H4K20me2 distinguishes pre-replicative from post-replicative chromatin to appropriately direct DNA repair pathway choice by 53BP1-RIF1-MAD2L2

The main pathways for the repair of DNA double strand breaks (DSBs) are non-homologous end-joining (NHEJ) and homologous recombination directed repair (HDR). These operate mutually exclusive and are activated by 53BP1 and BRCA1, respectively. As HDR can only succeed in the presence of an intact copy of replicated DNA, cells employ several mechanisms to inactivate HDR in the G1 phase of cell cycle. As cells enter S-phase, these inhibitory mechanisms are released and HDR becomes active. However, during DNA replication, NHEJ and HDR pathways are both functional and non-replicated and replicated DNA regions co-exist, with the risk of aberrant HDR activity at DSBs in non-replicated DNA. It has become clear that DNA repair pathway choice depends on inhibition of DNA end-resection by 53BP1 and its downstream factors RIF1 and MAD2L2. However, it is unknown how MAD2L2 accumulates at DSBs to participate in DNA repair pathway control and how the NHEJ and HDR repair pathways are appropriately activated at DSBs with respect to the replication status of the DNA, such that NHEJ acts at DSBs in pre-replicative DNA and HDR acts on DSBs in post-replicative DNA. Here we show that MAD2L2 is recruited to DSBs in H4K20 dimethylated chromatin by forming a protein complex with 53BP1 and RIF1 and that MAD2L2, similar to 53BP1 and RIF1, suppresses DSB accumulation of BRCA1. Furthermore, we show that the replication status of the DNA locally ensures the engagement of the correct DNA repair pathway, through epigenetics. In non-replicated DNA, saturating levels of the 53BP1 binding site, di-methylated lysine 20 of histone 4 (H4K20me2), lead to robust 53BP1-RIF1-MAD2L2 recruitment at DSBs, with consequent exclusion of BRCA1. Conversely, replication-associated 2-fold dilution of H4K20me2 promotes the release of the 53BP1-RIF1-MAD2L2 complex and favours the access of BRCA1. Thus, the differential H4K20 methylation status between pre-replicative and post-replicative DNA represents an intrinsic mechanism that locally ensures appropriate recruitment of the 53BP1-RIF1-MAD2L2 complex at DNA DSBs, to engage the correct DNA repair pathway

The CIP2A–TOPBP1 axis safeguards chromosome stability and is a synthetic lethal target for BRCA-mutated cancer

BRCA1/2-mutated cancer cells adapt to the genome instability caused by their deficiency in homologous recombination (HR). Identification of these adaptive mechanisms may provide therapeutic strategies to target tumors caused by the loss of these genes. In the present study, we report genome-scale CRISPR-Cas9 synthetic lethality screens in isogenic pairs of BRCA1- and BRCA2-deficient cells and identify CIP2A as an essential gene in BRCA1- and BRCA2-mutated cells. CIP2A is cytoplasmic in interphase but, in mitosis, accumulates at DNA lesions as part of a complex with TOPBP1, a multifunctional genome stability factor. Unlike PARP inhibition, CIP2A deficiency does not cause accumulation of replication-associated DNA lesions that require HR for their repair. In BRCA-deficient cells, the CIP2A-TOPBP1 complex prevents lethal mis-segregation of acentric chromosomes that arises from impaired DNA synthesis. Finally, physical disruption of the CIP2A-TOPBP1 complex is highly deleterious in BRCA-deficient tumors, indicating that CIP2A represents an attractive synthetic lethal therapeutic target for BRCA1- and BRCA2-mutated cancers

Identification and Characterization of Small Molecule Inhibitors of Polynucleotide Kinase 3'-Phosphatase

