Abstract Chromatin structure is extensively modified and remodeled to allow for efficient signaling and repair of DNA double-strand breaks (DSBs), a process which is influenced by the nature and properties of the damaged locus or the repair pathway involved. However, the extent of cell-to-cell variability in these DSB-induced chromatin modifications remains poorly characterized. Here, we report how chromatin accessibility is reshuffled at the damaged locus at high resolution. Notably, we identify a long-range increase in chromatin accessibility which is dependent on functional resection. We report that resection-dependent chromatin remodeling events at individual damaged loci display significant heterogeneity in single cells which are only partly explained by the cell cycle, and can frequently be detected as asymmetric and unidirectional. Moreover, we identify strong transcriptional phenotypes upon sustained DNA damage as well as subpopulations of S and G2/M cells for which similar resection-dependent chromatin accessibility patterns occur simultaneously at multiple DSBs. These coordinated chromatin accessibility patterns at multiple DSBs are associated with specific gene expression programs, including DNA damage checkpoint signaling, inflammation and apoptosis. Altogether, our results highlight the heterogeneity in chromatin remodeling during DSB repair and suggest unexpected links between DNA end resection and specific gene expression programs mounted in response to genotoxic stress.

Repair of DNA double-strand breaks (DSBs) produced in transcriptionally active chromatin occurs through a poorly characterized pathway called transcription-coupled DSB repair (TC-DSBR). Here, using a screening approach scoring multiple outputs in human cells, we identified proteins from the PERIOD complex, ensuring circadian oscillations, as previously unknown TC-DSBR players. We show that PER2 is recruited at TC-DSBs and contributes to their targeting to the nuclear envelope (NE), where SUN1 and the nuclear pore complex (NPC) act as docking sites. TC-DSB anchoring at the NE fosters RAD51 assembly and prevents DSB clustering and translocations. In agreement, the circadian clock regulates TC-DSB targeting to the NE, RAD51 assembly, and DSB clustering. Our study shows a direct link between the circadian rhythm and the response to DSBs in transcribed genes, opening strategies for chrono-chemotherapies based on topoisomerase poisons that induce DSBs in active loci.

RNA:DNA hybrids accumulate at DNA double-strand breaks (DSBs) and were shown to regulate homologous recombination repair. The mechanism responsible for the formation of these non-canonical RNA:DNA structures remains unclear although they were proposed to arise consequently to RNA polymerase II or III loading followed by DSB-induced de novo transcription at the break site. Here, we found no evidence of RNA polymerase recruitment at DSBs. Rather, strand-specific R-loop mapping revealed that RNA:DNA hybrids are mainly generated at DSBs occurring in transcribing loci, from the hybridization of pre-existing RNA to the 3' overhang left by DNA end resection. We further identified the H3K4me3 reader spindlin 1 and the transcriptional regulator PAF1 as factors promoting RNA:DNA hybrid accumulation at DSBs, through their role in mediating transcriptional repression in cis to DSBs. Altogether, we provide evidence that RNA:DNA hybrids accumulate at DSBs occurring in transcribing loci as a result of DSB-induced transcriptional shut down.

L'acide performique (PFA) est utilisé pour désinfecter les eaux résiduaires urbaines (ERU) en raison de son efficacité prouvée contre divers microorganismes pathogènes. Cependant, ses avantages par rapport à l’ozone, l’un des désinfectants principaux dans le traitement des ERU, ainsi que sa réactivité avec les molécules pharmaceutiques, restent peu documentés. Cette étude, menée sur un pilote alimenté en continu par de l'eau nitrifiée, compare l'efficacité du PFA, seul ou en couplage pour générer des procédés d’oxydation avancée (POAs), à celle de l'ozone pour éliminer trois microorganismes pathogènes (Escherichia coli, E. coli, entérocoques intestinaux, EI et spores de bactéries sulfito-réductrices, SBSR) et sept molécules pharmaceutiques couramment détectées dans les ERU de la région parisienne. Les résultats montrent que le PFA élimine efficacement E. coli et les EI (élimination croissante entre 0,8 et 2 mg PFA/L) et qu’il est plus efficace que l’ozone à toutes les doses spécifiques testées (0,14 à 1,00 gO3/gCOD). Les SBSR ont été mieux éliminées avec les POAs UV-C/PFA et ozone/PFA, grâce aux espèces réactives issues de la décomposition du PFA par l’ozone et/ou la photolyse UV-C. En outre, l'élimination des molécules pharmaceutiques par le PFA a montré des abattements moyens faibles (de 6 à 45 % pour 2 à 10 mg PFA/L). Par ailleurs, l’UV-C/PFA améliore les abattements moyens, en particulier à faible dose (22 % à 2 mg PFA/L), bien que cette amélioration ne soit pas due à un effet synergique. En revanche, comparé au PFA, l’ozone à fortes doses spécifiques a conduit à des abattements plus élevés (de 62 à 92 % pour 0,48 à 1,00 gO3/gCOD). De plus, à une faible dose d’ozone spécifique (0,29 gO3/gCOD), ozone/PFA s'est avéré plus efficace que l'utilisation individuelle du PFA ou de l'ozone.

