While both Homologous recombination (HR) and Non Homologous End Joining (NHEJ) can repair DNA double Strand Breaks (DSB), the mechanisms by which one or other of these pathways is chosen remain unclear. Here we show that transcriptionally active chromatin is preferentially repaired by HR. Using chromatin immunoprecipitation-sequencing (ChIP-seq), to analyse repair of multiple DSBs induced throughout the human genome, we identify an "HRprone" subset of DSBs that recruit the HR protein RAD51, undergo resection and rely on RAD51 for efficient repair. These DSBs are located in actively transcribed genes, and targeted to HR repair via the transcription-elongation associated histone mark, histone H3 lysine 36 trimethylation (H3K36me3). In agreement, depletion of SETD2, the main H3K36 trimethyltransferase, severely impedes HR at such DSBs. Our study thereby demonstrates a primary role of the chromatin context, in which a break occurs, in DSB repair.

Chromatin acts as a key regulator of DNA-related processes such as DNA damage repair. Although ChIP-chip is a powerful technique to provide high-resolution maps of protein-genome interactions, its use to study DNA double strand break (DSB) repair has been hindered by the limitations of the available damage induction methods. We have developed a human cell line that permits induction of multiple DSBs randomly distributed and unambiguously positioned within the genome. Using this system, we have generated the first genome-wide mapping of gammaH2AX around DSBs. We found that all DSBs trigger large gammaH2AX domains, which spread out from the DSB in a bidirectional, discontinuous and not necessarily symmetrical manner. The distribution of gammaH2AX within domains is influenced by gene transcription, as parallel mappings of RNA Polymerase II and strand-specific expression showed that gammaH2AX does not propagate on active genes. In addition, we showed that transcription is accurately maintained within gammaH2AX domains, indicating that mechanisms may exist to protect gene transcription from gammaH2AX spreading and from the chromatin rearrangements induced by DSBs.

DNA double-strand break (DSB) repair occurs within chromatin and can be modulated by chromatin modifying enzymes. Here we identify the related human histone deacetylases HDAC1 and HDAC2 as two participants in the DNA-damage response (DDR). We show that acetylation of histone H3 lysine 56 (H3K56) is regulated by HDAC1/2, and that HDAC1/2 are rapidly recruited to DNA-damage sites to promote H3K56 hypo-acetylation. Furthermore, we establish that HDAC1/2-depleted cells are hypersensitive to DNA-damaging agents and exhibit sustained DNA-damage signaling, phenotypes that reflect defective DSB repair, particularly by the pathway of non-homologous end-joining (NHEJ). Collectively, these results demonstrate that HDAC1 and HDAC2 function in the DDR by promoting DSB repair and thus provide important insights into the radio-sensitizing effects of HDAC inhibitors that are being developed as cancer therapies. KeywordsHDAC1; HDAC2; DNA damage; chromatin; non-homologous end-joining Because DNA damage represents a formidable challenge to the integrity of genetic material, cells have evolved multifaceted systems, collectively termed the DNA-damage response (DDR), to detect, signal and repair various types of DNA damage 1 , 2 . DNA double-strand breaks (DSBs) represent one of the most challenging forms of DNA damage which, if left unrepaired, can trigger cellular death and can contribute to human diseases, including cancer 1 . In eukaryotes, DSBs are repaired by two main pathways: non-homologous endjoining (NHEJ), which operates throughout the cell cycle, and homologous recombination (HR) which is limited to S and G2 cell-cycle phases 3 . For NHEJ, the Ku70/Ku80 complex loads onto free DNA ends where it helps recruit the DDR protein kinase DNA-PK (DNAdependent protein kinase) 4 , as well as other factors, including the nuclease Artemis and the ligase IV-XRCC4 complex, which are required for NHEJ to ensue 5 . NHEJ occurs rapidly within cells and mostly requires minimal processing of DNA ends. By contrast, HR requires extensive DNA end-resection to create stretches of single-stranded DNA (ssDNA) that bind factors such as RPA and RAD51 to promote the various steps in HR 3 . Since HR and NHEJ function together during certain phases of the cell cycle, mechanisms must exist to modulate In this study, our previous observation of a decrease of H3K56Ac upon DNA damage prompted us to evaluate the role of HDACs in the DDR. Here, we describe how human HDAC1 and HDAC2 respond to DNA damage and mediate changes in histone acetylation, including H3K56, following DNA-damage induction. Furthermore, by defining the effects of impairing HDAC1/2 function, we establish that these enzymes serve as important components of the DDR by promoting DSB signalling and repair, principally through their requirement for effective NHEJ. Europe PMC Funders Group RESULTS HDAC1 and HDAC2 localize to sites of DNA damageWe previously demonstrated that H3K56Ac levels are reduced by treatments that induce DNA damage, including the drug phleomycin that produces D...

