Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures

摘要

class="head no_bottom_margin" id="sec1title">IntroductionDNA double-strand breaks (DSBs) are extremely detrimental since they can lead to mutations and chromosomal rearrangements. DSBs arise from various environmental stresses and upon developmentally scheduled activation of endonucleases, but they can also arise physiologically during replication and transcription. Anomalies in the DSB repair apparatus are responsible for premature aging and neurodegenerative syndromes and are strongly implicated in cancer onset and progression.DSBs are mainly repaired by two partially redundant, yet profoundly different, pathways: homologous recombination (HR) and non-homologous end joining (NHEJ) (for review, see ). HR uses an intact copy of the damaged locus as a template and involves many factors for DSB detection, 5′ end resection, homology search, strand invasion, and resolution. In contrast, NHEJ repair machineries require no or limited resection and can join the two broken ends with no or minimal homology. Inaccuracy, failure, or misuse of each of these pathways can trigger very different consequences on the genome. DSB repair pathway choice can be influenced by cell cycle phase (), DNA end complexity (), and the type of damaged locus (, ).In eukaryotes, DSB repair occurs in the context of chromatin. Chromatin is a highly dynamic structure, affected by histone post-translational modifications, DNA methylation, or incorporation of histone variants (for review, see , ). Chromatin modifications can alter the stability of the histone octamer onto DNA but also be specifically recognized by “reader” modules found in chromosomal proteins as well as subunits of DNA transaction machineries. All together, nucleosome modifications regulate DNA accessibility; the stiffness, flexibility, and mobility of chromatin within the nucleus; and the recruitment of molecular machines ensuring transcription, replication, and repair.Key aspects in the interplay between DSB repair and chromatin environment have already emerged (). H2AX is rapidly phosphorylated by ATM (and named γH2AX) over several megabases surrounding the break (, , , ). In parallel, histone acetyltransferases and deacetylases tightly control acetylation levels of several residues of H3, H4, and H2A (, , , href="#bib62" rid="bib62" class=" bibr popnode">Lee et al., 2010, href="#bib72" rid="bib72" class=" bibr popnode">Miller et al., 2010, href="#bib81" rid="bib81" class=" bibr popnode">Ogiwara et al., 2011, href="#bib115" rid="bib115" class=" bibr popnode">Toiber et al., 2013) to regulate chromatin relaxation near DSBs. Acetylated histones also participate in the recruitment of nucleosome remodeling factors, enhancing DSB accessibility and facilitating resection (href="#bib8" rid="bib8" class=" bibr popnode">Bennett and Peterson, 2015, href="#bib62" rid="bib62" class=" bibr popnode">Lee et al., 2010, href="#bib115" rid="bib115" class=" bibr popnode">Toiber et al., 2013). Similarly, histone methyltransferases and demethylases can regulate recruitment and/or stabilization of repair proteins (CtIP, 53BP1, BRCA1 …) (href="#mmc1" rid="mmc1" class=" supplementary-material">Table S2). Moreover, ubiquitination and sumoylation pathways contribute to DSB-induced chromatin reorganization (for review, see href="#bib97" rid="bib97" class=" bibr popnode">Schwertman et al., 2016). All together, these specific chromatin modifications generate a chromatin state permissive for repair but also directly contribute to the recruitment of DSB repair machineries, repair pathway choice, and the activation of the DNA damage checkpoint.Yet the definite map of DSB-induced chromatin modifications and their respective involvement in DSB repair remain largely unknown. Furthermore, NHEJ and HR repair pathways conceivably require very different chromatin settings. Since chromatin structure plays a central role in DNA accessibility and flexibility, an in-depth characterization of the chromatin that assembles at DSBs represents a critical step in understanding how DSB repair machineries operate in the whole nucleus to restore the original DNA sequence and avoid deleterious genome rearrangements.Here, using high-throughput genomic approaches in a standardized system in which multiple DSBs are induced at defined positions across the human genome, we report the first comprehensive picture of the chromatin landscape induced around DSBs and its relationship with individual repair pathways.