The DNA damage response is essential to safeguard genome integrity. Although the contribution of chromatin in DNA repair has been investigated 1 , 2 , the contribution of chromosome folding to these processes remains unclear 3 . Here we report that, after the production of double-stranded breaks (DSBs) in mammalian cells, ATM drives the formation of a new chromatin compartment (D compartment) through the clustering of damaged topologically associating domains, decorated with γH2AX and 53BP1. This compartment forms by a mechanism that is consistent with polymer–polymer phase separation rather than liquid–liquid phase separation. The D compartment arises mostly in G1 phase, is independent of cohesin and is enhanced after pharmacological inhibition of DNA-dependent protein kinase (DNA-PK) or R-loop accumulation. Importantly, R-loop-enriched DNA-damage-responsive genes physically localize to the D compartment, and this contributes to their optimal activation, providing a function for DSB clustering in the DNA damage response. However, DSB-induced chromosome reorganization comes at the expense of an increased rate of translocations, also observed in cancer genomes. Overall, we characterize how DSB-induced compartmentalization orchestrates the DNA damage response and highlight the critical impact of chromosome architecture in genomic instability.

Repair of DNA Double-Strand Breaks (DSBs) produced in transcriptionally active chromatin occurs through a poorly characterized pathway called Transcription-Coupled DSB repair (TC-DSBR). Here, using a screening approach scoring multiple outputs in human cells, we identified proteins from the PERIOD complex, a key module ensuring circadian oscillations, as novel TC-DSBR players. We show that the core PER complex protein PER2 is recruited at TC-DSBs and that it contributes to the targeting of TC-DSBs at the nuclear envelope (NE). At the NE, SUN1 and the Nuclear Pore Complex (NPC) act as docking sites for TC-DSBs and TC-DSB anchoring fosters RAD51 assembly. Impaired DSB localization at the NE results in elevated DSB clustering and translocation rate. In agreement, the circadian clock regulates TC-DSB anchoring to the NE, RAD51 assembly, and DSB clustering. Our study shows that DSB localization to the NPC is a conserved molecular pathway that also occurs in human cells and provides a direct link between the circadian rhythm and the response to DSBs occurring in active genes. This opens new therapeutic strategies for chemotherapies based on drugs that are inducing DSBs in active loci such as topoisomerase poisons.

L’objectif de cet article est de proposer des protocoles de caractérisation thermo-rhéologique des boues digérées pour réaliser un dimensionnement pertinent des installations de traitement en station de traitement des eaux usées (STEU). Pour cela, tous les aspects rhéologiques pouvant impacter l’écoulement des boues dans les composants hydrauliques sont abordés, notamment le seuil d’écoulement, le comportement rhéofluidifiant, la thixotropie, la viscoélasticité ou encore la dépendance thermique des caractéristiques rhéologiques. Un protocole de détermination de chacune de ces propriétés rhéologiques adapté aux boues digérées issues de STEU est détaillé. Les lignes directrices proposées ici se basent sur les retours d’expériences de campagnes de caractérisation menées en laboratoire sur des boues digérées prélevées en STEU. Les résultats des caractérisations menées grâce à l’application des protocoles décrits confirment la présence d’un seuil d’écoulement et d’un caractère rhéofluidifiant pour des boues digérées issues de STEU. Ils montrent également que dans le cas d’une boue digérée, la thermo-rhéologie peut être simplifiée, car la thixotropie, la viscoélasticité et la dépendance thermique ont un impact négligeable sur l’écoulement de la boue digérée dans les conditions typiques des procédés en STEU.

