A Snapshot on the Cis Chromatin Response to DNA Double-Strand Breaks
Authors: Thomas Clouaire, Gaëlle Legube
Journal: Trends in Genetics (2019)

The development of specialized tools has recently provided unprecedented insights regarding the role of chromatin structure in the response to DNA double-strand breaks. High-resolution studies revealed that chromatin is modified on several different scales following DSB induction, ranging from a few kilobases to several megabases. Chromatin modifications at the megabase level may alter the physical properties of the chromatin fiber and regulate long-range chromosomal mobility upon damage. The nature and extent of local chromatin modifications are different upon repair by NHEJ or HR. Repair pathway-specific modifications have the potential to regulate the access of repair factors and the physical properties of the nucleosome, thereby participating in downstream repair events. In eukaryotes, detection and repair of DNA double-strand breaks (DSBs) operate within chromatin, an incredibly complex structure that tightly packages and regulates DNA metabolism. Chromatin participates in the repair of these lesions at multiple steps, from detection to genomic sequence recovery and chromatin is itself extensively modified during the repair process. In recent years, new methodologies and dedicated techniques have expanded the experimental toolbox, opening up a new era granting the high-resolution analysis of chromatin modifications at annotated DSBs in a genome-wide manner. A complex picture is starting to emerge whereby chromatin is altered at various scales around DSBs, in a manner that relates to the repair pathway used, hence defining a 'repair histone code'. Here, we review the recent advances regarding our knowledge of the chromatin landscape induced in cis around DSBs, with an emphasis on histone post-translational modifications and histone variants. In eukaryotes, detection and repair of DNA double-strand breaks (DSBs) operate within chromatin, an incredibly complex structure that tightly packages and regulates DNA metabolism. Chromatin participates in the repair of these lesions at multiple steps, from detection to genomic sequence recovery and chromatin is itself extensively modified during the repair process. In recent years, new methodologies and dedicated techniques have expanded the experimental toolbox, opening up a new era granting the high-resolution analysis of chromatin modifications at annotated DSBs in a genome-wide manner. A complex picture is starting to emerge whereby chromatin is altered at various scales around DSBs, in a manner that relates to the repair pathway used, hence defining a 'repair histone code'. Here, we review the recent advances regarding our knowledge of the chromatin landscape induced in cis around DSBs, with an emphasis on histone post-translational modifications and histone variants. DNA double-strand breaks (DSBs) constitute very dangerous lesions having the potential to produce harmful genetic alterations such as local DNA sequence modifications (insertions/deletions) or more substantial chromosomal rearrangements (e.g., translocations). To faithfully maintain genomic integrity, cells deploy a DNA damage response (DDR) comprising specialized machineries able to sense, signal, and repair DSBs by homologous recombination (HR) or non-homologous end joining (NHEJ) [1Mladenov E. et al.DNA double-strand-break repair in higher eukaryotes and its role in genomic instability and cancer: cell cycle and proliferation-dependent regulation.Semin. Cancer Biol. 2016; (51–64): 37-38Google Scholar] (Box 1). DSBs can occur upon exposure to a variety of genotoxic agents (e.g., ionizing radiation, chemotherapeutic drug) and under physiological conditions including developmentally programmed DSBs in lymphocytes or during meiosis but also during ubiquitous processes such as replication and transcription. Indeed, DSBs arise more frequently than previously anticipated, and their induction rate is not homogeneous along the genome. For example, transcribed genes (reviewed in [2Marnef A. et al.Transcription-coupled DNA double-strand break repair: active genes need special care.J. Mol. Biol. 2017; 429: 1277-1288Crossref PubMed