DNA lesions arise constantly in cells and are repaired by a variety of DNA repair pathways. Polynucleotide kinase 3’-phosphatase (PNKP) aids repair by phosphorylating 5’-hydroxyl DNA termini and dephosphorylating 3’-phosphate DNA termini for the completion of repair by DNA ligases. This activity is critical in vivo because DNA breaks do not usually possess ligatable termini. PNKP knockdown sensitizes cells to several DNA damaging agents, including the topoisomerase I (TOP1) inhibitor camptothecin - analogs of which are being developed into chemotherapeutic drugs - because the resolution of stalled TOP1-DNA complexes requires processing by PNKP. We hypothesize that small molecule inhibitors of PNKP could bolster the effects of radio- and chemotherapies on cancer cells. I have identified eight compounds that effectively inhibit human PNKP and, with reduced potency, T4 PNK in vitro. These compounds act by reversibly inhibiting the substrate-enzyme interaction but they do not appear to sensitize U2OS cells to camptothecin.MAS

Les modèles d'analyse des comportements à risque face à l'infection à VIH : une conception trop étroite de la rationalité

Moatti (Jean-Paul), Beltzer (Nathalie), Dab (William). - Analysing Unsafe Behaviour in Face of HIV Infection. The Limits of Rationality Social science research on the prevention of HIV infection has pointed to correlations between individuals' knowledge and beliefs about AIDS on the one hand, and their reactions - individual or collective - to infected persons and the risk of transmission, on the other. However, it has also shown that the connection between these correlations and actual behaviour has remained ambiguous. As is the case in other areas of preventive medicine, our results reinforce the view that improved information is not in itself sufficient to change individual behaviour, and thus reduce risk. The international literature that deals with the determinants of exposure to the risk of sexual transmission of HIV has been dominated by existing social-psychological models (the Health Belief Model, or social learning theory), and these models have been used with some success in the' community' implementation of preventive measures in San Fransisco's homosexual and bisexual communities. This paper points out the limitations of these models in explaining or predicting behaviour related to the risk of HIV transmission. It shows that they have relied implicitely on a reductionist notion of individual rationality, in which rationality is equated with complete avoidance of risk and with concern for absolute safety. Comparing these social- psychological models with models of expected utility - standard in the micro-economic analysis of behaviour under conditions of risks and uncertainty - and applying this theory to ACSF survey data, makes it possible to identify other underlying reasons for the persistence of individual exposure to the risk of sexually transmitted HIV, and to show that 'no- change' or 'intermediate change' strategies that do not give complete protection against HIV risks may yet prove to conform better with individual effective rationalities. Finally, the paper suggests some new directions for behavioural analyses that are less machinistic, and which would account better for the specific context and temporal dy- namics in which exposure to the risk of HIV occurs. Such analyses would benefit from the latest theoretical developments in micro-economics and social psychology.Moatti (Jean-Paul), Beltzer (Nathalie), Dab (William) - Les modèles d'analyse des comportements à risque face à l'infection à VIH : une conception trop étroite de la rationalité Les recherches en sciences sociales sur la prévention de l'infection à VIH ont établi des liens entre des connaissances et des croyances sur le SIDA d'une part, des attitudes individuelles et collectives face aux personnes infectées et au risque de transmission d'autre part ; mais elles ont également mis en évidence l'ambiguïté de ces relations avec les comportements déclarés. Comme dans de nombreux autres domaines de la prévention sanitaire, les recherches ont confirmé qu'une amélioration du niveau d'information sur le risque n'est clairement pas une condition suffisante pour provoquer des modifications de comportements individuels tendant à diminuer celui-ci. En matière d'analyse des déterminants de l'exposition au risque de transmission sexuelle du VIH, la littérature internationale est demeurée jusqu'à présent dominée par le recours à des modèles psychosociologiques préexistants (Health Belief Model ou « modèles de croyances pour la santé », théorie de l'apprentissage social, etc.), qui ont notamment servi de référence à l'intervention « communautaire » de prévention mise en œuvre