R-loops and PRC1 repress Polycomb-target genes in mouse embryonic stem cells. R-loops and PRC1 repress Polycomb-target genes in mouse embryonic stem cells

R-loops are three-stranded nucleic acid structures that form naturally during transcription, especially over unmethylated CpG-rich promoters. In mESC, such promoters of developmental regulator genes are occupied by the Polycomb-repressor complexes PRC1 and PRC2. Here we have explored the possibility that R-loops form over Polycomb-repressed genes and play a role in their transcriptional silencing. Using single gene and genome-wide analyses, we show that R-loops form at a specific subset of PRC-target genes and contribute to Polycomb occupancy on chromatin. Removal of R-loops leads to an up-regulation of nascent and processed transcripts and the appearance of the elongating form of RNA polymerase II. In contrast, removal of PRC2 does not influence R-loop formation, transcriptional repression and PRC1 recruitment. We finally show that R-loops and PRC1 can repress Polycomb-target genes in the absence of PRC2. Our results uncover an unanticipated synergy between R-loops and PRC1 in Polycomb repression mechanisms. Overall design: Genome-wide distribution of PRC2 subunits EZH2 and SUZ12 in mESC in presence or absence of R-loops

R环（R-loops）是一类天然形成于转录过程中的三链核酸结构，尤其富集于未甲基化的富含CpG的启动子区域。在小鼠胚胎干细胞（mESC）中，发育调控基因的此类启动子区域会被多梳抑制复合物PRC1与PRC2所占据。本研究探讨了R环在多梳抑制靶基因区域形成，并参与其转录沉默过程的可能性。通过单基因与全基因组分析，我们发现R环形成于PRC靶基因的特定子集，并有助于染色质上的多梳复合物占据。去除R环会导致新生转录本与加工后转录本的表达上调，同时出现RNA聚合酶II的延伸状态。与之相反，去除PRC2并不会影响R环形成、转录抑制以及PRC1的招募。我们最终证实，在PRC2缺失的情况下，R环与PRC1仍可抑制多梳靶基因的表达。本研究结果揭示了R环与PRC1在多梳抑制机制中未被预料到的协同作用。整体实验设计：在存在或缺失R环的小鼠胚胎干细胞（mESC）中，多梳复合物PRC2亚基EZH2与SUZ12的全基因组分布情况。