Le contrôle du cycle cellulaire, processus fondamental pour la prolifération cellulaire, est souvent altéré au cours de la tumorigenèse. Les facteurs de transcription E2Fs sont des régulateurs majeurs de l'expression de gènes impliqués dans le cycle cellulaire, la réplication de l'ADN, la mort cellulaire programmée ou encore la différenciation cellulaire. La famille des facteurs E2Fs contient des membres qui agissent comme activateurs ou répresseurs de la transcription et dont l'activité est régulée par un grand nombre de corégulateurs transcriptionnels, incluant notamment les protéines à poche pRb, p107, p130. RIP140 (Receptor Interacting Protein of 140kDa) a été identifié comme un corépresseur de nombreux récepteurs nucléaires, une autre grande famille de facteurs de transcription qui, pour certains, régulent positivement l'expression du gène RIP140.Ce travail de thèse a permis d'identifier RIP140 comme un nouveau répresseur de l'activité transcriptionnelle du facteur E2F1, dans des expériences de transfection transitoire ainsi que sur l'expression de gènes endogènes. Nous avons également montré que l'expression ectopique de RIP140 bloque la progression des cellules dans le cycle cellulaire. Dans les cancers du sein, le niveau d'expression de RIP140 présente une corrélation inverse avec celui de différents gènes cibles des facteurs E2Fs et semble discriminer les tumeurs luminales des tumeurs basales. Nous avons également démontré que le niveau d'ARNm RIP140 est régulé au cours du cycle cellulaire et que le promoteur du gène RIP140 est une cible directe des facteurs E2Fs. Cette régulation implique des sites de liaison des facteurs E2Fs et Sp1 de la région proximale du promoteur. La régulation de ce gène par E2F1 a également été observée au cours du processus de différenciation adipocytaire en utilisant un modèle murin E2F1-/-.En conclusion, ce travail a permis d'identifier RIP140 comme un nouvel acteur de la voie de signalisation par les facteurs E2Fs. / Cell cycle control, a fundamental process which controls cell proliferation, is frequently altered during tumorigenesis. The E2F transcription factors are central regulators of target gene expression involved in cell cycle regulation, DNA replication, apoptosis and differentiation. The E2F transcription factors family encompasses members which act as activators or repressors. Their activities are regulated by a large number of transcriptional coregulators, including in particular the pocket proteins pRb, p107, p130.The transcription coregulator RIP140 (Receptor Interacting Protein of 140kDa) has been identified as a partner of numerous nuclear receptors, another important transcription factor family. Some of these nuclear receptors positively regulate RIP140 gene expression.This work identified RIP140 as a new repressor of E2F1 transcriptional activity, both in transient transfection experiments and on the expression of endogenous target genes. We also showed that ectopic expression of RIP140 blocks cell cycle progression. In breast cancers, the level of RIP140 expression is inversely correlated with various target genes of E2Fs factors and seems to discriminate luminal from basal tumors. We also demonstrated that the RIP140 mRNA expression is regulated during cell cycle and that the RIP140 promoter is a direct target of E2F transcription factors. This regulation involves both E2F and Sp1 binding sites in the proximal region of the RIP140 promoter. The regulation of the RIP140 gene by E2F1 was also observed during adipocyte differentiation using an E2F1-/- mouse model.In conclusion, this study identified RIP140 as a new regulator of the E2F signalling pathway and as a novel E2F1 target gene. These results open new perspectives concerning the roles that this transcriptional coregulator might play in the control of cell proliferation and tumorigenesis.