SummaryModulating chromatin through histone methylation orchestrates numerous cellular processes. SETD2-dependent trimethylation of histone H3K36 is associated with active transcription. Here, we define a role for H3K36 trimethylation in homologous recombination (HR) repair in human cells. We find that depleting SETD2 generates a mutation signature resembling RAD51 depletion at I-SceI-induced DNA double-strand break (DSB) sites, with significantly increased deletions arising through microhomology-mediated end-joining. We establish a presynaptic role for SETD2 methyltransferase in HR, where it facilitates the recruitment of C-terminal binding protein interacting protein (CtIP) and promotes DSB resection, allowing Replication Protein A (RPA) and RAD51 binding to DNA damage sites. Furthermore, reducing H3K36me3 levels by overexpressing KDM4A/JMJD2A, an oncogene and H3K36me3/2 demethylase, or an H3.3K36M transgene also reduces HR repair events. We propose that error-free HR repair within H3K36me3-decorated transcriptionally active genomic regions promotes cell homeostasis. Moreover, these findings provide insights as to why oncogenic mutations cluster within the H3K36me3 axis.

Ataxia with oculomotor apraxia 2 (AOA-2) and amyotrophic lateral sclerosis (ALS4) are neurological disorders caused by mutations in the gene encoding for senataxin (SETX), a putative RNA:DNA helicase involved in transcription and in the maintenance of genome integrity. Here, using ChIP followed by high throughput sequencing (ChIP-seq), we report that senataxin is recruited at DNA double-strand breaks (DSBs) when they occur in transcriptionally active loci. Genome-wide mapping unveiled that RNA:DNA hybrids accumulate on DSB-flanking chromatin but display a narrow, DSB-induced, depletion near DNA ends coinciding with senataxin binding. Although neither required for resection nor for timely repair of DSBs, senataxin was found to promote Rad51 recruitment, to minimize illegitimate rejoining of distant DNA ends and to sustain cell viability following DSB production in active genes. Our data suggest that senataxin functions at DSBs in order to limit translocations and ensure cell viability, providing new insights on AOA2/ALS4 neuropathies.

How chromatin shapes pathways that promote genome-epigenome integrity in response to DNA damage is an issue of crucial importance. We report that human bromodomain (BRD)-containing proteins, the primary ''readers'' of acetylated chromatin, are vital for the DNA damage response (DDR). We discovered that more than one-third of all human BRD proteins change localization in response to DNA damage. We identified ZMYND8 (zinc finger and MYND [myeloid, Nervy, and DEAF-1] domain containing 8) as a novel DDR factor that recruits the nucleosome remodeling and histone deacetylation (NuRD) complex to damaged chromatin. Our data define a transcription-associated DDR pathway mediated by ZMYND8 and the NuRD complex that targets DNA damage, including when it occurs within transcriptionally active chromatin, to repress transcription and promote repair by homologous recombination. Thus, our data identify human BRD proteins as key chromatin modulators of the DDR and provide novel insights into how DNA damage within actively transcribed regions requires chromatin-binding proteins to orchestrate the appropriate response in concordance with the damage-associated chromatin context.

SummaryDouble-strand breaks (DSBs) are extremely detrimental DNA lesions that can lead to cancer-driving mutations and translocations. Non-homologous end joining (NHEJ) and homologous recombination (HR) represent the two main repair pathways operating in the context of chromatin to ensure genome stability. Despite extensive efforts, our knowledge of DSB-induced chromatin still remains fragmented. Here, we describe the distribution of 20 chromatin features at multiple DSBs spread throughout the human genome using ChIP-seq. We provide the most comprehensive picture of the chromatin landscape set up at DSBs and identify NHEJ- and HR-specific chromatin events. This study revealed the existence of a DSB-induced monoubiquitination-to-acetylation switch on histone H2B lysine 120, likely mediated by the SAGA complex, as well as higher-order signaling at HR-repaired DSBs whereby histone H1 is evicted while ubiquitin and 53BP1 accumulate over the entire γH2AX domains.

SUMMARY The NuA4/TIP60 acetyltransferase complex is a key regulator of genome expression and stability. Here, we identified MBTD1 as a new stable subunit of the complex and gleaned intriguing insights into TIP60’s function. Harboring a histone reader domain for H4K20me1/2, MBTD1 allows TIP60 to associate with specific gene promoters and to promote the repair of DNA double strand breaks by homologous recombination. Interestingly, the non-homologous end joining factor 53BP1 engages chromatin through simultaneous binding of H4K20me2 and H2AK15ub, and it was postulated that Tip60-dependent acetylation of H4 regulates this binding. Our findings now indicate that the TIP60 complex is a potent regulator of DNA damage repair pathways in part by targeting the same histone mark as 53BP1. In addition, deposition of H2AK15ub by RNF168 inhibits chromatin acetylation by TIP60, while this residue can be acetylated by TIP60 in vivo, blocking its ubiquitylation. Altogether, these results uncover an intricate mechanism orchestrated by the TIP60 complex which regulates 53BP1-dependent repair pathway selection through incompatible bivalent binding and modification of chromatin.