Scopus (20) Google Scholar]) or chromatin loop anchors [3Canela A. et al.Genome organization drives chromosome fragility.Cell. 2017; 170: 507-521.e18Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar] appear to represent regions of increased DSB frequency, and technological improvements will most likely identify other classes of endogenous DSB-prone regions in the future. It is therefore not surprising for DSB repair anomalies to be responsible for many disorders (premature aging, neurodegenerative syndromes) as well as the onset and progression of cancer.Box 1DSB Repair PathwaysThe two main DSB repair mechanisms are non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ functions by direct ligation of the two broken ends, requiring no or minimal processing of the DNA ends. This pathway operates throughout the cell cycle, and key NHEJ factors include Ku, DNA-PKcs, XRCC4, XLF, PAXX, and DNA ligase IV (recently reviewed in [140Chang H.H.Y. et al.Non-homologous DNA end joining and alternative pathways to double-strand break repair.Nat. Rev. Mol. Cell Biol. 2017; 18: 495-506Crossref PubMed Scopus (96) Google Scholar]). During HR, the undamaged copy of the broken chromosome will be used to initiate DNA synthesis and restore the nucleotide sequence (reviewed in [141Wright W.D. et al.Homologous recombination and the repair of DNA double-strand breaks.J. Biol. Chem. 2018; 293: 10524-10535Crossref PubMed Scopus (9) Google Scholar]). For this, 5′ to 3′ nucleolytic degradation of the broken end, called resection, will be initiated by the MRN (MRE11–RAD50–NBS1) complex and CtIP and is further extended by EXO1, DNA2, and accessory factors such as the helicase BLM. RPA will bind newly generated ssDNA to stabilize it. Subsequently, BRCA2 will stimulate the loading of the RAD51 recombinase to resected DNA, which results in a nucleofilament that will find the homologous sequence to initiate DNA synthesis. Under particular circumstances, such as HR deficiencies, alternative pathways such as single-strand annealing (SSA) or microhomology-mediated end joining (MMEJ) can be mobilized, at a cost for genome integrity since these pathways tend to be highly mutagenic [140Chang H.H.Y. et al.Non-homologous DNA end joining and alternative pathways to double-strand break repair.Nat. Rev. Mol. Cell Biol. 2017; 18: 495-506Crossref PubMed Scopus (96) Google Scholar]. The initiation and extent of resection are key aspects governing DSB repair pathway choice since it is a requirement for HR (and alternative mutagenic pathways) and will inhibit the execution of NHEJ. Key DSB repair factors such as 53BP1 and BRCA1 play very important roles in controlling resection. The two main DSB repair mechanisms are non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ functions by direct ligation of the two broken ends, requiring no or minimal processing of the DNA ends. This pathway operates throughout the cell cycle, and key NHEJ factors include Ku, DNA-PKcs, XRCC4, XLF, PAXX, and DNA ligase IV (recently reviewed in [140Chang H.H.Y. et al.Non-homologous DNA end joining and alternative pathways to double-strand break repair.Nat. Rev. Mol. Cell Biol. 2017; 18: 495-506Crossref PubMed Scopus (96) Google Scholar]). During HR, the undamaged copy of the broken chromosome will be used to initiate DNA synthesis and restore the nucleotide sequence (reviewed in [141Wright W.D. et al.Homologous recombination and the repair of DNA double-strand breaks.J. Biol. Chem. 2018; 293: 10524-10535Crossref PubMed Scopus (9) Google Scholar]). For this, 5′ to 3′ nucleolytic degradation of the broken end, called resection, will be initiated by the MRN (MRE11–RAD50–NBS1) complex and CtIP and is further extended by EXO1, DNA2, and accessory factors such as the helicase BLM. RPA will bind newly generated ssDNA to stabilize it. Subsequently, BRCA2 will stimulate the loading of the RAD51 recombinase to resected DNA, which results in a nucleofilament that will find the homologous sequence to initiate DNA synthesis. Under particular circumstances, such as HR deficiencies, alternative pathways such as single-strand annealing (SSA) or microhomology-mediated end joining (MMEJ) can be mobilized, at a cost for genome integrity since these pathways