β-Casein is a major milk protein, which is synthesized in mammary alveolar secretory epithelial cells (MECs) upon the stimulation of lactogenic hormones, mainly prolactin and glucocorticoids (HP). Previous studies revealed that the proximal promoter (-258 bp to +7 bp) of the β-casein gene is sufficient for induction of the promoter activity by HP. This proximal region contains the binding sites for the signal transducer and activator of transcription 5 (STAT5), glucocorticoid receptor (GR), and octamer transcription factors (Oct). STAT5 and GR are essential downstream mediators of prolactin and glucocorticoid signaling, respectively. This study investigated the functions of Oct-1 and Oct-2 in HP induction of β-casein gene expression. By transiently transfection experiment, we showed that individual overexpression of Oct-1 and Oct-2 further enhanced HP-induced β-casein promoter activity, respectively, while Oct-1 and Oct-2 knockdown significantly inhibited the HP-induced β-casein promoter activity, respectively. HP rapidly induced the binding of both Oct-1 and Oct-2 to the β-casein promoter, and this induction was not mediated by either increasing their expression or inducing their translocation to the nucleus. In MECs, Oct-2 was found to physically interact with Oct-1 regardless of HP treatment. However, HP induced physical interactions of Oct-1 or Oct-2 with both STAT5 and GR. Although the interaction between Oct-1 and Oct-2 did not synergistically stimulate HP-induced β-casein gene promoter activity, the synergistic effect was observed for the interactions of Oct-1 or Oct-2 with STAT5 and GR. The interactions of Oct-1 with STAT5 and GR enhanced or stabilized the binding of STAT5 and GR to the promoter. Abolishing the interaction between Oct-1 and STAT5 significantly reduced the hormonal induction of β-casein gene transcription. Thus, our study indicates that HP activate β-casein gene expression by inducing the physical interactions of Oct-1 and Oct-2 with STAT5 and GR in mouse MECs. There is a high and increasing demand for insulin because of the rapid increase in diabetes incidence worldwide. However, the current manufacturing capacities can barely meet the increasing global demand for insulin, and the cost of insulin production keeps rising. The mammary glands of dairy animals have been regarded as ideal bioreactors for mass production of therapeutically important human proteins. We tested the feasibility of producing human proinsulin in the milk of transgenic mice. In this study, four lines of transgenic mice were generated to harbor the human insulin gene driven by the goat β-casein gene promoter. The recombinant human proinsulin was detected in the milk by Western blotting and enzyme-linked immunosorbent assay. The highest expression level of human proinsulin was as high as 8.1 μg/µl in milk of transgenic mice at mid-lactation. The expression of the transgene was only detected in the mammary gland during lactation. The transgene expression profile throughout lactation resembled the milk yield curve, with higher expression level at middle lactation and lower expression level at early and late lactation. The blood glucose and insulin levels and major milk compositions of transgenic mice were not changed. The mature insulin derived from the milk proinsulin retained biological activity. Thus, our study indicates that it is practical to produce high levels of human proinsulin in the milk of dairy animals, such as dairy cattle and goat.

La famille des gènes Hox code pour des facteurs de transcription connus pour leur contribution essentielle à l’élaboration de l’architecture du corps et ce, au sein de tout le règne animal. Au cours de l’évolution chez les vertébrés, les gènes Hox ont été redéfinis pour générer toute une variété de nouveaux tissus/organes. Souvent, cette diversification s’est effectuée via des changements quant au contrôle transcriptionnel des gènes Hox. Chez les mammifères, la fonction de Hoxa13 n’est pas restreinte qu’à l’embryon même, mais s’avère également essentielle pour le développement de la vascularisation fœtale au sein du labyrinthe placentaire, suggérant ainsi que sa fonction au sein de cette structure aurait accompagné l’émergence des espèces placentaires. Au chapitre 2, nous mettons en lumière le recrutement de deux autres gènes Hoxa, soient Hoxa10 et Hoxa11, au compartiment extra-embryonnaire. Nous démontrons que l’expression de Hoxa10, Hoxa11 et Hoxa13 est requise au sein de l’allantoïde, précurseur du cordon ombilical et du système vasculaire fœtal au sein du labyrinthe placentaire. De façon intéressante, nous avons découvert que l’expression des gènes Hoxa10-13 dans l’allantoïde n’est pas restreinte qu’aux mammifères placentaires, mais est également présente chez un vertébré non-placentaire, indiquant que le recrutement des ces gènes dans l’allantoïde précède fort probablement l’émergence des espèces placentaires. Nous avons généré des réarrangements génétiques et utilisé des essais transgéniques pour étudier les mécanismes régulant l’expression des gènes Hoxa dans l’allantoïde. Nous avons identifié un fragment intergénique de 50 kb capable d’induire