Projet de thèse en Sciences de la vie et de la santé
Sous la direction de Sarah Lambert.
Thèses en préparation à université Paris-Saclay , dans le cadre de École doctorale Structure et Dynamique des Systèmes Vivants , en partenariat avec Integrite du genome, ARN et cancer (laboratoire) et de Faculté des sciences d'Orsay (référent) depuis le 30-09-2019 .
La capacité des cellules à survivre et à proliférer dépend de la duplication fidèle et précise du génome. Les échecs dans ce processus conduisent à des mutations provoquant des troubles génomiques, avec un impact profond sur les processus physiologiques tels que le développement, l'homéostasie des tissus, les fonctions neurologiques et le vieillissement. La réplication de l'ADN est continuellement menacée par un large spectre de barrières de fourche de réplication intrinsèques inévitables (RFB) ou par des drogues exogènes endommageant l'ADN et bloquant la réplication. Les cellules activent différentes réponses aux dommages de l'ADN (DDR) pour coordonner la signalisation des fourches stressées avec leur stabilité, leur réparation et leur redémarrage afin d'éviter une réplication incomplète de l'ADN, des non-disjonction chromosomiques et un raccourcissement des télomères. Néanmoins, la régulation spatio-temporelle des mécanismes de réparation des fourches de réplication reste peu connue. Le complexe des pores nucléaires (NPC) est composé d'environ 30 nucléoporines individuelles pour former des structures macromoléculaires hautement conservées dans l'enveloppe nucléaire. Sa fonction principale est le transport nucléocytoplasmique et l'exportation d'ARN, mais plusieurs nucléoporines ont été impliquées dans la réparation de l'ADN. Les lésions de l'ADN difficiles à réparer, y compris les cassures double brin (DSB) irréparables, les télomères érodés et les fourches de réplication effondrées sont connues pour relocaliser aux pores nucléaires afin de faciliter des voies de réparation alternatives. se déplacer vers le NPC au cours duquel d'autres voies de réparation ont lieu3. L'équipe a identifié que des fourches de réplication bloquées par des obstacles spécifiques se délocalisent vers les pores nucléaires afin de favoriser leur prise en charge, notamment leur redémarrage et leur stabilité (données non publiées). Quels sont les mécanismes responsables de cette délocalisation et comment les pores nucléaires contrôlent la réparation des fourches sont des questions en suspens. Pour combler cette lacune, l'objectif général est de développer une approche interdisciplinaire combinant des essais génomiques novateurs, à des techniques d'imagerie sur cellules vivantes. Le projet portera sur : 1) la cartographie à l'échelle du génome des sites de stress de réplication s'associant aux pores nucléaires; 2) les mécanismes qui sont activés aux pores nucléaires afin de réparer les fourches de réplication.
Contribution of the Nuclear Pore Complex to the resolution of replication stress
Outline of the project Flaws in the DNA replication process, known as replication stress, result in inaccurate chromosome duplication and subsequent mitotic abnormalities. Replication stress has emerged as a major source of genome instability contributing to genomic disorders, neurological diseases, aging and cancer. The causes of replication stress are many and varied but ultimately result in stressed replication forks that are fragile DNA structures prone to chromosomal rearrangements. The main research line of the team is to investigate the spatial and temporal organization of molecular circuits that prevent stressed forks to be converted into pathological DNA structures and the inheritance of DNA lesions and epi-genetic changes to the progeny. A combined genetic, biochemical and molecular approach allow us to explore how recombination, repair and chromatin-based processes resolve replication stress within the sub-nuclear architecture of the genome. The main objective of this PhD proposal is to investigate how the nuclear architecture contributes to the regulation of the processing of stressed replication forks to maintain their stability and ensure the resumption of DNA synthesis. Scientific context The ability of cells to survive and proliferate depends on the faithful and accurate duplication of the genome. Failures in this process lead to mutations causing genomic disorders, with profound impact on physiological processes such as development, tissue homeostasis, neurological functions and aging. DNA replication is continuously threatened by a broad spectrum of unavoidable intrinsic