tend to be highly mutagenic [140Chang H.H.Y. et al.Non-homologous DNA end joining and alternative pathways to double-strand break repair.Nat. Rev. Mol. Cell Biol. 2017; 18: 495-506Crossref PubMed Scopus (96) Google Scholar]. The initiation and extent of resection are key aspects governing DSB repair pathway choice since it is a requirement for HR (and alternative mutagenic pathways) and will inhibit the execution of NHEJ. Key DSB repair factors such as 53BP1 and BRCA1 play very important roles in controlling resection. Like any other DNA-related process, DSB repair occurs within a precisely organized and dynamic chromatin environment (Box 2). Histone post-translational modifications or variants can alter nucleosome structure and therefore control many properties of the chromatin fiber (such as DNA accessibility, secondary structures, topological constraints, stiffness, and mobility) but can also directly act as recruitment platforms for the chromatin reader (see Glossary) proteins, emphasizing the pivotal function of chromatin during transcription, replication, and repair.Box 2Chromatin Is Organized at Many Different LevelsThe primary level of chromatin organization consists of DNA winding around octamers of core histones (H2A, H2B, H3, and H4), forming arrays of nucleosomes that is usually conceptualized as 'beads on a string'. In living cells, this basic chromatin fiber further folds and adopts a range of configurations and compaction states at many different scales, providing the foundation for the 3D organization of chromosomes in the nuclear space. Modifications at the nucleosomal level include DNA methylation, histone post-translational modifications (such as phosphorylation, acetylation, methylation, and ubiquitination), or incorporation of histone variants [142Talbert P.B. Henikoff S. Histone variants on the move: substrates for chromatin dynamics.Nat. Rev. Mol. Cell Biol. 2017; 18: 115-126Crossref PubMed Scopus (53) Google Scholar, 143Soshnev A.A. et al.Greater than the sum of parts: complexity of the dynamic epigenome.Mol. Cell. 2016; 62: 681-694Abstract Full Text Full Text PDF PubMed Google Scholar]. Nucleosomes themselves are also very dynamic in their positioning, occupancy, and turnover. Furthermore, linker histones such as H1 can directly interact with the nucleosome and participate in the folding of nucleosome arrays. These chromatin marks do not occur randomly on the genome but follow a stereotyped pattern depending on the function of the underlying locus [4Zhou V.W. et al.Charting histone modifications and the functional organization of mammalian genomes.Nat. Rev. Genet. 2011; 12: 7-18Crossref PubMed Scopus (636) Google Scholar]. Indeed, specific chromatin features associate and characterize promoters, active gene bodies, regulatory elements (enhancers, domain boundaries), Polycomb-repressed domains, and heterochromatin (pericentromeres, subtelomeric regions, lamina-associated domains, or nucleolus-associated domains). Furthermore, recent data suggest that genome folding in 3D is governed by both the segregation of active or inactive chromatin into compartmental domains as well as the formation of long-range chromatin loops by CTCF and cohesin, leading to the formation of topologically associating domains [144Rowley M.J. Corces V.G. Organizational principles of 3D genome architecture.Nat. Rev. Genet. 2018; 19: 789-800Crossref PubMed Scopus (1) Google Scholar]. The primary level of chromatin organization consists of DNA winding around octamers of core histones (H2A, H2B, H3, and H4), forming arrays of nucleosomes that is usually conceptualized as 'beads on a string'. In living cells, this basic chromatin fiber further folds and adopts a range of configurations and compaction states at many different scales, providing the foundation for the 3D organization of chromosomes in the nuclear space. Modifications at the nucleosomal level include DNA methylation, histone post-translational modifications (such as phosphorylation, acetylation, methylation, and ubiquitination), or incorporation of histone variants [142Talbert P.B. Henikoff S. Histone variants on the move: substrates for chromatin dynamics.Nat. Rev. Mol. Cell Biol. 2017; 18: 115-126Crossref PubMed Scopus (53) Google Scholar, 143Soshnev A.A. et al.Greater than the sum of parts: complexity of the dynamic epigenome.Mol. Cell. 2016; 62: 681-694Abstract Full Text Full Text PDF PubMed Google Scholar]. Nucleosomes themselves are also very dynamic in their positioning, occupancy, and turnover. Furthermore, linker histones such as H1 can directly interact with the nucleosome and participate in the folding of nucleosome arrays. These chromatin marks do not occur randomly on the genome but follow a stereotyped pattern depending on the function of the underlying locus [4Zhou V.W. et al.Charting histone modifications and the functional organization of mammalian genomes.Nat. Rev. Genet. 2011; 12: 7-18Crossref PubMed Scopus (636) Google Scholar]. Indeed, specific chromatin features associate and characterize promoters, active gene bodies, regulatory elements (enhancers, domain boundaries), Polycomb-repressed domains, and heterochromatin (pericentromeres, subtelomeric regions, lamina-associated domains, or nucleolus-associated domains). Furthermore, recent data suggest that genome folding in 3D is governed by both the segregation of active or inactive chromatin into compartmental domains as well as the formation of long-range chromatin loops by CTCF and cohesin, leading to the formation of topologically associating domains [144Rowley M.J. Corces V.G. Organizational principles of 3D genome architecture.Nat. Rev. Genet. 2018; 19: 789-800Crossref PubMed Scopus (1) Google Scholar]. During DSB repair, chromatin modifications have the potential to contribute to the accurate execution of repair pathways by regulating DNA end accessibility, synapsis, or single-strand DNA (ssDNA) generation and also to participate in DSB signaling or the control of DSB mobility within the nucleus. Yet, while the genome-wide sequencing revolution strongly helped to understand the role of chromatin in transcription [4Zhou V.W. et al.Charting histone modifications and the functional organization of mammalian genomes.Nat. Rev. Genet. 2011; 12: 7-18Crossref PubMed Scopus (636) Google Scholar], its detailed contribution to DSB repair has long been impeded by the inability to induce DSBs at defined positions across entire cell populations. This precluded the use of dedicated methods such as ChIP-seq to analyze DSB-induced chromatin changes at high resolution or to compare DSB repair events occurring in different chromatin contexts. The recent development of tools to induce DSBs at annotated positions (Box 3), combined with high-throughput sequencing approaches and/or super-resolution microscopy, revolutionized our capability to study the nature and function of chromatin at DSBs. These new methods could make it possible to tackle new questions; (i) to what extent is the chromatin response to DSB universal, that is, how does it change depending on the genomic position of the break and (ii) what is the nature of DSB-induced chromatin and how does this chromatin landscape contribute to repair reactions and to the maintenance of genomic integrity? While it is now clearly established that the position of the break, and its associated chromatin features, plays a critical role in its subsequent repair (reviewed in [2Marnef A. et al.Transcription-coupled DNA double-strand break repair: active genes need special care.J. Mol. Biol. 2017; 429: 1277-1288Crossref PubMed Scopus (20) Google Scholar, 5Fortuny A. Polo S.E. The response to DNA damage in heterochromatin domains.Chromosoma. 2018; 127: 291-300Crossref PubMed Scopus (0) Google Scholar, 6van Sluis M. McStay B. Nucleolar reorganization in response to rDNA damage.Curr. Opin. Cell Biol. 2017; 46: 81-86Crossref PubMed Scopus (6) Google Scholar]), the precise chromatin landscape induced around DSBs has only recently started to be deciphered.Box 3Pros and Cons of Prevalent DSB Induction MethodsCommon DSB induction methods include ionizing radiations (γ- or X-rays), crosslinking agents (mitomycin C, cisplatin), radiomimetic compounds (bleomycin, neocarzinostatin), topoisomerase inhibitors (camptothecin, etoposide), replication inhibitors (hydroxyurea, aphidicolin), chemicals (such as hydrogen peroxide), and localized laser micro-irradiation (damaging only a stripe in the nucleus). Since no new