l’expression d’un gène rapporteur dans l’allantoïde. Cependant, nous avons trouvé que le mécanisme de régulation contrôlant l’expression du gène Hoxa au sein du compartiment extra-embryonnaire est fort complexe et repose sur plus qu’un seul élément cis-régulateur. Au chapitre 3, nous avons utilisé la cartographie génétique du destin cellulaire pour évaluer la contribution globale des cellules exprimant Hoxa13 aux différentes structures embryonnaires. Plus particulièrement, nous avons examiné plus en détail l’analyse de la cartographie du destin cellulaire de Hoxa13 dans les pattes antérieures en développement. Nous avons pu déterminer que, dans le squelette du membre, tous les éléments squelettiques de l’autopode (main), à l’exception de quelques cellules dans les éléments carpiens les plus proximaux, proviennent des cellules exprimant Hoxa13. En contraste, nous avons découvert que, au sein du compartiment musculaire, les cellules exprimant Hoxa13 et leurs descendantes (Hoxa13lin+) s’étendent à des domaines plus proximaux du membre, où ils contribuent à générer la plupart des masses musculaires de l’avant-bras et, en partie, du triceps. De façon intéressante, nous avons découvert que les cellules exprimant Hoxa13 et leurs descendantes ne sont pas distribuées uniformément parmi les différents muscles. Au sein d’une même masse musculaire, les fibres avec une contribution Hoxa13lin+ différente peuvent être identifiées et les fibres avec une contribution semblable sont souvent regroupées ensemble. Ce résultat évoque la possibilité que Hoxa13 soit impliqué dans la mise en place de caractéristiques spécifiques des groupes musculaires, ou la mise en place de connections nerf-muscle. Prises dans leur ensemble, les données ici présentées permettent de mieux comprendre le rôle de Hoxa13 au sein des compartiments embryonnaires et extra-embryonnaires. Par ailleurs, nos résultats seront d’une importance primordiale pour soutenir les futures études visant à expliquer les mécanismes transcriptionnels soutenant la régulation des gènes Hoxa dans les tissus extra-embryonnaires. / The Hox family of transcription factors is well known for its key contribution in the establishment of the body architecture in all the animal kingdom. During vertebrate evolution, Hox genes have been co-opted to pattern a variety of novel tissues/organs. Often, this diversification has been achieved by changes in Hox transcriptional control. In mammals, Hoxa13 function is not restricted to the embryo proper, but is also essential for the proper development of the fetal vasculature within the placental labyrinth, suggesting that its function in this structure accompanied the emergence of placental species. In chapter 2, we report on the recruitment of two other Hoxa genes, namely Hoxa10 and Hoxa11, in the extra embryonic compartment. We show that Hoxa10, Hoxa11 and Hoxa13 expression is required in the allantois, the precursor of the umbilical cord and fetal vasculature within the placental labyrinth. Interestingly, we found that Hoxa10-13 gene expression in the allantois is not restricted to placental mammals, but is also present in a non-placental vertebrate, indicating that the recruitment of these genes in the allantois most likely predates the emergence of placental species. We generated genetic rearrangements and used transgenic assays to investigate the regulatory mechanisms underlying Hoxa gene expression in the allantois. We identified a 50 kb intergenic fragment able to drive reporter gene expression in the allantois. However, we found that the regulatory mechanism controlling Hoxa gene expression in the extra-embryonic compartment is very complex and relies on more than one cis-regulatory element. In chapter 3, we used genetic fate mapping to assess the overall contribution of Hoxa13 expressing cells to the different embryonic structures. In particular, we focused on Hoxa13 fate-mapping analysis in the developing forelimbs. We could determine that, in the limb skeleton, all autopod (hand) skeletal elements, with the exception of a few cells in the most proximal carpal elements, originate from Hoxa13 expressing cells. In contrast, we found that, in the muscle compartment, Hoxa13 expressing cells and their descendants extend to more proximal limb domains, where they contribute to most of the muscle masses of the forearm and, in part, to the triceps. Interestingly we found that Hoxa13 expressing cells and their descendants are not identically distributed among different muscles. Within the same muscular mass, fibres with different Hoxa13lin+ contribution can be identified, and fibers with similar contribution are often clustered together. This result raises the possibility that Hoxa13 might be involved in establishing specific features of muscle groups, or in establishing nerve-muscle connectivity. Altogether, the data presented herein provide a better understanding of the role of Hoxa13 in both the embryonic and extra-embryonic compartment. Moreover, our results will be of key importance for further investigations aimed at unravelling transcriptional mechanisms underlying Hoxa gene regulation in extra embryonic tissues.