replication fork barriers (RFBs) or by exogenous DNA damaging and replication-blocking drugs. Cells activate distinct DNA Damage Responses (DDR) to coordinate the signalling of stressed forks with their stability, repair and restart to avoid incomplete DNA replication, chromosome non-disjunction and telomere shortening. Stressed forks are repaired by mechanisms with variable capacities to preserve genome stability. Although, dedicated DNA repair pathways are spatially localized in the nucleus, the temporal and spatial regulation of distinct fork repair pathways remain largely elusive. The nuclear pore complex (NPC) are composed of 30 individual nucleoporins to form highly conserved macromolecular structures in the nuclear envelop. Its core function is the nucleocytoplasmic transport and RNA export, but several individual nucleoporins have been involved in DNA repair. Hard to repair DNA lesions, including irreparable double strand breaks (DSBs), eroded telomeres and collapsed replication forks are known to relocate to NPC at which alternative repair pathways take place. The team has identified that replication forks stalled by specific RFB relocate to NPC to favor the stability and the resumption of DNA synthesis (unpublished data). However, it remains far from clear how stressed forks are detected to relocate to NPC and how NPC contributes to the dynamic resolution of stressed forks. To fill this gap, the overall objective is to develop a cross disciplinary approach that combines novel and cutting edge functional genomics assays, and live cell imaging. The proposal will address: 1) the genome wide-mapping of replication stress sites that relocate to NPC; 2) the mechanisms that are activated at NPC to preserve fork integrity. Experimental system model The team has developed conditional RFB to block a replisome in a polar manner. Induction of the RFB results in dysfunctional forks. Forks stalled at the RFB are processed and restarted by a Recombination-Dependent Replication (RDR) pathway that the team has previously described at an unprecedented resolution[5,6]. Indeed, the team has developed unique physical and genetic assays to allow the analysis of the different steps of RDR, from factors recruitment (by ChIP and cell imaging) to the analysis of fork structures (by bi-dimensional gel electrophoresis, 2DGE), combined with a quantitative measure of replication-restart efficiency. Furthermore, we have developed fluorescently tagged RFBs (LacO-marked RFB) to track in vivo the fate of a single dysfunctional fork during cell cycle progression and within nucleus compartmentalization, in individual cell. Those tools will be employed to investigate the of NPC components in the processing of stressed replication forks. Preliminary data The team has identified that the active RFB transiently relocates and associates with NPC during S-phase. SUMO (a type of post-transcriptional modification that regulates the activity of several DNA repair factors) and STUBL (Sumo-targeted Ubiquitin Ligase, Human RFN4) pathways are necessary RFB relocation and efficient RDR. Moreover, 2 individual nucleoporins were identified as contributing to RDR and the processing of replication forks. Altogether, the data indicate that NPC is an integral part of the RDR pathway. This PhD proposal address: 1) the genome wide-mapping of replication stress sites that relocate to NPC; 2) the mechanisms that are activated at NPC to preserve fork integrity and replication competence. Objectives 1) Mapping of replication stress sites that relocate to NPC. The objective is to implement a DAM-ID approach to NPC components, a proximity methylation-based method to mark genomic binding site of protein of interested by adenine methylation. A NPC component (Npp106) will be fused to the Dam domain. Adenine methylation will be detected by PCR-based technics and/or by expressing a GFP tracer. Previous DAM-ID approaches have proven to be successful in fission yeast. This DAM-ID approach will be first combined with the site specific RFB to validate the system and to allow the detection of interaction between stressed forks and NPC in single cell and at a single fork-resolution level. A previous report has shown that collapsed forks relocate to NPC in budding yeast. To extent our observations, we will map at the genome wide level the sites that undergo adenine methylation by Npp106-Dam in response to various replication stress. These experiments will be done in cells synchronized in S-phase and treated with drugs that induce stalled forks (HU), damaged forks (CPT and MMS). synchronized S-phase cells The genomic features of the identified genomic sites will be determined (replication origins location, transcriptional and chromatin landscape, ). The identified genomic loci will be validated by cell-imaging based approach in single cell. Finally, based on the data obtained with the site-specific RFB, we will define which factors are necessary to relocate stressed forks to NPC (SUMO, STUBL, DNA repair pathways) and what are the consequences of the lack of relocation on the maintenance of genome stability at stressed forks. 