Double-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.
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•DSB-chromatin landscape and HR/NHEJ chromatin signatures uncovered by ChIP-seq•H2BK120 undergoes a switch from ubiquitination to acetylation at a local scale•H1 is removed and ubiquitin accumulates on entire γH2AX domains, mainly at HR DSB•53BP1 spreads over megabase-sized domains, mostly in G1 at HR-prone DSBs
Using ChIP-seq in a cell line where multiple annotated DNA double-strand breaks can be induced on the human genome, Clouaire et al. report a comprehensive view of the chromatin landscape set up at DSBs and decipher the chromatin signature associated with HR and NHEJ repair.
Although both homologous recombination (HR) and nonhomologous end joining can repair DNA double-strand breaks (DSBs), the mechanisms by which one of these pathways is chosen over the other remain ...unclear. Here we show that transcriptionally active chromatin is preferentially repaired by HR. Using chromatin immunoprecipitation-sequencing (ChIP-seq) to analyze repair of multiple DSBs induced throughout the human genome, we identify an HR-prone 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 are targeted to HR repair via the transcription elongation-associated mark trimethylated histone H3 K36. Concordantly, depletion of SETD2, the main H3 K36 trimethyltransferase, severely impedes HR at such DSBs. Our study thereby demonstrates a primary role in DSB repair of the chromatin context in which a break occurs.
The ability of DNA double-strand breaks (DSBs) to cluster in mammalian cells has been a subject of intense debate in recent years. Here we used a high-throughput chromosome conformation capture assay ...(capture Hi-C) to investigate clustering of DSBs induced at defined loci in the human genome. The results unambiguously demonstrated that DSBs cluster, but only when they are induced within transcriptionally active genes. Clustering of damaged genes occurs primarily during the G1 cell-cycle phase and coincides with delayed repair. Moreover, DSB clustering depends on the MRN complex as well as the Formin 2 (FMN2) nuclear actin organizer and the linker of nuclear and cytoplasmic skeleton (LINC) complex, thus suggesting that active mechanisms promote clustering. This work reveals that, when damaged, active genes, compared with the rest of the genome, exhibit a distinctive behavior, remaining largely unrepaired and clustered in G1, and being repaired via homologous recombination in postreplicative cells.
Modulating 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.
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•A role for SETD2 in DSB resection and homologous recombination repair•Histone H3K36me3 is required for homologous recombination•SETD2 and RAD51 suppress mutations arising from microhomology-mediated end-joining•Mutations affecting H3K36me3 levels may promote tumorigenesis
The SETD2 gene encodes the histone H3K36 trimethyltransferase. Pfister et al. now show that human SETD2-dependent H3K36me3 maintains genome stability by promoting error-free DNA repair through homologous recombination (HR). Upon DNA damage, SETD2-depleted cells exhibit reduced DNA resection, impaired recruitment of early HR factors, and increased utilization of the error-prone microhomology-mediated end-joining repair pathway. Eliminating H3K36me3 by overexpressing the oncogene KDM4A also impairs HR. Thus, H3K36me3 suppresses tumorigenesis by promoting accurate DNA repair.
Recent advances in our understanding of the management and repair of DNA double-strand breaks (DSBs) rely on the study of targeted DSBs that have been induced in living cells by the controlled ...activity of site-specific endonucleases, usually recombinant restriction enzymes. Here we describe a protocol for quantifying these endonuclease-induced DSBs; this quantification is essential to an interpretation of how DSBs are managed and repaired. A biotinylated double-stranded oligonucleotide is ligated to enzyme-cleaved genomic DNA, allowing the purification of the cleaved DNA on streptavidin beads. The extent of cleavage is then quantified either by quantitative PCR (qPCR) at a given site or at multiple sites by genome-wide techniques (e.g., microarrays or high-throughput sequencing). This technique, named ligation-mediated purification, can be performed in 2 d. It is more accurate and sensitive than existing alternative methods, and it is compatible with genome-wide analysis. It allows the amount of endonuclease-mediated breaks to be precisely compared between two conditions or across the genome, thereby giving insight into the influence of a given factor or of various chromatin contexts on local repair parameters.
In mammalian cells, DNA double-strand breaks (DSB) can be repaired by 2 main pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). To give access to DNA damage to the repair ...machinery the chromatin structure needs to be relaxed, and chromatin modifications play major roles in the control of these processes. Among the chromatin modifications, changes in nucleosome composition can influence DNA damage response as observed with the H2A.Z histone variant in yeast. In mammals, p400, an ATPase of the SWI/SNF family able to incorporate H2A.Z in chromatin, was found to be important for histone ubiquitination and BRCA1 recruitment around DSB or for HR in cooperation with Rad51. Recent data with 293T cells showed that mammalian H2A.Z is recruited to DSBs and is important to control DNA resection, therefore participating both in HR and NHEJ. Here we show that depletion of H2A.Z in the osteosarcoma U2OS cell line and in immortalized human fibroblasts does not change parameters of DNA DSB repair while affecting clonogenic ability and cell cycle distribution. In addition, no recruitment of H2A.Z around DSB can be detected in U2OS cells either after local laser irradiation or by chromatin immunoprecipitation. These data suggest that the role of H2A.Z in DSB repair is not ubiquitous in mammals. In addition, given that important cellular parameters, such as cell viability and cell cycle distribution, are more sensitive to H2A.Z depletion than DNA repair, our results underline the difficulty to investigate the role of versatile factors such as H2A.Z.
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