DSBs should occur after treatment, repair events are synchronous and can be kinetically resolved. Yet, while very potent and in many cases clinically relevant, these methods suffer drawbacks, limiting their use in high-resolution chromatin studies. First, most of these methods induce DSB randomly throughout the genome, with the exception of topoisomerase poisons and replicative stress inducers, which produce DSB within 'hot spots' (i.e., respectively at sites of topoisomerase binding, or at sites where replication forks collapse). However even in the latter cases, where the positions of the DSBs are known, it is still unclear how often breakage occurs across the cell population, which will effectively dilute any high-resolution signal obtained at the population level. Furthermore, most methods induce other types of damage (oxidized bases, abasic sites, ssDNA breaks) or may only effectively trigger DSBs in some cells in the population (e.g., in S phase). A variety of sequence-specific DSBs induction systems have been developed over the years. This includes the HO endonuclease in yeast, TALE nucleases, Zinc Finger nucleases (e.g., FokI), CRISPR/Cas9, homing endonucleases (I-SceI or I-PpoI), and restriction enzymes such as AsiSI (reviewed in [145Mladenova V. et al.Novel biological approaches for testing the contributions of single dsbs and DSB clusters to the biological effects of high LET radiation.Front. Oncol. 2016; 6: 163Crossref PubMed Scopus (3) Google Scholar]). These tools allow induction of DSBs at known positions on the genome, and importantly in a homogenous manner in the cell population, making them amenable for high-resolution studies such as ChIP-seq. Furthermore, some systems (including AsiSI, I-PpoI, or CRISPR/Cas9) allow one to induce multiple DSBs scattered across the genome, allowing the comparison of repair at various endogenous loci, while HO (unless used at its original Mat locus) or I-SceI require the enzyme recognition site to be artificially introduced in the genome. By design, the main disadvantage of these methods stems from the ability of nuclease to recut properly repaired sites, impairing efficient kinetic deconvolution of repair reactions, in sharp contrast with localized laser micro-irradiations, which can resolve events occurring a few seconds apart. Instead, the cell population represents a mixture of events ranging from early cleavage to late repair, which may impede the efficient detection of transient events. Lastly, it is important to stress that these annotated DSBs induction systems may not all be equivalent. For example, AsiSI is unable to create DSBs in heterochromatin (being inhibited by DNA methylation) and Cas9-induced breaks display an inherent propensity to be repaired by mutagenic pathways [146Brinkman E.K. et al.Kinetics and fidelity of the repair of Cas9-induced double-strand DNA breaks.Mol. Cell. 2018; 70: 801-813.e6Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. Common DSB induction methods include ionizing radiations (γ- or X-rays), crosslinking agents (mitomycin C, cisplatin), radiomimetic compounds (bleomycin, neocarzinostatin), topoisomerase inhibitors (camptothecin, etoposide), replication inhibitors (hydroxyurea, aphidicolin), chemicals (such as hydrogen peroxide), and localized laser micro-irradiation (damaging only a stripe in the nucleus). Since no new DSBs should occur after treatment, repair events are synchronous and can be kinetically resolved. Yet, while very potent and in many cases clinically relevant, these methods suffer drawbacks, limiting their use in high-resolution chromatin studies. First, most of these methods induce DSB randomly throughout the genome, with the exception of topoisomerase poisons and replicative stress inducers, which produce DSB within 'hot spots' (i.e., respectively at sites of topoisomerase binding, or at sites where replication forks collapse). However even in the latter cases, where the positions of the DSBs are known, it is still unclear how often breakage occurs across the cell population, which will effectively dilute any high-resolution signal obtained at the population level. Furthermore, most methods induce other types of damage (oxidized bases, abasic sites, ssDNA breaks) or may only effectively trigger DSBs in some cells in the population (e.g., in S phase). A variety of sequence-specific DSBs induction systems have been developed over the years. This includes the HO endonuclease in yeast, TALE nucleases, Zinc Finger nucleases (e.g., FokI), CRISPR/Cas9, homing endonucleases (I-SceI or I-PpoI), and restriction enzymes such as AsiSI (reviewed in [145Mladenova V. et al.Novel biological approaches for testing the contributions of single dsbs and DSB clusters to the biological effects of high LET radiation.Front. Oncol. 