EIF4E, le facteur d’initiation de la traduction chez les eucaryotes est un oncogène puissant et qui se trouve induit dans plusieurs types de cancers, parmi lesquels les sous-types M4 et M5 de la leucémie aiguë myéloblastique (LAM). EIF4E est régulé à plusieurs niveaux cependant, la régulation transcriptionnelle de ce gène est peu connue. Mes résultats montrent que EIF4E est une cible transcriptionnelle directe du facteur nucléaire « kappa-light- chain- enhancer of activated B cells » (NF-κB).Dans les cellules hématopoïétiques primaires et les lignées cellulaires, les niveaux de EIF4E sont induits par des inducteurs de NF-κB. En effet, l’inactivation pharmaceutique ou génétique de NF-κB réprime l’activation de EIF4E. En effet, suite à l’activation de NF-κB chez l’humain, le promoteur endogène de EIF4E recrute p65 (RelA) et c-Rel aux sites évolutionnaires conservés κB in vitro et in vivo en même temps que p300 ainsi que la forme phosphorylée de Pol II. De plus, p65 est sélectivement associé au promoteur de EIF4E dans les sous-types LAM M4/M5 mais non pas dans les autres sous-types LAM ou dans les cellules hématopoïétiques primaires normales. Ceci indique que ce processus représente un facteur essentiel qui détermine l’expression différentielle de EIF4E dans la LAM. Les analyses de données d’expressions par séquençage de l’ARN provenant du « Cancer Genome Atlas » (TCGA) suggèrent que les niveaux d’ARNm de EIF4E et RELA se trouvent augmentés dans les cas LAM à pronostic intermédiaire ou faible mais non pas dans les groupes cytogénétiquement favorables. De plus, des niveaux élevés d’ARNm de EIF4E et RELA sont significativement associés avec un taux de survie relativement bas chez les patients. En effet, les sites uniques κB se trouvant dans le promoteur de EIF4E recrutent le régulateur de transcription NF-κB p65 dans 47 nouvelles cibles prévues. Finalement, 6 nouveaux facteurs de transcription potentiellement impliqués dans la régulation du gène EIF4E ont été prédits par des analyses de données ChIP-Seq provenant de l’encyclopédie des éléments d’ADN (ENCODE). Collectivement, ces résultats fournissent de nouveaux aperçus sur le control transcriptionnel de EIF4E et offrent une nouvelle base moléculaire pour sa dérégulation dans au moins un sous-groupe de spécimens de LAM. L’étude et la compréhension de ce niveau de régulation dans le contexte de spécimens de patients s’avère important pour le développement de nouvelles stratégies thérapeutiques ciblant l’expression du gène EIF4E moyennant des inhibiteurs de NF-κB en combinaison avec la ribavirine. / The eukaryotic translation initiation factor EIF4E is a powerful oncogene that is overexpressed in cancers, including the M4 and M5 subtypes of acute myeloid leukemia (AML). EIF4E is regulated at multiple levels; however not much is known about the transcriptional regulation of this gene. My findings show that the nuclear factor kappa-light- chain-enhancer of activated B cells (NF-κB) is a direct transcriptional regulator of EIF4E. EIF4E levels are induced in primary hematopoietic cells and in cell lines in response to NF-κB activating stimuli. Pharmacological and genetic inhibition of NF-κB suppresses EIF4E levels. NF-κB factors RelA (p65) and c-Rel are recruited to evolutionarily conserved κB sites in the EIF4E promoter in vitro and in vivo following NF-κB activation concurrent with the recruitment of p300 and phosphorylated Pol II. Furthermore, p65 is selectively associated with the EIF4E promoter in M4/M5 AML subtypes but not in other AML subtypes or normal primary hematopoietic cells and thus represents an underlying factor in determining the differential expression of EIF4E in AML. Analysis of gene expression RNA-Seq data from The Cancer Genome Atlas (TCGA) suggests that EIF4E and RELA mRNA levels are upregulated in intermediate and poor prognosis AML but not in the cytogenetically favorable group. Additionally, elevated EIF4E and RELA mRNA levels are significantly associated with worst patient survival outcome. Furthermore, 8 new putative NF-κB target genes that may be regulated with a pattern similar to EIF4E in poor prognosis AML were in silico predicted from Chip-Seq data. Finally, 6 new transcription factors that may be implicated in EIF4E gene regulation were predicted from the analysis of ChIP-Seq data from the encyclopedia of DNA elements (ENCODE). Collectively, these findings could offer novel insights into the transcriptional regulation of EIF4E and a novel molecular basis for its dysregulation in AML. Understanding this level of regulation within the context of patient specimens is important for the development of novel therapeutic strategies to target EIF4E gene expression with specific NF-κB inhibitors combined with ribavirin.