avec un certain succès dans la communauté homosexuelle et bisexuelle de San Francisco. L'article développe une critique des limites de ces modèles en termes d'explication et de prédiction des comportements face au risque de transmission du VIH, et s'efforce de démontrer qu'ils reposent implicitement sur une conception réductrice de la rationalité individuelle qui assimile celle-ci à la seule exposition nulle au risque et à la recherche de la sécurité absolue. Une confrontation de ces modèles psychosociologiques avec la théorie de l'utilité espérée, modélisation classique en micro-économie des comportements des agents face au risque et à l'incertitude, ainsi qu'une application empirique de cette théorie aux données de l'enquête ACSF, permet de souligner d'autres logiques sous-jacentes à la persistance d'expositions individuelles au risque de transmission sexuelle du VIH ; et d'illustrer le fait que des stratégies de non changement, ou de changement «intermédiaire», ne donnant aucune garantie réelle de protection face au risque VIH, puissent néanmoins s'avérer comme les plus cohérentes avec les rationalités effectives des individus. En conclusion, l'article suggère quelques pistes pour des analyses moins mécanistes des comportements qui seraient susceptibles de mieux tenir compte du contexte spécifique et de la dynamique temporelle dans laquelle s'inscrivent les expositions au risque d'infection par le VIH ; ces analyses pourraient utilement s'appuyer sur les développements théoriques les plus récents tant de la recherche micro-économique que la recherche psychosociologique.Moatti (Jean-Paul), Beltzer (Nathalie), Dab (William). - Los modèles de análisis de comportamientos a riesgo concernientes a la infección del virus del sida: una concepción limitada de la racionalidad La investigación en ciencias sociales sobre la prevención del virus del sida ha esta- blecido vínculos entre los conocimientos y opiniones sobre la enfermedad por un lado, y las actitudes individuales y colectivas hacia las personas infectadas y el riesgo de transmisión por otro; también han puesto en evidencia la ambigiiedad de estos vínculos en relación con los comportamientos declarados. Como ya ha sucedido en otras esferas de la prevención sanitaria, las investigaciones han confirmado que una mejor información sobre los riesgos no es una condición suficiente para la modificación de los comportamientos individuales ten- dientes a disminuirlos. En el análisis de los déterminantes de la exposición al riesgo de transmisión sexual del virus, la literatura internacional ha estado dominada por el recurso a modelos psico-sociológicos preexistentes (Health Belief Model o "modelo de creencia en la salud", teoria del aprendizaje sexual, etc); estos modelos han servido de referencia para la intervención "comunitaria" de prevención llevada a cabo con cierto éxito en el seno de las comunidades homosexual y bisexual de San Francisco. El articulo hace una critica de los limites de estos modelos a la hora de explicar y predecir los comportamientos ante el riesgo de transmisión del virus, y prétende demostrar que están implícitamente basados en una concepción reductora de la racionalidad individual, la cual se limitaría a una exposición nula al riesgo y a la búsqueda de la seguridad ab- soluta. Una confrontación de estos modelos psico-sociológicos con la teoria de la utilidad esperada, modelización clásica del comportamiento de los agentes hacia el riesgo y la incer- tidumbre en microeconomia, y una aplicación empírica de esta teoria a los datos de la en- cuesta ACSF, permiten destacar otra logica subyacente a la persistencia de exposiciones individuales al riesgo de transmisión sexual del virus. Este procedimiento también permite ilustrar el hecho de que las estrategias que no implican cambio de actitud, о que implican un cambio "intermedio", sin garantía real de protección frente al riesgo de infección, son las más cohérentes con las racionalidades efectivas de los individuos. En conclusion, el articulo sugiere algunas pistas para análisis menos mecanicistas de los comportamientos, que serian susceptibles de tener más en cuenta el contexto especifico y la dinámica temporal en la cual se inscriben las exposiciones a riesgo de infección del virus. Estos análisis podrían apoyarse en los avances teóricos más recientes tanto de la investigación microeconómica como de la investigación psico-sociológica.Moatti Jean-Paul, Beltzer Nathalie, Dab William. Les modèles d'analyse des comportements à risque face à l'infection à VIH : une conception trop étroite de la rationalité. In: Population, 48ᵉ année, n°5, 1993. Sexualité et sciences sociales : les apports d'une enquête, sous la direction de Michel Bozon et Henri Leridon. pp. 1505-1534

