CDYL1-dependent decrease in lysine crotonylation at DNA double-strand break sites functionally uncouples transcriptional silencing and repair.

Previously, we showed that CDYL1 is recruited to DNA double-strand breaks (DSBs) to promote homologous recombination (HR) repair and foster transcriptional silencing. However, how CDYL1 elicits DSB-induced silencing is not fully understood. Here, we identify a CDYL1-dependent local decrease in the transcriptionally active marks histone lysine crotonylation (Kcr) and crotonylated lysine 9 of H3 (H3K9cr) at AsiSI-induced DSBs, which correlates with transcriptional silencing. Mechanistically, we reveal that CDYL1 crotonyl-CoA hydratase activity counteracts Kcr and H3K9cr at DSB sites, which triggers the eviction of the transcription elongation factor ENL and fosters transcriptional silencing. Furthermore, genetic inhibition of CDYL1 hydratase activity blocks the reduction in H3K9cr and alleviates DSB-induced silencing, whereas HR efficiency unexpectedly remains intact. Therefore, our results functionally uncouple the repair and silencing activity of CDYL1 at DSBs. In a broader context, we address a long-standing question concerning the functional relationship between HR repair and DSB-induced silencing, suggesting that they may occur independently.

RBM6 splicing factor promotes homologous recombination repair of double-strand breaks and modulates sensitivity to chemotherapeutic drugs.

RNA-binding proteins regulate mRNA processing and translation and are often aberrantly expressed in cancer. The RNA-binding motif protein 6, RBM6, is a known alternative splicing factor that harbors tumor suppressor activity and is frequently mutated in human cancer. Here, we identify RBM6 as a novel regulator of homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Mechanistically, we show that RBM6 regulates alternative splicing-coupled nonstop-decay of a positive HR regulator, Fe65/APBB1. RBM6 knockdown leads to a severe reduction in Fe65 protein levels and consequently impairs HR of DSBs. Accordingly, RBM6-deficient cancer cells are vulnerable to ATM and PARP inhibition and show remarkable sensitivity to cisplatin. Concordantly, cisplatin administration inhibits the growth of breast tumor devoid of RBM6 in mouse xenograft model. Furthermore, we observe that RBM6 protein is significantly lost in metastatic breast tumors compared with primary tumors, thus suggesting RBM6 as a potential therapeutic target of advanced breast cancer. Collectively, our results elucidate the link between the multifaceted roles of RBM6 in regulating alternative splicing and HR of DSBs that may contribute to tumorigenesis, and pave the way for new avenues of therapy for RBM6-deficient tumors.

NELF-E is recruited to DNA double-strand break sites to promote transcriptional repression and repair.

Double-strand breaks (DSBs) trigger rapid and transient transcription pause to prevent collisions between repair and transcription machineries at damage sites. Little is known about the mechanisms that ensure transcriptional block after DNA damage. Here, we reveal a novel role of the negative elongation factor NELF in blocking transcription activity nearby DSBs. We show that NELF-E and NELF-A are rapidly recruited to DSB sites. Furthermore, NELF-E recruitment and its repressive activity are both required for switching off transcription at DSBs. Remarkably, using I-SceI endonuclease and CRISPR-Cas9 systems, we observe that NELF-E is preferentially recruited, in a PARP1-dependent manner, to DSBs induced upstream of transcriptionally active rather than inactive genes. Moreover, the presence of RNA polymerase II is a prerequisite for the preferential recruitment of NELF-E to DNA break sites. Additionally, we demonstrate that NELF-E is required for intact repair of DSBs. Altogether, our data identify the NELF complex as a new component in the DNA damage response.

CDYL1 fosters double-strand break-induced transcription silencing and promotes homology-directed repair.

Cells have evolved DNA damage response (DDR) to repair DNA lesions and thus preserving genomic stability and impeding carcinogenesis. DNA damage induction is accompanied by transient transcription repression. Here, we describe a previously unrecognized role of chromodomain Y-like (CDYL1) protein in fortifying double-strand break (DSB)-induced transcription repression and repair. We showed that CDYL1 is rapidly recruited to damaged euchromatic regions in a poly (ADP-ribose) polymerase 1 (PARP1)-dependent, but ataxia telangiectasia mutated (ATM)-independent, manner. While the C-terminal region, containing the enoyl-CoA hydratase like (ECH) domain, of CDYL1 binds to poly (ADP-ribose) (PAR) moieties and mediates CDYL1 accumulation at DNA damage sites, the chromodomain and histone H3 trimethylated on lysine 9 (H3K9me3) mark are dispensable for its recruitment. Furthermore, CDYL1 promotes the recruitment of enhancer of zeste homolog 2 (EZH2), stimulates local increase of the repressive methyl mark H3K27me3, and promotes transcription silencing at DSB sites. In addition, following DNA damage induction, CDYL1 depletion causes persistent G2/M arrest and alters H2AX and replication protein A (RPA2) phosphorylation. Remarkably, the 'traffic-light reporter' system revealed that CDYL1 mainly promotes homology-directed repair (HDR) of DSBs in vivo. Consequently, CDYL1-knockout cells display synthetic lethality with the chemotherapeutic agent, cisplatin. Altogether, our findings identify CDYL1 as a new component of the DDR and suggest that the HDR-defective 'BRCAness' phenotype of CDYL1-deficient cells could be exploited for eradicating cancer cells harboring CDYL1 mutations.

A role of human RNase P subunits, Rpp29 and Rpp21, in homology directed-repair of double-strand breaks.

DNA damage response (DDR) is needed to repair damaged DNA for genomic integrity preservation. Defective DDR causes accumulation of deleterious mutations and DNA lesions that can lead to genomic instabilities and carcinogenesis. Identifying new players in the DDR, therefore, is essential to advance the understanding of the molecular mechanisms by which cells keep their genetic material intact. Here, we show that the core protein subunits Rpp29 and Rpp21 of human RNase P complex are implicated in DDR. We demonstrate that Rpp29 and Rpp21 depletion impairs double-strand break (DSB) repair by homology-directed repair (HDR), but has no deleterious effect on the integrity of non-homologous end joining. We also demonstrate that Rpp29 and Rpp21, but not Rpp14, Rpp25 and Rpp38, are rapidly and transiently recruited to laser-microirradiated sites. Rpp29 and Rpp21 bind poly ADP-ribose moieties and are recruited to DNA damage sites in a PARP1-dependent manner. Remarkably, depletion of the catalytic H1 RNA subunit diminishes their recruitment to laser-microirradiated regions. Moreover, RNase P activity is augmented after DNA damage in a PARP1-dependent manner. Altogether, our results describe a previously unrecognized function of the RNase P subunits, Rpp29 and Rpp21, in fine-tuning HDR of DSBs.