DROSHA, DICER and Damage-Induced long ncRNA control BMI1-dependent transcriptional repression at DNA double-strand break. Esposito&Capozzo et al.

At DNA Double strand break sites, local chromatin modifications and damage-induced transcriptional silencing in cis (DISC) occur concomitantly with RNA PolII dependent de novo transcription of damage-induced long non-coding RNAs (dilncRNAs). We hypothesized that the two events could be reconciled in a unique mechanism. We discovered that the two RNAi machinery factors DICER, DROSHA, together with dilncRNAs and the shorter products DDRNAs, sustain the recruitment of the Polycomb protein BMI1 at the sites of break, fostering local H2AK119Ub deposition and DISC. We studied the process mainly in the DIvA cellular system, where endogenous annotated DSBs are enzymatically induced. We analyzed transcriptional repression of 6 DSB-bearing genes via RT-qPCR and investigated BMI1 recruitment at damage sites via ChIP, PLA or DI-PLA in cells depleted for DICER and DROSHA or where dilncRNAs-DDRNAs axis was impaired via antisense oligonucleotides; dissected BMI1-DRSOHA, BMI1-dilncRNAs and BMI1-DDRNAs interaction at the site of break; generated transcriptomic data in DIvA cells depleted for DICER/DROSHA.在DNA双链断裂（DNA Double Strand Break, DSB）位点，局部染色质修饰与顺式损伤诱导转录沉默（damage-induced transcriptional silencing in cis, DISC）可与依赖RNA Pol II的损伤诱导长链非编码RNA（damage-induced long non-coding RNAs, dilncRNAs）从头转录伴随发生。我们提出假说，这两类事件可通过单一机制实现统一阐释。本研究发现，RNA干扰（RNA interference, RNAi）机制因子DICER、DROSHA，连同dilncRNAs及其较短产物损伤诱导小RNA（damage-induced small RNAs, DDRNAs），可促进多梳蛋白BMI1（Polycomb protein BMI1）在断裂位点的招募，进而推动局部组蛋白H2A赖氨酸119位泛素化（H2AK119 ubiquitination, H2AK119Ub）修饰沉积与DISC的形成。本研究主要依托DIvA细胞系统开展实验，该系统可通过酶促手段诱导内源性注释DSBs的产生。我们通过实时定量聚合酶链反应（Real-Time Quantitative Polymerase Chain Reaction, RT-qPCR）分析了6个携带DSBs的基因的转录抑制水平，并在DICER与DROSHA敲低的细胞，或通过反义寡核苷酸破坏dilncRNAs-DDRNAs调控轴的细胞中，借助染色质免疫沉淀（Chromatin Immunoprecipitation, ChIP）、邻近连接测定（Proximity Ligation Assay, PLA）或原位DNA邻近连接测定（DNA in situ Proximity Ligation Assay, DI-PLA）探究了BMI1在损伤位点的招募情况；我们还解析了断裂位点处BMI1与DROSHA、BMI1与dilncRNAs以及BMI1与DDRNAs的相互作用；并在DICER/DROSHA双敲低的DIvA细胞中生成了转录组数据。