Dysregulations of lipid metabolism in the liver may trigger steatosis progression, leading to potentially severe clinical consequences such as nonalcoholic fatty liver diseases (NAFLDs). Molecular mechanisms underlying liver lipogenesis are very complex and fine-tuned by chromatin dynamics and multiple key transcription factors. Here, we demonstrate that the nuclear factor HMGB1 acts as a strong repressor of liver lipogenesis. Mice with liver-specific Hmgb1 deficiency display exacerbated liver steatosis, while Hmgb1 -overexpressing mice exhibited a protection from fatty liver progression when subjected to nutritional stress. Global transcriptome and functional analysis revealed that the deletion of Hmgb1 gene enhances LXRα and PPARγ activity. HMGB1 repression is not mediated through nucleosome landscape reorganization but rather via a preferential DNA occupation in a region carrying genes regulated by LXRα and PPARγ. Together, these findings suggest that hepatocellular HMGB1 protects from liver steatosis development. HMGB1 may constitute a new attractive option to therapeutically target the LXRα-PPARγ axis during NAFLD.

The repair of DNA double-strand breaks (DSBs) is essential for safeguarding genome integrity. When a DSB forms, the PI3K-related ATM kinase rapidly triggers the establishment of megabase-sized, chromatin domains decorated with phosphorylated histone H2AX (γH2AX), which act as seeds for the formation of DNA-damage response foci1. It is unclear how these foci are rapidly assembled to establish a 'repair-prone' environment within the nucleus. Topologically associating domains are a key feature of 3D genome organization that compartmentalize transcription and replication, but little is known about their contribution to DNA repair processes2,3. Here we show that topologically associating domains are functional units of the DNA damage response, and are instrumental for the correct establishment of γH2AX-53BP1 chromatin domains in a manner that involves one-sided cohesin-mediated loop extrusion on both sides of the DSB. We propose a model in which H2AX-containing nucleosomes are rapidly phosphorylated as they actively pass by DSB-anchored cohesin. Our work highlights the importance of chromosome conformation in the maintenance of genome integrity and demonstrates the establishment of a chromatin modification by loop extrusion.

DNA is a complex molecule carrying the instructions an organism needs to develop, live and reproduce. In 1953, Watson and Crick discovered that DNA is composed of two chains forming a double-helix. Later on, other structures of DNA were discovered and shown to play important roles in the cell, in particular G-quadruplex (G4). Following genome sequencing, several bioinformatic algorithms were developed to map G4s in vitro based on a canonical sequence motif, G-richness and G-skewness or alternatively sequence features including k-mers, and more recently machine/deep learning. Recently, new sequencing techniques were developed to map G4s in vitro (G4-seq) and G4s in vivo (G4 ChIP-seq) at few hundred base resolution. Here, we propose a novel convolutional neural network (DeepG4) to map cell-type specific active G4 regions ( e.g . regions within which G4s form both in vitro and in vivo). DeepG4 is very accurate to predict active G4 regions in different cell types. Moreover, DeepG4 identifies key DNA motifs that are predictive of G4 region activity. We found that such motifs do not follow a very flexible sequence pattern as current algorithms seek for. Instead, active G4 regions are determined by numerous specific motifs. Moreover, among those motifs, we identified known transcription factors (TFs) which could play important roles in G4 activity by contributing either directly to G4 structures themselves or indirectly by participating in G4 formation in the vicinity. In addition, we used DeepG4 to predict active G4 regions in a large number of tissues and cancers, thereby providing a comprehensive resource for researchers. Availability: https://github.com/morphos30/DeepG4 .

