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. Overall design: 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).