Genome-wide association studies in multifactorial diseases have contributed to our understanding of genetic risk and defined specific disease-associated loci in particular populations. However, risk cannot be fully explained by genetics and evidence points to both genetic and environmental factors being important in causation and pathophysiology. The role of vitamin D in calcium homeostasis is well established. Over the last 30 years it has become clear that vitamin D has a wider role in physiology and disease, notably in autoimmune, cancer and infectious conditions. However, the molecular mechanisms and possible causal role of these associations are poorly understood. Here I propose that the role of vitamin D in immune and inflammatory responses is significant, that genetic variation partly determines the response to vitamin D and that integrative analysis can yield important insights for disease mechanisms. For this I investigate the relationship between vitamin D and genetic risk involving the immune system by focusing on multiple sclerosis and sepsis, conditions classically defined as autoimmune and inflammatory respectively. I describe data resolving genetic variation associated with autoimmune diseases in vitamin D receptor binding sites; the association to multiple sclerosis of a genetic variant lying within a VDR binding site; the correlation of plasma vitamin D with genotype and cell specific transcriptomes in healthy volunteers; and the extent of vitamin D deficiency in severe sepsis and septic shock, its association with survival, correlation with gene expression and use in sub-classification to identify patients at higher risk of death. The limitations of each study and future work are discussed. Integrating epidemiological and clinical observations with genetic and functional genomics techniques has the potential to reveal interactions in population specific disease susceptibility that may lead to an improved understanding of disease mechanisms and clinical translation. The work I present here bridges molecular analysis, candidate and genome- wide, with phenotypic observations that are important in our understanding of disease.

Le Glypican-3 (GPC3) est surexprimé dans 72% des carcinomes hépatocellulaire (CHC). C’est un co-récepteur membranaire du récepteur WNT, qui appartient à la famille des protéoglycanes à sulfates d'héparane. L'objectif général de ma thèse vise à étudier les mécanismes de régulation post-transcriptionnelle de l’expression du GPC3 dans le CHC. Pour cela, j’ai développé un test fonctionnel qui m’a permis de cribler une bibliothèque de 876 microARNs humains. Ceci a conduit à l’identification de 5 microARNs régulateurs de l’expression de l’ARNm codant pour le GPC3 via sa région 3’ non traduite (NT). Mon travail de thèse porte plus particulièrement sur le miR-1271 et le miR-1291 car ils sont dérégulés dans le CHC et sont respectivement inhibiteur et inducteur de l’expression du GPC3. Dans un premier projet, j’ai démontré que le miR-1271 cible directement la région 3’NT du GPC3 et diminue la stabilité de son ARNm. Ce microARN est sous-exprimé dans le CHC et son expression corrèle négativement avec celle de l'ARNm du GPC3 dans les CHC associés à une infection par le virus de l’hépatite B. Dans un deuxième projet, j’ai démontré que le miR-1291 régule positivement l’expression du GPC3 en inhibant un facteur intermédiaire. Une analyse in silico a permis d’identifier IRE1α comme candidat. IRE1α est une protéine transmembranaire du réticulum endoplasmique (RE) qui participe à « l’Unfolded Protein Response », une réponse adaptative activée lors de l’accumulation de protéines mal conformées dans le RE. J’ai démontré qu’IRE1α clive l’ARNm codant pour le GPC3 grâce à son activité endoribonucléase. D’autre part, le miR-1291 cible directement l’ARNm codant pour IRE1α dans sa région 5’NT ce qui inhibe son expression et induit une surexpression du GPC3. Le miR-1291 est surexprimé dans le CHC et son expression corrèle positivement avec celle de l’ARNm du GPC3. En conclusion, mon travail de thèse m’a permis de mettre en évidence et de caractériser deux nouveaux microARNs (miR-1271 et miR-1291) contrôlant l’expression du GPC3 par des mécanismes directs ou indirects. La pertinence physiopathologique de ces régulations dans le CHC est en accord avec les niveaux d’expression respectifs de ces microARNs, qui pourraient contribuer à la surexpression du GPC3 dans ces tumeurs. / Glypican-3 (GPC3) is overexpressed in 72% of hepatocellular carcinoma (HCC). It is a co-receptor for WNT receptor and belongs to the heparan sulfate proteoglycans family. The general objective of my