2016; 6: 163Crossref PubMed Scopus (3) Google Scholar]). These tools allow induction of DSBs at known positions on the genome, and importantly in a homogenous manner in the cell population, making them amenable for high-resolution studies such as ChIP-seq. Furthermore, some systems (including AsiSI, I-PpoI, or CRISPR/Cas9) allow one to induce multiple DSBs scattered across the genome, allowing the comparison of repair at various endogenous loci, while HO (unless used at its original Mat locus) or I-SceI require the enzyme recognition site to be artificially introduced in the genome. By design, the main disadvantage of these methods stems from the ability of nuclease to recut properly repaired sites, impairing efficient kinetic deconvolution of repair reactions, in sharp contrast with localized laser micro-irradiations, which can resolve events occurring a few seconds apart. Instead, the cell population represents a mixture of events ranging from early cleavage to late repair, which may impede the efficient detection of transient events. Lastly, it is important to stress that these annotated DSBs induction systems may not all be equivalent. For example, AsiSI is unable to create DSBs in heterochromatin (being inhibited by DNA methylation) and Cas9-induced breaks display an inherent propensity to be repaired by mutagenic pathways [146Brinkman E.K. et al.Kinetics and fidelity of the repair of Cas9-induced double-strand DNA breaks.Mol. Cell. 2018; 70: 801-813.e6Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. Elegant studies, following the recruitment kinetics for many repair factors upon DSB induction at localized micro-irradiation (e.g., [7Aleksandrov R. et al.Protein dynamics in complex DNA lesions.Mol. Cell. 2018; 69: 1046-1061.e5Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar, 8Kochan J.A. et al.Meta-analysis of DNA double-strand break response kinetics.Nucleic Acids Res. 2017; 45: 12625-12637Crossref PubMed Scopus (3) Google Scholar]) have started to accurately establish the spatio-temporal organization of the DDR. In addition, the use of phospho-activatable green fluorescent protein (GFP)-tagged core histone identified that chromatin is rapidly relaxed following laser micro-irradiation in a manner that depends on poly(ADP-ribose) polymerase 1 (PARP1) and chromatin remodelers (e.g., [9Sellou H. et al.The poly(ADP-ribose)-dependent chromatin remodeler Alc1 induces local chromatin relaxation upon DNA damage.Mol. Biol. Cell. 2016; 27: 3791-3799Crossref PubMed Scopus (20) Google Scholar, 10Smith R. et al.CHD3 and CHD4 recruitment and chromatin remodeling activity at DNA breaks is promoted by early poly(ADP-ribose)-dependent chromatin relaxation.Nucleic Acids Res. 2018; 46: 6087-6098Crossref PubMed Scopus (2) Google Scholar, 11Luijsterburg M.S. et al.PARP1 links CHD2-mediated chromatin expansion and H3.3 deposition to DNA repair by non-homologous end-joining.Mol. Cell. 2016; 61: 547-562Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 12Burgess R.C. et al.Activation of DNA damage response signaling by condensed chromatin.Cell Rep. 2014; 9: 1703-1717Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar]), followed by a recompaction phase, necessary for full DDR activation [12Burgess R.C. et al.Activation of DNA damage response signaling by condensed chromatin.Cell Rep. 2014; 9: 1703-1717Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar] (Figure 1, Key Figure). Altogether, this revealed that chromatin structure is altered in an ordered manner following DSB (reviewed in [13Dantuma N.P. van Attikum H. Spatiotemporal regulation of posttranslational modifications in the DNA damage response.EMBO J. 2016; 35: 