ATM and CDK2 control chromatin remodeler CSB to inhibit RIF1 in DSB repair pathway choice

Cockayne syndrome group B protein (CSB) is a multifunctional chromatin remodeler involved in double-strand break repair. Here the authors investigate the molecular post-translational signals regulating CSB activity

H4K20me2 distinguishes pre-replicative from post-replicative chromatin to appropriately direct DNA repair pathway choice by 53BP1-RIF1-MAD2L2

The main pathways for the repair of DNA double strand breaks (DSBs) are non-homologous end-joining (NHEJ) and homologous recombination directed repair (HDR). These operate mutually exclusive and are activated by 53BP1 and BRCA1, respectively. As HDR can only succeed in the presence of an intact copy of replicated DNA, cells employ several mechanisms to inactivate HDR in the G1 phase of cell cycle. As cells enter S-phase, these inhibitory mechanisms are released and HDR becomes active. However, during DNA replication, NHEJ and HDR pathways are both functional and non-replicated and replicated DNA regions co-exist, with the risk of aberrant HDR activity at DSBs in non-replicated DNA. It has become clear that DNA repair pathway choice depends on inhibition of DNA end-resection by 53BP1 and its downstream factors RIF1 and MAD2L2. However, it is unknown how MAD2L2 accumulates at DSBs to participate in DNA repair pathway control and how the NHEJ and HDR repair pathways are appropriately activated at DSBs with respect to the replication status of the DNA, such that NHEJ acts at DSBs in pre-replicative DNA and HDR acts on DSBs in post-replicative DNA. Here we show that MAD2L2 is recruited to DSBs in H4K20 dimethylated chromatin by forming a protein complex with 53BP1 and RIF1 and that MAD2L2, similar to 53BP1 and RIF1, suppresses DSB accumulation of BRCA1. Furthermore, we show that the replication status of the DNA locally ensures the engagement of the correct DNA repair pathway, through epigenetics. In non-replicated DNA, saturating levels of the 53BP1 binding site, di-methylated lysine 20 of histone 4 (H4K20me2), lead to robust 53BP1-RIF1-MAD2L2 recruitment at DSBs, with consequent exclusion of BRCA1. Conversely, replication-associated 2-fold dilution of H4K20me2 promotes the release of the 53BP1-RIF1-MAD2L2 complex and favours the access of BRCA1. Thus, the differential H4K20 methylation status between pre-replicative and post-replicative DNA represents an intrinsic mechanism that locally ensures appropriate recruitment of the 53BP1-RIF1-MAD2L2 complex at DNA DSBs, to engage the correct DNA repair pathway

Logement, les priorités pour 2027. Rapport du Groupe logement Terra Nova

International audienceLe logement est un poste de dépense important pour les ménages, surtout les plus jeunes qui cherchent à s’installer. Le choix du logement pèse sur le pouvoir d’achat et les difficultés d’accès accentuent les inégalités. L’accès au logement doit donc rester une priorité des politiques publiques pour les années qui viennent. Au nom de son devoir de solidarité, l’État doit garder un rôle central pour répondre au besoin de logements mais aussi prendre en compte de nouveaux enjeux comme la lutte contre les émissions de GES. Pour autant, une approche centralisée, centrée sur la construction et territorialement uniforme n’apparaît plus pertinente aujourd’hui. Les principaux défis actuels appellent en effet des différenciations territoriales ainsi que l’engagement de l’ensemble des acteurs concernés par ce secteur sur le terrain, des collectivités locales aux organismes HLM. Les collectivités locales disposent depuis plusieurs années de compétences importantes pour le logement. Qu’attendre encore de l’État, dans ce nouveau contexte de coresponsabilité ? L’État doit garder un rôle de garant du cadre législatif et réglementaire et assurer une péréquation entre les collectivités au nom de la solidarité nationale. Il encourage en parallèle de nouvelles formes de contractualisation reposant sur l’initiative des acteurs locaux, par exemple avec la formulation de « projets de territoire ». Celle-ci doit néanmoins se développer dans un cadre indiquant clairement une orientation générale d’une politique qui mobilise des financements importants. Nous défendons ici la perspective d’une politique publique du logement moins centralisée mais gardant une forte ambitio

