L’organisation non-aléatoire du noyau des cellules eucaryotes et la compaction de l’ADN en chromatine plus ou dense peuvent influencer de nombreuses fonctions liées au métabolisme de l’ADN, y compris la stabilité du génome. Les cassures double-brin sont les dommages à l’ADN les plus néfastes pour la cellule. Pour préserver l’intégrité de leur génome, les cellules eucaryotes ont développé des mécanismes de réparation des cassures double-brin qui sont conservés de la levure à l’homme. Parmi ceux-ci, la recombinaison homologue utilise une séquence homologue intacte présente ailleurs dans le génome et peut se diviser en deux sous voies de réparation. La conversion génique transfère l’information génétique d’une molécule à son homologue, tandis que le Break Induced Replication (BIR) établit une fourche de réplication qui peut procéder jusqu’à la fin du chromosome.Mon travail de thèse s’est attaché à caractériser la contribution du statut chromatinien et de l’organisation tridimensionnelle du génome à la réparation des cassures double-brin. L’organisation du noyau de la levure S. cerevisiae ainsi que la propagation de l’hétérochromatine au niveau des régions subtélomériques peuvent être modifiées via la surexpression des protéines Sir3 et sir3A2Q. Nous avons montré que le groupement des télomères accroit la conversion génique entre deux séquences subtélomériques, soulignant le rôle clé de la proximité spatiale et de la recherche d’homologie. Nous avons également constaté que la présence d’hétérochromatine au niveau du site de cassure limite la résection, ce qui permet une disparition plus lente des extrémités, qui resteraient disponibles plus longtemps pour réaliser la recherche d’homologie et achever la réparation. Enfin, nous avons observé que la présence d’hétérochromatine au site donneur diminue l’efficacité de recombinaison et qu’elle doit moduler une étape commune aux deux voies de réparation, à savoir l’invasion de brin. Ces travaux nous ont permis de décrire de nouvelles voies de régulation de la réparation de l’ADN. / The non-random organization of the eukaryotic cell nucleus and the folding of genome in chromatin more or less condensed can influence many functions related to DNA metabolism, including genome stability. Double-strand breaks (DSBs) are the most deleterious DNA damages for the cells. To preserve genome integrity, eukaryotic cells thus developed DSB repair mechanisms conserved from yeast to human, among which homologous recombination (HR) that uses an intact homologous sequence to repair a broken chromosome. HR can be separated in two sub-pathways: Gene Conversion (GC) transfers genetic information from one molecule to its homologous and Break Induced Replication (BIR) establishes a replication fork than can proceed until the chromosome end.My doctorate work was focused on the contribution of the chromatin context and 3D genome organization on DSB repair. In S. cerevisiae, nuclear organization and heterochromatin spreading at subtelomeres can be modified through the overexpression of the Sir3 or sir3A2Q mutant proteins. We demonstrated that reducing the physical distance between homologous sequences increased GC rates, reinforcing the notion that homology search is a limiting step for recombination. We also showed that heterochromatinization of DSB site fine-tunes DSB resection, limiting the loss of the DSB ends required to perform homology search and complete HR. Finally, we noticed that the presence of heterochromatin at the donor locus decreased both GC and BIR efficiencies, probably by affecting strand invasion. This work highlights new regulatory pathways of DNA repair.