PhD thesis was to study the mechanisms by which GPC3 is post-transcriptionnally regulated in HCC. To this end, I developed a functional test that allowed me to screen a library of 876 human microRNAs. This led me to identify 5 microRNAs that regulate the expression of GPC3 mRNA through its 3’Untranslated Region (UTR). The work presented in this thesis particulary focuses on miR-1271 and miR-1291 as both microRNAs present a deregulated expression in HCC and are respectively inhibitor and activator of GPC3 mRNA expression. In a first project, I demonstrated that miR-1271 directly binds to GPC3 mRNA 3’UTR and affects its stability. This microRNA is underexpressed in HCC and its expression negatively correlates with that of GPC3 mRNA in a subgroup of HCC corresponding to those associated with hepatitis B virus infection. In a second project, I demonstrated that miR-1291 postively regulates the expression of GPC3 mRNA by targeting an intermediate factor. An in silico analysis led to the identification of the Inositol Requiring Enzyme 1 alpha (IRE1α) as a potential candidate. IRE1α is an endoplasmic reticulum (ER) resident type I transmembrane protein and contributes to the signaling of the Unfolded Protein Response (UPR). The UPR is an adaptive response activated upon accumulation of improperly folded proteins in the ER. I showed that IRE1α cleaves GPC3 mRNA through its endoribonuclease activity. Moreover I demonstrated that miR-1291 directly targets IRE1α mRNA through its 5’UTR, thereby decreasing its expression and contributing to GPC3 mRNA overexpression. MiR-1291 is overexpressed in HCC and its expression positively correlates with that of GPC3 mRNA. In summary, the work carried out during my PhD allowed the identification and the characterization of two new microRNAs (miR-1271 and miR-1291) that control the expression of GPC3 mRNA through direct or indirect mechanisms. The pathophysiological relevance of these regulatory mechanisms is in agreement with the respective expression levels of these microRNAs in HCC, which could therefore contribute to the overexpression of GPC3 in those tumors.

La protéine multifonctionnelle E4F1 fut initialement identifiée comme une cible de l'oncoprotéine virale E1A au cours de l'infection par l'adénovirus. Nos données, ainsi que celles d'autres laboratoires, montrent qu'E4F1 intervient à de multiples niveaux de régulation de la voie p53, une voie de signalisation très fréquemment inactivée au cours de la tumorigenèse. Au cours de ma thèse, j'ai mis en évidence et caractérisé un nouveau niveau de régulation impliquant la protéine E4F1 et un autre régulateur important de la voie p53 : l'oncoprotéine Mdm2. Mes travaux ont permis de montrer, pour la première fois, qu'E4F1 et Mdm2 sont présents dans un même complexe multiprotéique associé à la chromatine et dont le recrutement est indépendant de p53. La formation du complexe E4F1-Mdm2 est sous la dépendance de certains stress cellulaires, telle que la réponse aux stress oxydatifs. De plus, Mdm2 et E4F1 sont capables de s'ubiquitinyler mutuellement sans promouvoir leur protéolyse. Nos résultats préliminaires suggèrent que ce complexe chromatinien Mdm2-E4F1 est impliqué dans le contrôle d'un programme transcriptionnel apparenté à une réponse au stress oxydatif. L'ensemble de ces données suggère des fonctions de Mdm2 au niveau de la chromatine, indépendantes de sa fonction bien décrite de régulateur du suppresseur de tumeur p53. / E4F1 is a multifunctional protein that was first identified as a cellular target of the viral oncoprotein E1A during adenoviral infection. We, and others, have shown that E4F1 impinges at different level of the p53 pathway, which is frequently inactivated during tumorigenesis. During my PhD, I have highlighted and characterized a novel level of regulation implicating E4F1 and an other key regulator of the p53 pathway: the Mdm2 oncoprotein.My work have shown for the first time that Mdm2 and E4F1 directly interact in a same multiprotein complex associated with chromatin, and this recruitment occurs independently of p53. The Mdm2-E4F1 complex formation is dependant of some cellular stresses, such as oxidative stresses reponses. Our preliminary data also indicate that E4F1 and Mdm2 ubiquitylate each other without promoting their proteolysis, and influence their stability on chromatin. Our preliminary results indicate that this Mdm2-E4F1 chromatinian complex is involved in the regulation of a transcriptional program dependant of oxidative stresses. These data support the notion that Mdm2 presents unexpected functions on chromatin, independently of its very well described p53 regulation.

