The interaction of PRC2 with RNA or chromatin is mutually antagonistic [ChIP-Seq]

Polycomb repressive complex 2 (PRC2) maintains developmental regulator genes in a repressed state through methylation of histone H3 at lysine 27 (H3K27me3) and is necessary for cell differentiation. We and others have previously found that the PRC2 subunit Suz12 interacts with RNA in vitro and other studies have shown that Ezh2 and Jarid2 also possess RNA binding function. The interaction of PRC2 with RNA has been suggested to regulate PRC2 targeting or enzymatic activity, but the RNAs directly bound by PRC2 in cells, and the role of each PRC2 RNA binding subunit, remain unclear. We have used different CLIP techniques, which use UV-crosslinking to allow detection of direct Suz12-RNA interactions as they occur in living mouse ES cells. Suz12 binds nascent RNA and has a preference for interaction with the 3'UTR, showing it does have binding specificity in cells. RNAs bound by Suz12 at the 3'UTR encode developmental regulator genes. Suz12 remains bound to RNA upon deletion of Ezh2 or Jarid2 showing that it binds RNA independently of other PRC2 subunits. We also show that binding of Suz12 to RNA or chromatin is mutually inhibitory. Although Ezh2 and Jarid2 also bind RNA, Ezh2 and Jarid2 deletion causes an increase in Suz12 RNA binding, without changing its specificity, which reflects the loss of Suz12 from chromatin. Similarly, disruption of Suz12-RNA interactions by RNA polymerase II inhibition or RNase treatment increases Suz12 binding to chromatin. These results therefore suggest that Suz12 acts as an RNA sensor, binding to the 3'UTR of nascent RNAs and modulating the interaction of PRC2 with chromatin. Overall design: Suz12, and Ezh2 ChIP-seq experiments before and after Tamoxifen treatment of Mus musculus Ezh2 fl/fl Stem Cells.

多梳抑制复合体2（Polycomb repressive complex 2, PRC2）通过对组蛋白H3赖氨酸27位点进行甲基化修饰（H3K27me3），将发育调控基因维持在抑制状态，且该复合体对细胞分化至关重要。本研究团队与其他课题组此前均发现，PRC2的亚基SUZ12可在体外与RNA结合；另有研究表明，EZH2与JARID2同样具备RNA结合能力。已有研究推测PRC2与RNA的互作可调控PRC2的靶向定位或酶活性，但目前学界仍未明确细胞内PRC2直接结合的RNA种类，以及每个PRC2 RNA结合亚基的具体功能。本研究采用了多种紫外交联免疫沉淀技术（cross-linking immunoprecipitation, CLIP），该技术借助紫外交联手段，可在活体小鼠胚胎干细胞中直接检测SUZ12与RNA的天然互作。SUZ12可结合新生RNA，且对3'非翻译区（3' untranslated region, 3'UTR）存在结合偏好性，这表明其在细胞内确实具备结合特异性。SUZ12通过3'UTR结合的RNA，其编码产物均为发育调控基因。在敲除EZH2或JARID2后，SUZ12仍可与RNA结合，这说明SUZ12无需依赖PRC2其他亚基即可独立结合RNA。本研究同时证实，SUZ12与RNA的结合和其与染色质的结合存在相互抑制效应。尽管EZH2与JARID2同样可结合RNA，但敲除EZH2或JARID2会提升SUZ12与RNA的结合水平，且不会改变其结合特异性，这一现象反映了SUZ12从染色质上的解离。类似地，通过抑制RNA聚合酶II活性或核糖核酸酶（RNase）处理破坏SUZ12与RNA的互作，会增强SUZ12与染色质的结合能力。综上，上述结果表明SUZ12可作为RNA感受器，通过结合新生RNA的3'UTR，调控PRC2与染色质的互作。实验总体设计：对小家鼠Ezh2 fl/fl干细胞进行他莫昔芬处理前后，分别开展SUZ12与EZH2的染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）实验。