Em Trypanosoma cruzi os sítios de replicação estão localizados na periferia nuclear. A fim de entender a dinâmica das moléculas envolvidas na replicação, Orc/Cdc6 foi usado como marcador da maquinaria de pré-replicação e PCNA como marcador da maquinaria de replicação durante o ciclo celular da forma epimastigota. Ambas as moléculas apresentaram dois padrões de localização nuclear: padrão disperso e periférico. Em ensaio de dupla marcação três combinações dos padrões de Orc/Cdc6 e TcPCNA foram encontrados durante G1/S: Orc/Cdc6 periférico e PCNA disperso, ambos dispersos e ambos periféricos. Por meio destes resultados pudemos concluir que durante G1 ambas as moléculas se encontram dispersas. Ao final desta fase, Orc/Cdc6 migra para periferia nuclear enquanto PCNA permanece disperso, migrando para a periferia nuclear quando a célula entra em S, quando a replicação do DNA irá ocorrer. Assim, a replicação na periferia nuclear não se deve à localização prévia das moléculas de replicação nesta região, mas sim à migração destas moléculas para os sítios apropriados. / In Trypanosoma cruzi the replication sites are located at nuclear periphery. In order to analyse the dynamics of molecules involved in replication, Orc/Cdc6 was used as a marker of pre-replication machinery and PCNA as a marker of replication machinery during the cell cycle of epimastigote form. Both molecules presented two nuclear patterns: dispersed pattern and peripheral pattern. Double-labeling assay showed three different patterns of Orc/Cdc6 and PCNA in the nucleus: Orc/Cdc6 at nuclear periphery and PCNA dispersed, both dispersed and both at nuclear periphery. This data allowed us to conclude that during early G1 phase both molecules are dispersed in the nucleus and during late G1 Orc/Cdc6 goes to nuclear periphery while TcPCNA remains dispersed, moving to nuclear periphery, where DNA replication will take place, when S phase starts. Thus, the replication of DNA at nuclear periphery is not due to localization of replications factors at nuclear periphery; instead it depends on the movement of these factors to the appropriated sites.

Le développement embryonnaire précoce des mammifères femelles s’accompagne de l’inactivation transcriptionnelle d’un de leurs deux chromosomes X. Cet évènement est initié suite à l’expression mono-allélique de l’ARN non codant Xist, qui est contrôlée par de nombreux éléments cis-régulateurs présents dans le centre d’inactivation du chromosome X (Xic) – tel son anti-sens répresseur Tsix. Mon travail de thèse a consisté à développer des approches permettant d’appréhender le paysage structural dans lequel s’exerce cette régulation. La caractérisation de l’architecture tridimensionnelle du Xic, par des techniques basées sur la capture de conformation chromosomique (3C) et l’hybridation in situ en fluorescence (FISH), m’a permis de mettre en évidence que les promoteurs respectifs de Xist et Tsix sont engagés dans des interactions physiques intimes avec des loci distaux, localisés au sein du Xic, et de montrer qu’au moins certaines de ces régions exercent un effets régulateurs à longue-distance. Les éléments du Xic contactés par les régions promotrices de Xist et de Tsix sont en outre fondamentalement différents, chacune engageant des associations chromosomiques sur plusieurs centaines de kilobases dans leur direction 5’ respective.Ce travail a également permis de révéler des propriétés insoupçonnées de l’architecture chromosomiques. En effet, le Xic apparaît scindé en plusieurs sous-régions, couvrant chacune entre 200kb et 1Mb, à l’intérieur desquelles les interactions chromosomiques sont préférentiellement établies. L’existence de ces domaines d’interaction s’intègre avec d’autres propriétés structurales du génome, tels la composition de la chromatine sous-jacente et l’association à la lamine nucléaire, mais n’apparaît pas en dépendre directement. En étudiant la dynamique de la conformation chromosomique du Xic au cours de la différenciation cellulaire, j’ai pu constater la robustesse de cette organisation, sauf sur le chromosome X inactif, qui se distingue par la perte des contacts chromosomiques préférentiels détectables sur son homologue actif.Enfin, j’ai pu mettre en évidence que la variabilité du repliement général du chromosome X amène à un instant donné chaque allèle de Tsix à contacter physiquement des jeux de séquences distales différents, suggérant que l’environnement structural instantané de chacun de ces allèles à l’orée de l’activation mono-allélique de Xist est différent. Ce travail, combinant des approches à l’échelle de la population cellulaire d’une part et de la fibre de chromatine unique d’autre part, apporte une nouvelle vision du paysage structural et régulateur dans lequel s’inscrit le contrôle de l’activité transcriptionnelle de Xist, et fourni de nouvelles perspectives concernant les principes fondamentaux de l’organisation topologique