Among the types of damage, DNA double-strand breaks (DSBs) (provoked by various environmental stresses, but also during normal cell metabolic activity) are the most deleterious, as illustrated by the variety of human diseases associated with DSB repair defects. DSBs are repaired by two groups of pathways: homologous recombination (HR) and nonhomologous end joining. These pathways do not trigger the same mutational signatures, and multiple factors, such as cell cycle stage, the complexity of the lesion and also the genomic location, contribute to the choice between these repair pathways. To study the usage of the HR machinery at DSBs, we propose a genome-wide method based on the chromatin immunoprecipitation of the HR core component Rad51, followed by high-throughput sequencing.

DNA Double-Strand Breaks (DSBs) repair is essential to safeguard genome integrity but the contribution of chromosome folding into this process remains elusive. Here we unveiled basic principles of chromosome dynamics upon DSBs in mammalian cells, controlled by key kinases from the DNA Damage Response. We report that ATM is responsible for the reinforcement of topologically associating domains (TAD) that experience a DSB. ATM further drives the formation of a new chromatin sub-compartment (“D” compartment) upon clustering of damaged TADs decorated with γH2AX and 53BP1. “D” compartment formation mostly occurs in G1, is independent of cohesin and is enhanced upon DNA-PK pharmacological inhibition. Importantly, a subset of DNA damage responsive genes that are upregulated following DSBs also physically localize in the D sub-compartment and this ensures their optimal activation, providing a function for DSB clustering in activating the DNA Damage Response. However, these DSB-induced changes in genome organization also come at the expense of an increased translocations rate, which we could also detect on cancer genomes. Overall, our work provides a function for DSB-induced compartmentalization in orchestrating the DNA Damage Response and highlights the critical impact of chromosome architecture in genomic instability. ### Competing Interest Statement The authors have declared no competing interest.

DNA Double-Strand Breaks (DSBs) repair is essential to safeguard genome integrity. Upon DSBs, the ATM PI3K kinase rapidly triggers the establishment of megabase-sized, γH2AX-decorated chromatin domains which further act as seeds for the formation of DNA Damage Response (DDR) foci 1 . How these foci are rapidly assembled in order to establish a “repair-prone” environment within the nucleus is yet unclear. Topologically Associating Domains (TADs) are a key feature of 3D genome organization that regulate transcription and replication, but little is known about their contribution to DNA repair processes 2,3 . Here we found that TADs are functional units of the DDR, instrumental for the correct establishment of γH2AX/53BP1 chromatin domains in a manner that involves one-sided cohesin-mediated loop extrusion on both sides of the DSB. We propose a model whereby H2AX-containing nucleosomes are rapidly phosphorylated as they actively pass by DSB-anchored cohesin. Our work highlights the critical impact of chromosome conformation in the maintenance of genome integrity and provides the first example of a chromatin modification established by loop extrusion.

DNA is a complex molecule carrying the instructions an organism needs to develop, live and reproduce. In 1953, Watson and Crick discovered that DNA is composed of two chains forming a double-helix. Later on, other structures of DNA were discovered and shown to play important roles in the cell, in particular G-quadruplex (G4). Following genome sequencing, several bioinformatic algorithms were developed to map G4s in vitro based on a canonical sequence motif, G-richness and G-skewness or alternatively sequence features including k-mers, and more recently machine/deep learning. Here, we propose a novel convolutional neural network (DeepG4) to map active G4s (forming both in vitro and in vivo). DeepG4 is very accurate to predict active G4s, while most state-of-the-art algorithms fail. Moreover, DeepG4 identifies key DNA motifs that are predictive of G4 activity. We found that active G4 motifs do not follow a very flexible sequence pattern as current algorithms seek for. Instead, active G4s are determined by numerous specific motifs. Moreover, among those motifs, we identified known transcription factors (TFs) which could play important roles in G4 activity by contributing either directly to G4 structures themselves or indirectly by participating in G4 formation in the vicinity. Moreover, we showed that specific TFs might explain G4 activity depending on cell type. Lastly, variant analysis suggests that SNPs altering predicted G4 activity could affect transcription and chromatin, e.g . gene expression, H3K4me3 mark and DNA methylation. Thus, DeepG4 paves the way for future studies assessing the impact of known disease-associated variants on DNA secondary structure by providing a mechanistic interpretation of SNP impact on transcription and chromatin. Availability: . Author summary DNA is a molecule carrying genetic information and found in all living cells. In 1953, Watson and Crick found that DNA has a double helix structure. However, other DNA structures were later identified, and most notably, G-quadruplex (G4). In 2000, the Human Genome Project revealed the widespread presence of G4s in the genome using algorithms. To date, all G4 mapping algorithms were developed to map G4s on naked DNA, without knowing if they could be formed in the cell. Here, we designed a novel artificial intelligence algorithm that could map G4s active in the cell from the DNA sequence. We showed its better accuracy compared to existing algorithms. Moreover, we identified key transcriptional factor motifs that could explain G4 activity depending on cell type. Lastly, we demonstrated the existence of mutations that could alter G4 activity and therefore impact molecular processes, such as transcription, in the cell. Such results could provide a novel mechanistic interpretation of known disease-associated mutations. ### Competing Interest Statement The authors have declared no competing interest.