UBE2D3 facilitates NHEJ by orchestrating ATM signalling through multi-level control of RNF168

Abstract Maintenance of genome integrity requires tight control of DNA damage response (DDR) signalling and repair, with phosphorylation and ubiquitination representing key elements. How these events are coordinated to achieve productive DNA repair remains elusive. Here we identify the ubiquitin-conjugating enzyme UBE2D3 as a regulator of ATM kinase-induced DDR that promotes non-homologous end-joining (NHEJ) at telomeres. UBE2D3 contributes to DDR-induced chromatin ubiquitination and recruitment of the NHEJ-promoting factor 53BP1, both mediated by RNF168 upon ATM activation. Additionally, UBE2D3 promotes NHEJ by limiting RNF168 accumulation and facilitating ATM-mediated phosphorylation of KAP1-S824. Mechanistically, defective KAP1-S824 phosphorylation and telomeric NHEJ upon UBE2D3-deficiency are linked to RNF168 hyperaccumulation and aberrant PP2A phosphatase activity. Together, our results identify UBE2D3 as a multi-level regulator of NHEJ that orchestrates ATM and RNF168 activities. Moreover, they reveal a negative regulatory circuit in the DDR that is constrained by UBE2D3 and consists of RNF168- and phosphatase-mediated restriction of KAP1 phosphorylation

A genetic map of the response to DNA damage in human cells

Versión postprint próximamente disponible en: http://hdl.handle.net/10261/228202The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 28 CRISPR/Cas9 screens against 25 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 840 genes whose loss causes either sensitivity or resistance to DNA damaging agents. Mining this dataset, we uncovered that ERCC6L2, which is mutated in a bone-marrow failure syndrome, codes for a canonical non-homologous end-joining pathway factor; that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.AAQ, GSM and AMH are recipients of long-term EMBO fellowships (ALTF 910-2017, 795-2017 under a CC-BY-NC-ND 4.0 International license. not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available bioRxiv preprint doi: https://doi.org/10.1101/845446; this version posted November 18, 2019. The copyright holder for this preprint (which was 27 and 124-2019, respectively), NH was supported by a Human Frontier Science Program long-term Fellowship, SER is supported by a fellowship from AIRC and SA was a Banting post-doctoral fellow. ASB was supported by a PhD Studentship from AEFAT (Asociación Española Familia Ataxia Telangiectasia) and an EMBO short-term fellowship for a visit to the DD laboratory. The ICRF187 screen in FCL laboratory was funded by grants from the Spanish Government (SAF2017- 89619-R, European Regional Development Fund) and the European Research Council (ERC-CoG2014-647359). Work in the RSW laboratory was supported in part by the US National Institute of Health Intramural Program, US National Institute of Environmental Health Sciences (NIEHS, 1Z01ES102765). DD is a Canada Research Chair (Tier I) and the work was supported from grants from the CIHR (FDN143343 to DD; FRN 123518, PJT-156330 to AM) Canadian Cancer Society grant 705644 (to DD) with additional support to DD from the Krembil Foundation.N

A Genetic Map of the Response to DNA Damage in Human Cells

Versión preprint disponible en: http://hdl.handle.net/10261/228200The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity, and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 31 CRISPR-Cas9 screens against 27 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 890 genes whose loss causes either sensitivity or resistance to DNA-damaging agents. Mining this dataset, we discovered that ERCC6L2 (which is mutated in a bone-marrow failure syndrome) codes for a canonical non-homologous end-joining pathway factor, that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents, and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.A set of CRISPR screens in cells treated with different genotoxic agents illuminates the cellular response to DNA damage, identifying new factors in several repair pathways and pinpointing a novel drug mechanism-of-action