des chromosomes chez les mammifères. / Early development of female mammals is accompanied by transcriptional inactivation of one of their two X chromosomes. This event is initiated following mono-allelic expression of the Xist non-coding RNA – what is achieved by the interplay of numerous cis-regulatory elements present within the X inactivation center (Xic), such as its repressive antisense Tsix. Our work aimed at throwing light on the structural landscape that underlies such long-range regulation. Characterization of the three-dimensional architecture of the Xic, by the means of Chromosome Conformation Capture (3C)-based techniques and in situ fluorescence hybridization (FISH), revealed that the respective promoters of Xist and Tsix contact many distal genomic elements within the Xic, and that at least one of such interacting region exerts long-range cis-transcriptional control. Noticeably, Xist and Tsix promoters associate with different sets of elements in their respective 5’ direction that are spread out over several hundreds of kilobases These experiments also revealed unforeseen properties of chromatin architecture. Indeed, the Xic appears to be partitioned in several sub-regions, each spanning between 200kb and 1Mb, inside which chromosomal interactions are preferentially established. The existence of these interaction domains integrates with other structural features of the genome, such as underlying chromatin composition and association with the nuclear lamina, but does not seem to directly depend on them. By analyzing chromosome conformation of the Xic during cell differentiation we document the robustness of this organizational principle, with the noticeable exception of the inactive X chromosome that assumes a folding pattern that is more random than its active homolog. Finally we also bring evidence that variability in the folding pattern of the two X chromosomes in the same cell brings each Tsix allele in association with different sets of chromosomal partners at a given moment, suggesting that the instantaneous structural environment of each allele at the onset of mono-allelic Xist up-regulation is different.By combining approaches at the scale of cell populations on the one hand, and at the single chromatin fiber level on the other, this study provides a first vision of the structural landscape in which Xist regulation takes place, and brings new insights concerning fundamental properties of chromosome organization in mammals.

It is becoming increasingly clear that long-range control of gene expression is mediated through direct physical interactions between genes and regulatory elements, either intra- or interchromosomally. In addition to transcriptional initiation, formation of active chromatin hubs seem to be crucial for increased transcriptional efficiency as well as insulation from neighbouring heterochromatic environment. Regulatory factors apparently have an important role in organization of such functional modules in a development and differentiated- dependent fashion. The relevance of trans-acting factors in the ‘choice’ process of X-Chromosome Inactivation (XCI) was highlighted by our observations where CTCF was shown to occupy a homologous position on the active mouse and human Xist/XIST promoters and its binding affinity was altered in familial cases of opposite skewed X-inactivation patterns. The paradigm of genomic imprinting, i.e. the Igf2-H19 locus, manifests its imprinted states through the H19 Imprinting Control Region (ICR). The repression of the maternal Igf2 allele depends on the insulator properties of the H19 ICR when this interacts with CTCF. The studies here detected a novel kind of CTCF-dependent tightly closed pocket- like higher order structure exclusively on maternal allele which was found to be essential for imprinted Igf2 expression as well as maintenance of precise epigenetic marks at various Differentially Methylated Regions (DMRs) across this locus. Despite the highly condensed state of the mitotic chromosome, the insulator protein CTCF was found to constitutively occupy its known target sites. Furthermore, pivotal CTCF-dependent long-range regulatory loops within Igf2-H19 locus were found to survive mitotic compaction and such mechanisms might serve as a novel kind of epigenetic memory to minimize transcriptional chaos and to reset proper expression domains in the daughter cells as soon as cells exit mitosis. Our observations also suggest that the epigenetic reprogramming of H19 ICR during spermatogenesis is initiated by a CTCF-dependent recruitment of chromatin remodeling factor Lsh to the H19 ICR followed by completion of the imprint acquisition process by a replacement of CTCF with its closely related paralogue termed BORIS. Overall, this thesis unravels the novel roles for CTCF as an architectural factor in the organization of higher order chromatin conformations and transcriptional regulation.

