H3K9me2 and BRCA1 cooperate to prevent satellite repeat transcription, R-loop formation and germline sterility [ChIP-seq]

Repetitive sequences, transposable elements and silent tissue-specific genes in C. elegans are differentially enriched for di- and tri-methyl H3 lysine 9 (H3K9). SET-25 (SUV39h1/h2) catalyzes H3K9me3, while MET-2 (SetDB1) deposits only H3K9me1/me2. RNA-seq and genome-wide H3K9 methylation mapping in met-2 and set-25 single mutants showed that H3K9me2-mediated repression of satellite repeats by MET-2 correlates with germline integrity. Aberrant transcription of repeats and DNA transposons generates R-loops, loss of fertility and hydroxyurea hypersensitivity. In a genome-wide synthetic lethal screen, we identified the BRCA1 complex and factors implicated in the degradation of nuclear RNA as essential for germline integrity in met-2 mutants. Highly additive with met-2, the loss of the BRCA1 complex triggers satellite repeat transcription, generating R-loops on transcribed repeats. Supporting direct causality between satellite repeat transcription and BRCA1-mediated genome integrity, the targeted induction of MSAT1 transcripts at endogenous sites leads to damage-induced loss of fertility in wild-type C. elegans. Libraries were prepared from chromatin IP and input samples using the NEBNext ultra DNA library prep kit for Illumina (NEB # 7370) and the NEBNext Multiplex Oligos for Illumina (NEB # E7335), according to the manufacturer’s recommendations. No size selection was performed during sample preparation and the libraries were indexed and amplified using 12 PCR cycles, using the recommended conditions. After a final cleanup with Agencourt AmPure XP beads (Beckman # A63881), the library size distribution and concentrations were determined using a BioAnalyzer 2100 (Agilent technologies) and Qubit (Invitrogen) instrument, respectively. The final pools were prepared by mixing equimolar amounts of all individual indexed libraries and then sequenced on a HiSeq 2500 (Illumina) in Rapid mode (Paired-End 50).

秀丽隐杆线虫（C. elegans）基因组中的重复序列、转座元件以及沉默组织特异性基因，在H3赖氨酸9二甲基化与三甲基化修饰上存在差异富集。SET-25（SUV39h1/h2）可催化H3K9me3的形成，而MET-2（SetDB1）仅能介导H3K9me1/me2的修饰沉积。对met-2与set-25单基因突变体开展RNA测序（RNA-seq）及全基因组H3K9甲基化图谱分析后发现，MET-2通过H3K9me2介导的卫星重复序列沉默，与生殖系完整性密切相关。重复序列及DNA转座子的异常转录会生成R环（R-loops），进而引发生育力丧失及羟基脲超敏反应。在全基因组合成致死筛选实验中，我们鉴定出BRCA1复合物及参与核RNA降解的相关因子，是met-2突变体维持生殖系完整性所必需的因子。与met-2突变存在极强的加性遗传效应，BRCA1复合物的缺失会触发卫星重复序列的转录，并在已转录的重复序列区域形成R环。为验证卫星重复序列转录与BRCA1介导的基因组完整性之间的直接因果关系，我们在内源位点靶向诱导MSAT1转录本的表达，结果在野生型秀丽隐杆线虫中引发了损伤诱导的生育力丧失。本研究按照制造商推荐的操作流程，采用适配Illumina平台的NEBNext Ultra DNA文库制备试剂盒（NEB #7370）与NEBNext多重适配引物（NEB #E7335），对染色质免疫沉淀（ChIP）样本及输入样本完成文库构建。样本制备过程中未进行片段筛选，且遵循推荐反应条件，采用12个PCR循环完成文库的索引标记与扩增。最终使用Agencourt AMPure XP磁珠（Beckman #A63881）完成最后一轮纯化，随后分别采用2100生物分析仪（Agilent Technologies）与Qubit荧光定量仪（Invitrogen）测定文库的片段分布与浓度。将所有已完成索引标记的单个文库以等摩尔浓度混合构建最终测序混合池，随后在HiSeq 2500测序仪（Illumina）上以快速运行模式开展50bp双端测序。