Common fragile sites (CFSs) are breakage-prone genomic loci, and are considered to be hotspots for genomic rearrangements frequently observed in cancers. Understanding the underlying mechanisms for CFS instability will lead to better insight on cancer etiology. Here we show that Polycomb group proteins BMI1 and RNF2 are suppressors of transcription-replication conflicts (TRCs) and CFS instability. Cells depleted of BMI1 or RNF2 showed slower replication forks and elevated fork stalling. These phenotypes are associated with increase occupancy of RNA Pol II (RNAPII) at CFSs, suggesting that the BMI1-RNF2 complex regulate RNAPII elongation at these fragile regions. Using proximity ligase assays, we showed that depleting BMI1 or RNF2 causes increased associations between RNAPII with EdU-labeled nascent forks and replisomes, suggesting increased TRC incidences. Increased occupancy of a fork protective factor FANCD2 and R-loop resolvase RNH1 at CFSs are observed in RNF2 CRISPR-KO cells, which are consistent with increased transcription-associated replication stress in RNF2-deficient cells. Depleting FANCD2 or FANCI proteins further increased genomic instability and cell death of the RNF2-deficient cells, suggesting that in the absence of RNF2, cells depend on these fork-protective factors for survival. These data suggest that the Polycomb proteins have non-canonical roles in suppressing TRC and preserving genomic integrity.

常见的易碎位点（CFS）是易断裂的基因组位点，被认为是癌症中经常观察到的基因组重排的热点。了解CFS不稳定性的潜在机制将导致对癌症病因学的更好认识。在这里，我们显示Polycomb组蛋白BMI1和RNF2是转录复制冲突（TRC）和CFS不稳定性的抑制剂。耗尽BMI1或RNF2的细胞显示出较慢的复制叉和升高的叉停滞。这些表型与CFS处RNA Pol II（RNAPII）的增加占有率有关，表明BMI1-RNF2复合物调节这些脆弱区域的RNAPII延伸。使用邻近连接酶分析法，我们发现消耗BMI1或RNF2会导致RNAPII与EdU标记的新生叉和复制体之间的关联增加，提示TRC发生率增加。在RNF2 CRISPR-KO细胞中观察到叉保护因子FANCD2和R环溶解酶RNH1在CFS处的占用增加，这与RNF2缺陷细胞中转录相关的复制应激增加有关。耗尽FANCD2或FANCI蛋白会进一步增加RNF2缺陷细胞的基因组不稳定性和细胞死亡，这表明在缺乏RNF2的情况下，细胞的存活依赖于这些叉保护因子。这些数据表明，Polycomb蛋白在抑制TRC和保持基因组完整性方面具有非典型作用。耗尽FANCD2或FANCI蛋白会进一步增加RNF2缺陷细胞的基因组不稳定性和细胞死亡，这表明在缺乏RNF2的情况下，细胞的存活依赖于这些叉保护因子。这些数据表明，Polycomb蛋白在抑制TRC和保持基因组完整性方面具有非典型作用。耗尽FANCD2或FANCI蛋白会进一步增加RNF2缺陷细胞的基因组不稳定性和细胞死亡，这表明在缺乏RNF2的情况下，细胞的存活依赖于这些叉保护因子。这些数据表明，Polycomb蛋白在抑制TRC和保持基因组完整性方面具有非典型作用。