Em Trypanosoma cruzi os sítios de replicação estão localizados na periferia nuclear. A fim de entender a dinâmica das moléculas envolvidas na replicação, Orc/Cdc6 foi usado como marcador da maquinaria de pré-replicação e PCNA como marcador da maquinaria de replicação durante o ciclo celular da forma epimastigota. Ambas as moléculas apresentaram dois padrões de localização nuclear: padrão disperso e periférico. Em ensaio de dupla marcação três combinações dos padrões de Orc/Cdc6 e TcPCNA foram encontrados durante G1/S: Orc/Cdc6 periférico e PCNA disperso, ambos dispersos e ambos periféricos. Por meio destes resultados pudemos concluir que durante G1 ambas as moléculas se encontram dispersas. Ao final desta fase, Orc/Cdc6 migra para periferia nuclear enquanto PCNA permanece disperso, migrando para a periferia nuclear quando a célula entra em S, quando a replicação do DNA irá ocorrer. Assim, a replicação na periferia nuclear não se deve à localização prévia das moléculas de replicação nesta região, mas sim à migração destas moléculas para os sítios apropriados. / In Trypanosoma cruzi the replication sites are located at nuclear periphery. In order to analyse the dynamics of molecules involved in replication, Orc/Cdc6 was used as a marker of pre-replication machinery and PCNA as a marker of replication machinery during the cell cycle of epimastigote form. Both molecules presented two nuclear patterns: dispersed pattern and peripheral pattern. Double-labeling assay showed three different patterns of Orc/Cdc6 and PCNA in the nucleus: Orc/Cdc6 at nuclear periphery and PCNA dispersed, both dispersed and both at nuclear periphery. This data allowed us to conclude that during early G1 phase both molecules are dispersed in the nucleus and during late G1 Orc/Cdc6 goes to nuclear periphery while TcPCNA remains dispersed, moving to nuclear periphery, where DNA replication will take place, when S phase starts. Thus, the replication of DNA at nuclear periphery is not due to localization of replications factors at nuclear periphery; instead it depends on the movement of these factors to the appropriated sites.

Gene transfer techniques such as retroviral transduction have many applications such as cell marking, cell reprogramming, and therapeutics. Transgene expression, however, is often variable and maintaining long-term expression is problematic in progenitor cell types. To better control transgene expression, research has focused on the optimized use of cis-regulatory elements, such as promoters, enhancers and insulators. In addition to controlling gene expression, these regulatory elements modulate the nuclear organization of the transgene. The integration site also exerts significant effects on steady state and temporal transgene expression via the neighbouring chromatin environment. The first part of this thesis describes the co-operation of modified β-globin intronic elements in providing high-level expression and favorable nuclear localization. I demonstrate that these elements are compatible with efficient lentivirus transduction for globin gene therapy purposes. In the second chapter, I examine high-expressing EGFP retroviral transgenes and show that such steady state expression may exhibit rapid transcriptional fluctuations, which is modulated by different transcriptional dynamics at different integration sites. Finally, in the last chapter, I evaluate the use of a 3’D4Z4 insulator element in maintaining long-term EGFP transgene expression in ES cells, and discover integration-site specific temporal dynamics in retroviral vector expression. Overall, my results demonstrate that using multiple regulatory elements and insulating these elements from different types of genomic loci optimize transgene expression and dynamics in progenitor cells.

Gene transfer techniques such as retroviral transduction have many applications such as cell marking, cell reprogramming, and therapeutics. Transgene expression, however, is often variable and maintaining long-term expression is problematic in progenitor cell types. To better control transgene expression, research has focused on the optimized use of cis-regulatory elements, such as promoters, enhancers and insulators. In addition to controlling gene expression, these regulatory elements modulate the nuclear organization of the transgene. The integration site also exerts significant effects on steady state and temporal transgene expression via the neighbouring chromatin environment. The first part of this thesis describes the co-operation of modified β-globin intronic elements in providing high-level expression and favorable nuclear localization. I demonstrate that these elements are compatible with efficient lentivirus transduction for globin gene therapy purposes. In the second chapter, I examine high-expressing EGFP retroviral transgenes and show that such steady state expression may exhibit rapid transcriptional fluctuations, which is modulated by different transcriptional dynamics at different integration sites. Finally, in the last chapter, I evaluate the use of a 3’D4Z4 insulator element in maintaining long-term EGFP transgene expression in ES cells, and discover integration-site specific temporal dynamics in retroviral vector expression. Overall, my results demonstrate that using multiple regulatory elements and insulating these elements from different types of genomic loci optimize transgene expression and dynamics in progenitor cells.