The ribosomal DNA (rDNA) represents a particularly unstable locus undergoing frequent breakage. DNA double-strand breaks (DSBs) within rDNA induce both rDNA transcriptional repression and nucleolar segregation, but the link between the two events remains unclear. Here we found that DSBs induced on rDNA trigger transcriptional repression in a cohesin- and HUSH (human silencing hub) complex-dependent manner throughout the cell cycle. In S/G2 cells, transcriptional repression is further followed by extended resection within the interior of the nucleolus, DSB mobilization at the nucleolar periphery within nucleolar caps, and repair by homologous recombination. We showed that nuclear envelope invaginations frequently connect the nucleolus and that rDNA DSB mobilization, but not transcriptional repression, involves the nuclear envelope-associated LINC complex and the actin pathway. Altogether, our data indicate that rDNA break localization at the nucleolar periphery is not a direct consequence of transcriptional repression but rather is an active process that shares features with the mobilization of persistent DSB in active genes and heterochromatin.

The histone demethylase LSD1 is a key chromatin regulator that is often deregulated in cancer. Its ortholog, dLsd1 plays a crucial role in Drosophila oogenesis; however, our knowledge of dLsd1 function is insufficient to explain its role in the ovary. Here, we have performed genome-wide analysis of dLsd1 binding in the ovary, and we document that dLsd1 is preferentially associated to the transcription start site of developmental genes. We uncovered an unanticipated interplay between dLsd1 and the GATA transcription factor Serpent and we report an unexpected role for Serpent in oogenesis. Besides, our transcriptomic data show that reducing dLsd1 levels results in ectopic transposable elements (TE) expression correlated with changes in H3K4me2 and H3K9me2 at TE loci. In addition, our results suggest that dLsd1 is required for Piwi dependent TE silencing. Hence, we propose that dLsd1 plays crucial roles in establishing specific gene expression programs and in repressing transposons during oogenesis.

Le mercredi 3 juillet 2019 vers 16h45, un incendie s’est declare sur l’unite de clarifloculation de la station d'epuration de Seine Aval du SIAAP (Yvelines). Les performances de traitement ont ete temporairement degradees pendant 3 jours ; impliquant un apport important de flux de matiere organique et de nutriments dans le milieu naturel. Ces apports ont conduit a une desoxygenation forte de la Seine jusqu’a 30 km a l’aval de la station, avec une mortalite piscicole estimee a 7,5 tonnes (poissons, algues et dechets) sur ce bief de la Seine. Les reseaux de suivi de la qualite de la Seine ont ete immediatement mobilises pour suivre l’impact de l’incident sur la qualite du milieu naturel. La mobilisation des equipes du SIAAP, de Sorbonne Universite (PirenSeine), de Normandie Universite et du GIP-Seine Aval, a permis de mettre en place un suivi environnemental, allant de Conflans-Sainte-Honorine a Tancarville, soit 280 km de lineaire de Seine suivis. Cet article discute de l’impact de l’incident sur le milieu naturel et de son attenuation lors du transit de l’eau sur le bassin versant Seine Normandie, a l’aval de l’agglomeration parisienne.

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.

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.