2) Role of the NPC in the processing of stressed forks. The team has identified individual nucleoporins that are necessary to promote RDR. Moreover, the team has identified several genetics conditions in which the defective relocation of the LacO-marked RFB to NPC correlates with an uncontrolled resection of nascent strands by the nuclease Exo1, a detrimental process for genome stability and chromosome disjunction, as previously reported by the team[5, 6]. These data put forward a working hypothesis in which NPC build-up an environment that allows 1) the resumption of DNA synthesis at stressed forks and 2) that protect stressed forks from uncontrolled resection by Exo1. We will tether the LacO-marked RFB to NPC and address the functional consequences of this tethering on RDR and fork resection. We will ask if such tethering can prevent uncontrolled fork resection and associated marks of genome instability in the identified genetics backgrounds. Desumoylation events and protein degradation take place at the NPC or the nuclear periphery. We will test if such events are required to promote RDR and to negatively regulate the Exo1-mediated fork resection in appropriate genetics backgrounds. Perspective Replication stress-induced chromosomal instability is a driving force of cancer development. Deciphering the temporal and spatial regulation of molecular circuits necessary to solve replication stress and evaluate their impact on genome instability is crucial to the understanding of cancer etiology. Stressed forks are targeted by several nucleases activities through mechanisms that remain largely under appreciated. Unprotected forks, in which nascent strands are extensively resected, are fragile DNA structures prone to chromosomal breakage and rearrangements. In this proposal, we will investigate how the nuclear architecture and in particular NPCs components are involved in the maintenance of genome stability at stressed forks. References 1. Magdalou I et al. The causes of replication stress and their consequences on genome stability and cell fate. Semin Cell Dev Biol. 2014 Jun;30:154-64. 2. Freudenreich CH, Su XA. Relocalization of DNA lesions to the nuclear pore complex. FEMS Yeast Res. 2016 Dec 1;16(8). 3. Géli V, Lisby M. Recombinational DNA repair is regulated by compartmentalization of DNA lesions at the nuclear pore complex. Bioessays. 2015 Dec;37(12):1287-92. 4. Lambert et al. Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange. Mol Cell. 2010 Aug 13;39(3):346-59. 5. Ait Saada A, Teixeira-Silva A, Iraqui I, Costes A, Hardy J, Paoletti G, Fréon K, Lambert SAE. Unprotected Replication Forks Are Converted into Mitotic Sister Chromatid Bridges. Mol Cell. 2017; 4;66(3):398-410 6. Teixeira-Silva A, Ait Saada A, Hardy J, Iraqui I, Nocente MC, Fréon K, Lambert SAE. The end-joining factor Ku acts in the end-resection of double strand break-free arrested replication forks. Nat Commun. 2017 Dec 7;8(1):1982. 7. Vogel MJ, Peric-Hupkes D, van Steensel B. Detection of in vivo protein-DNA interactions using DamID in mammalian cells. Nat Protoc. 2007;2(6):1467-78. 8. Steglich B, Filion GJ, van Steensel B, Ekwall K. The inner nuclear membrane proteins Man1 and Ima1 link to two different types of chromatin at the nuclear periphery in S. pombe. Nucleus. 2012 Jan-Feb;3(1):77-87. 9. Nagai S, Dubrana K, Tsai-Pflugfelder M, Davidson MB, Roberts TM, Brown GW, Varela E, Hediger F, Gasser SM, Krogan NJ. Functional targeting of DNA damage to a nuclear pore-associated SUMO-dependent ubiquitin ligase. Science. 2008 Oct 24;322(5901):597-602. 10. Ray Chaudhuri A, Callen E, Ding X, Gogola E, Duarte AA, Lee JE, Wong N, Lafarga V, Calvo JA, Panzarino NJ, John S, Day A, Crespo AV, Shen B, Starnes LM, de Ruiter JR, Daniel JA, Konstantinopoulos PA, Cortez D, Cantor SB, Fernandez-Capetillo O, Ge K, Jonkers J, Rottenberg S, Sharan SK, Nussenzweig A. Replication fork stability confers chemoresistance in BRCA-deficient cells. Nature. 2016 Jul 21;535(7612):382-7.