L'ADN chromosomique des cellules eucaryotes est fortement condensé au sein d'un complexe nucléoprotéïque, la chromatine. Aussi bien l'organisation spatiale que la composition biochimique (état “épigénomique”) de la chromatine jouent un rôle fondamental dans la régulation des gènes. Grâce aux récents développements des techniques de séquençage à haut-débit, il est possible de déterminer l'état épigénomique local de la chromatine ainsi que la probabilité de contact entre deux sites génomiques (technique dite de “Hi-C”). Ces deux techniques ont permis de mettre en évidence l’existence de domaines d’interaction dont les positions corrèlent fortement avec la segmentation épigénomique de la chromatine. Cependant, les mécanismes responsables de ce couplage sont encore mal compris. L’objectif de cette thèse est de bâtir des modèles physiques permettant de valider l’hypothèse que l’épigénome est un acteur majeur dans le repliement 3D de la chromatine. Pour cela, nous avons tout d’abord développé “IC-Finder”, un algorithme permettant de segmenter les cartes Hi-C en domaines d’interaction. Nous avons alors pu quantifier précisément l’association entre épigénome et organisation de la chromatine. Les corrélations trouvées justifient l’idée de modéliser la chromatine par un copolymère par bloc dont les monomères ont chacun un état épigénomique. Dans ce cadre, nous avons développé une méthode d’inférence des potentiels d'interaction entre sites génomiques à partir des cartes Hi-C expérimentales. Ce travail permettra à plus long terme de prévoir l’organisation de la chromatine sous différentes conditions, ce qui permettra d’étudier en particulier les changements de structure résultant de l’altération de l’épigénome. / DNA of eukaryotes is highly condensed in a nucleoprotein complex called chromatin. Both the spatial organization and the biochemical composition (“epigenomic” state) of the chromatin are fundamental for gene regulation. Remarkably, recent studies indicate that1D epigenomic domains tend to fold into 3D topologically associated domains (TADs) forming specialized nuclear chromatin compartments. In this thesis, we address the question of the coupling between chromatin folding and epigenome. We first built a software called IC-finder to segment HiC maps into interacting domains. We next used it to quantify correlations between the TADs and epigenomic partitions of the genome. This led us to develop a physical model of the chromatin with the working hypothesis that chromatin organization is driven by physical interactions between epigenomic loci. We modeled chromatin as a block copolymer where each block corresponds to an epigenomic domain. With this framework, we developed a method to infer interaction parameters between chromatin loci from experimental Hi-C map. An outcome of such inference process would be a powerful tool to predict chromatin organization in various conditions, allowing investigating in silico changes in TAD formations and long-range contacts when altering the epigenome.

