mM EDTA pH 8.0) pulsed PIC. The lysate was pelleted by spinning at 1400 rpm for 5 minutes at room temperature. With discarding the supernatant, the chromatin pellet was suspended in 0.33% SDS incubation buffer (0.3% SDS, 1.6% Triton X-100, 100mM NaCl, 50mM Tris pH 8.0, 5mM EDTA pH 8.0) pulsed PIC at a concentration of ~30 million cells per ml, followed by Branson Sfx150 Sonifier sonication for total ON 4 minutes (1?second ON, 4?seconds OFF; 50% amplitude) to achieve enrichment of 200~500?bp DNA fragments. Sonicated chromatin was checked by agarose gel electrophoresis or TapeStation 2000 (Agilent) and quantitated with Qubit. For regular ChIP, 10~20 µg chromatin and specific antibodies (e.g. SUZ12, JARID2, MTF2, FLAG, H3K27me3, and H2AK119ub) were incubated overnight with rotation at 4 °C. Following morning, 30 µl protein A/G magnetic beads were added to each ChIP sample, and incubated with rotation at 4?°C for 2 hours. After incubation, the beads were washed 5 times; once with low salt buffer (2?mM EDTA, 20?mM Tris, 1% Triton, 0.1% SDS and 150?mM NaCl), twice with high salt buffer (2?mM EDTA, 20?mM Tris, 1% Triton, 0.1% SDS and 500?mM NaCl) and twice with TE (1?mM EDTA and 10?mM Tris). After the last wash, beads were suspended in 200 µl elution buffer (1% SDS, 0.1 M NaHCO3) and incubated for 15 minutes with rotation at room temperature fallow incubation in the thermomixer for 10 minutes with gentle shaking (500 rpm) at 37?°C. The eluted ChIP supernatant was transferred to a new EP tube and incubated in a thermomixer overnight with 1000 rpm shaking at 65°C to remove the crosslinks. Then, RNase A was added and incubated for 1 hour at 37 °C with shaking; following Proteinase K treatment for 2 hours at 55 °C with shaking. ChIP DNA was purified using the Qiagen MinElute PCR purification kit and measured with the Qubit High-Sensitive DNA assay kit. Quantitative PCR was performed to check the ChIP efficacy with suitable primers. The qPCR primer sequences were shown in SuppTable. In addition, quantitative ChIP-Rx was performed using a modified published approach (Orlando et al., 2014, Healy et al., 2019). Briefly, 10% human NT2 chromatin or Drosophila S2 chromatin was added to mouse chromatin lysate. Mixed chromatin was treated as single regular ChIP-seq experiment until the completion of DNA sequencing.