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.

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.

The ability of DNA Double Strand Breaks (DSBs) to cluster in mammalian cells has been subjected to intense debate over the past few 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. We unambiguously found that DSBs do cluster but only when induced in transcriptionally active genes. Clustering of damaged genes mainly occurs 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 LINC (LInker of Nuclear and Cytoplasmic skeleton) complex, suggesting that active mechanisms promote DSB clustering. This work reveals that when damaged, active genes exhibit a very peculiar behavior compared to the rest of the genome, being mostly left unrepaired and clustered in G1 while being repaired by homologous recombination in post-replicative cells.

Upon DNA damage, histone modifications are reshaped to accommodate DNA damage signaling and repair. Gong et al. report that the histone demethylase KDM5A promotes loading of the chromatin remodeling complex ZMYND8–NuRD to double-strand DNA breaks through H3K4me3 demethylation, thereby allowing repair of the lesion.

SummaryDNA double-strand breaks (DSBs) elicit the so-called DNA damage response (DDR), largely relying on ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PKcs), two members of the PI3K-like kinase family, whose respective functions during the sequential steps of the DDR remains controversial. Using the DIvA system (DSB inducible via AsiSI) combined with high-resolution mapping and advanced microscopy, we uncovered that both ATM and DNA-PKcs spread in cis on a confined region surrounding DSBs, independently of the pathway used for repair. However, once recruited, these kinases exhibit non-overlapping functions on end joining and γH2AX domain establishment. More specifically, we found that ATM is required to ensure the association of multiple DSBs within “repair foci.” Our results suggest that ATM acts not only on chromatin marks but also on higher-order chromatin organization to ensure repair accuracy and survival.

Non-coding RNAs (ncRNAs) play major roles in proper chromatin organization and function. Senescence, a strong anti-proliferative process and a major anticancer barrier, is associated with dramatic chromatin reorganization in heterochromatin foci. Here we analyze strand-specific transcriptome changes during oncogene-induced human senescence. Strikingly, while differentially expressed RNAs are mostly repressed during senescence, ncRNAs belonging to the recently described vlincRNA (very long intergenic ncRNA) class are mainly activated. We show that VAD, a novel antisense vlincRNA strongly induced during senescence, is required for the maintenance of senescence features. VAD modulates chromatin structure in cis and activates gene expression in trans at the INK4 locus, which encodes cell cycle inhibitors important for senescence-associated cell proliferation arrest. Importantly, VAD inhibits the incorporation of the repressive histone variant H2A.Z at INK4 gene promoters in senescent cells. Our data underline the importance of vlincRNAs as sensors of cellular environment changes and as mediators of the correct transcriptional response.

Antisense RNAs are non-coding RNAs that can regulate their corresponding sense RNAs and are generally produced from specific promoters. We uncover here a family of antisense RNAs, named START RNAs, produced during cellular senescence by transcriptional read-through at convergent protein-coding genes. Importantly, START RNAs repress the expression of their corresponding sense RNAs. In proliferative cells, we found that the Pol II elongation rate is limited downstream of TTS at START loci, allowing transcription termination to occur before Pol II reaches the convergent genes, thus preventing antisense RNA production and interference with the expression of the convergent genes. START RNAs are repressed by H2A.Z histone variant, whose local occupancy decreases in senescence. Our results thus uncover a mechanism of gene expression regulation relying on read-through antisense transcript production at convergent genes, underlining the functional importance of chromatin regulation in the control of RNA pol II elongation rate at intergenic regions.

The H2A.Z histone variant plays major roles in the control of gene expression. In human, H2A.Z is encoded by two genes expressing two isoforms, H2A.Z.1 and H2A.Z.2 differing by three amino acids. Here, we undertook an integrated analysis of their functions in gene expression using endogenously-tagged proteins. RNA-Seq analysis in untransformed cells showed that they can regulate both distinct and overlapping sets of genes positively or negatively in a context-dependent manner. Furthermore, they have similar or antagonistic function depending on genes. H2A.Z.1 and H2A.Z.2 can replace each other at Transcription Start Sites, providing a molecular explanation for this interplay. Mass spectrometry analysis showed that H2A.Z.1 and H2A.Z.2 have specific interactors, which can mediate their functional antagonism. Our data indicate that the balance between H2A.Z.1 and H2A.Z.2 at promoters is critically important to regulate specific gene expression, providing an additional layer of complexity to the control of gene expression by histone variants.