Enfermer le porteur de l’information génétique dans le noyau a obligée la cellule a créé un système de transport complexe, qui permet l’export d’un ARNm du noyau au cytoplasme. Le mécanisme général de l’export des ARNm est encore mal connu, même si les facteurs principaux ont été découverts il y a longtemps. De récents progrès en microscopie nous ont permis d’étudier directement le comportement des ARNm durant le processus d’export. Durant ma maitrise, nous avons été capables de localiser et suivre des ARNm en temps réel pour la première fois chez Saccharomyces cerevisiae. Nous avons créé un gène rapporteur en mettant le gène GLT1 sous le contrôle du promoteur GAL1. Nous avons aussi marqué l’ARNm de GLT1 avec plusieurs boucles PP7. L’ARNm sera visible après l’attachement de plusieurs protéines PP7-GFP aux boucles. En utilisant la technique d’imagerie en cellules vivantes, nous sommes capable de visualiser et suivre chaque ARNm, depuis son relâchement du site de transcription jusqu’à l’export. Une fois relâché du site de transcription, l’ARNm diffuse librement dans le nucléoplasme, mais une fois à la périphérie nucléaire, il commence à « scanner » l’enveloppe nucléaire avant d’être exporté. Nous avons trouvé que le « scanning » dépend de la présence des Myosin Like Proteins (Mlp1p et Mlp2p), protéines qui forment le panier nucléaire, car suite à la délétion de MLP1 et MLP2, les ARNm n’étaient plus capable de « scanner ». Nous avons également trouvé que la partie C-terminale de Mlp1p était nécessaire au « scanning ». De plus, suite à la délétion du gène TOM1, gène codant pour une ubiquitine ligase, les ARNm ont un comportement similaire aux ARNm d’une souche ∆mlp1/mlp2, suggérant que le « scanning » permet à Tom1p d’ubiquitiner Yra1p, ce qui causera son relâchement de l’ARNm. Également, nous avons montré que les ARNm endogènes MDN1 et CBL2 scannent aussi la périphérie nucléaire. Ensemble, nos résultats suggèrent que le scanning est un processus par lequel passent tout les ARNm nucléaire lorsqu’ils se retrouvent à la périphérie du noyau, pour initier plusieurs étapes de réarrangements nécessaires à leurs export. De plus, nous avons examiné le rôle de Yhr127p, une protéine nouvellement identifiée qui se lie à l’ARN. Après avoir marqué cette protéine avec la GFP, nous avons montré qu’elle forme des foci dans le noyau et que ces derniers vont disparaitre suite à l’arrêt de la transcription. La délétion de YHR127 à conduit à une augmentation de la transcription de quelques gènes spécifiques, mais n’affecte pas la capacité de la cellule à exporter les ARNm. Nos résultats suggèrent que cette protéine joue un rôle dans la régulation de la transcription et/ou dans la stabilité de l’ARNm. / In eukaryotic cells, the processes of RNA and protein synthesis are spatially separated into two distinct compartments. With this division, a complex pathway of nucleocytoplasmic RNA export has evolved, which to date remains poorly understood. Recent advances in single-molecule microscopy have enabled direct studies focused on investigating the dynamics and kinetics of RNA export. In this Master thesis, we present the first real time visualization of mRNA export in the yeast Saccharomyces cerevisiae. We first generated a GLT1 reporter under the control of the inducible GAL1 promoter, in which the GLT1 mRNA was tagged with an array of PP7 repeats and detected by exogenous PP7-GFP binding protein. Using a single-molecule live cell imaging approach, we were able to visualize and track the behavior of individual mRNAs from the site of transcription to the point of export. Interestingly, we found that once released from the transcription site, single mRNAs diffuse freely in the nucleoplasm, but once they reach the nuclear periphery, they scan the periphery before being exported to the cytoplasm. This scanning behavior was dependent on Myosin Like Proteins (Mlp1p and Mlp2p), which form the basket of the Nuclear Pore Complex (NPC), as mRNAs were not retained at the periphery and were rapidly released into the nucleoplasm in mlp1p/mlp2p double mutant cells. Specifically, we found that the C-terminal part of Mlp1p was important for scanning. Furthermore, mRNAs from cells depleted of the E3 ubiquitin ligase TOM1 had a similar phenotype to mRNAs in mlp1p/mlp2p double mutant cells, suggesting a role for scanning in the Tom1p-mediated release of Yra1p from the RNA. Lastly, we confirmed that endogenous MDN1 and CBL2 mRNAs also exhibit scanning behaviour. Taken together, our results suggest that mRNAs scanning the nuclear periphery is a general behaviour for all mRNAs to initiate the mRNA export process, allowing mRNP arrangement required for export to occur at the nuclear periphery. In addition, we investigated the role of YHR127, a newly identified RNA binding protein, in RNA biogenesis. Notably, we show that GFP-tagged YHR127p formed distinct foci in the nucleus, which were lost upon transcription arrest. Deletion of YHR127 led to an increase in transcript levels of specific genes, but not to a global accumulation of mRNAs in the nucleus, suggesting a role for this protein in regulating transcription and/or mRNA stability.

No description available.