6-23Crossref PubMed Google Scholar]). Yet, the exact molecular nature of in cis chromatin modifications that accounts for this DSB-induced chromatin behavior visualized by microscopy is unknown. Since chromatin is organized at many different scales, it is important to precisely understand how DSBs can reshape this structure at each level, from the nucleosomes adjacent to the broken sites up to entire chromosomal domains, and this starts with the molecular characterization of the chromatin landscape induced at DSB. To this aim, a systematic and high-resolution ChIP-seq mapping of histone modifications induced at multiple DSBs was performed in human cells [14Clouaire T. et al.Comprehensive mapping of histone modifications at DNA double-strand breaks deciphers repair pathway chromatin signatures.Mol. Cell. 2018; 72: 250-262.e6Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. This study took advantage from the DIvA system (Box 3), which allows for the controlled induction of about 100 DSBs, in both transcriptionally active and inactive regions (with exception to heterochromatin), within a short time window of 4 h in cultured U2OS cells. This revealed a multiscale and complex pattern of induced histone modifications around DSBs, with few large-scale (megabase-wide) chromatin changes, as well as local (<10 kb) alterations [14Clouaire T. et al.Comprehensive mapping of histone modifications at DNA double-strand breaks deciphers repair pathway chromatin signatures.Mol. Cell. 2018; 72: 250-262.e6Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar] (Figure 1). In addition, mapping of repair proteins, such as those involved in HR or NHEJ, allowed assignment of a specific chromatin landscape to a particular repair pathway. These local alterations of the chromatin landscape are both involved in recruiting repair proteins (such as pro- or anti-resection factors) and in modulating the nucleosome stability onto DNA, thereby regulating DSB processing. Below, we provide a recent update on the nature and (potential) function of the chromatin-induced DSB landscape, with a focus on histone post-translational modifications occurring during either HR or NHEJ and thereby defining the 'repair pathway histone code' (Figure 2).Figure 2A DSB Repair Histone Code. Recent studies revealed that nucleosomes exhibit a specific composition and post-translational signature during repair by non-homologous end joining (NHEJ; left) and homologous recombination (HR; right) repair. Yet, whether, how, and when these histone modifications, individually or collectively, are actually required for repair still remains to be determined. The cross on top of a histone modification indicates the disappearance of this particular modification upon damage. N-term, N terminus; Ac, acetyl-; P, phospho-; Me, methyl-; Ub, ubiquitin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The first histone modification shown to be specifically induced in cis to DSB was the phosphorylation of the H2AX histone variant on serine 139 also known as γH2AX [15Rogakou E.P. et al.DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139.J. Biol. Chem. 1998; 273: 5858-5868Crossref PubMed Scopus (3319) Google Scholar]. This modification, mainly catalyzed by kinases such as ataxia telangiectasia mutated (ATM) or ataxia telangiectasia and Rad3-related (ATR), displays the striking behavior to spread over incredibly large domains since it can cover up to 2 Mb of DSB-surrounding chromatin in mammals [16Iacovoni J.S. et al.High-resolution profiling of gammaH2AX around DNA double strand breaks in the mammalian genome.EMBO J. 2010; 29: 1446-1457Crossref PubMed Scopus (211) Google Scholar, 17Caron P. et al.Non-redundant functions of ATM and DNA-PKcs in response to DNA double-strand breaks.Cell Rep. 2015; 13: 1598-1609Abstract Full Text Full Text PDF PubMed Google Scholar, 18Savic V. et al.Formation of dynamic gamma-H2AX domains along broken DNA strands is distinctly regulated by ATM and MDC1 and dependent upon H2AX densities in chromatin.Mol. Cell. 2009; 34: 298-310Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar]. Interestingly, γH2AX signaling may not only rely on modification of pre-existing H2AX but also on de novo deposition of this variant at d