3 domaines cellulaires définissent l'épiderme de la queue de Ciona intestinalis. Le domaine des lignes médianes, donne naissance à deux structures différenciées larvaires, la nageoire et système nerveux périphérique. Il a été montré que les voies de signalisation BMP et FGF sont respectivement les inducteurs des lignes médianes ventrale et dorsale. Ce travail a consisté en la caractérisation des mécanismes moléculaires, situés à l'interface des signaux inducteurs et des processus de différenciation, définissant l'identité cellulaire des lignes médianes. L'identification des relations épistatiques reliant 7 facteurs de transcription impliqués dans ce processus suggère un fonctionnement en réseau hiérarchisé et place Msxb comme gène clé. Cette hiérarchie est validée par: un atlas d'expression spatio-temporelle, la perte de fonction de Msxb, l'analyse des régions cis-régulatrices de la transcription. Nous avons identifié 2 types d'enhancers, contrôlant respectivement les gènes précoces et tardifs du réseau. L'expression précoce de Msxb dans les précurseurs dorsaux est controlée par un enhancer distinct régulé par la voie FGF relayée par Otx et Nodal. Cette signature transcriptionnelle est retrouvée dans les enhancers de gènes co-exprimés, et chez l'orthologue de Msxb chez une autre ascidie. Enfin, nous montrons que la partie la plus postérieure des lignes médianes constitue un troisième compartiment, déployant un programme génétique distinct. Ce travail nous renseigne sur la structure, les mécanismes moléculaires de formation des lignes médianes, l'existence d'une signature transcriptionnelle évolutivement conservée pour le gène clé de l'acquisition de ce destin cellulaire. / Ciona intestinalis tail epidermis has 8 rows of cells defining 3 domains. One of them, the midline domain, gives rise to differentiated cells which form the larval fin and part of peripheral nervous system. Previous work has shown that BMP and FGF signalling are the inducers of ventral and dorsal midlines respectively. My work consisted in the identification of molecular events which lead epidermal cells to adopt midline fate, from induction to tail differentiation. We identified 7 transcription factors involved in this process. Identification of epistatic relationships suggest that these genes are in a hierarchical network where Msxb is a key gene. This hierarchy is validated by 1) a spatio-temporal expression atlas, 2) loss of function of Msxb, 3) cis-regulatory regions analysis for each network gene. We identified 2 types of enhancers, one capable to decouple ventral / dorsal signals used by early genes, and the other used by later genes, acting as a global response in both midlines. We showed that the early expression of Msxb in dorsal precursors is controled by a distinct enhancer, regulated by FGF9/16/20 via Otx and Nodal. This transcriptional signature is found in enhancers of co-expressed genes and in Msxb orthologue in another ascidian. Finally, we showed the most posterior part of the midlines is controlled by a distinct genetic program than the one used in dorsal and ventral midlines. This work gives insight into midlines structure, the mechanisms involved in their formation and a conserved transcriptional signature for the key gene involved in midline cell fate.

No description available.

Diabetes é uma doença metabólica caracterizada por hiperglicemia associada a prejuízos na captação e utilização de glicose, em que reduções na expressão da proteína GLUT4 (codificada pelo gene SLC2A4), bem como das enzimas Hexokinase-2 e Glycogen synthase (codificadas pelos genes HK2 e GYS1), desempenham papel importante. Recentemente, um novo elemento vem sendo relacionado à etiopatogenia e à fisiopatologia do diabetes, os microRNAs (miRNAs), que são pequenos RNAs envolvidos na regulação da expressão gênica, geralmente afetando a degradação de mRNAs. Entretanto, a participação de miRNAs envolvidos na redução da expressão de mRNAs relacionados a proteínas envolvidas na captação e utilização de glicose, sobretudo em músculo esquelético, permanece desconhecida. O objetivo desse estudo foi investigar a expressão de miRNAs potencialmente reguladores da expressão de Slc2a4/GLUT4, Hk2/HK2 e Gys1/GYS1 em músculo esquelético de ratos diabéticos. Utilizamos ratos Wistar machos que foram tornados diabéticos pela administração de estreptozotocina. Após 13 dias, 3 grupos foram formados: não-diabético (ND) e diabético tratado com placebo (DP) ou insulina (DI). O tratamento foi conduzido por 7 dias, totalizando 21 dias de diabetes. Variáveis metabólicas foram avaliadas e os músculos sóleos foram removidos para avaliar a expressão de mRNAs, miRNAs e proteínas. Uma abrangente análise in silico foi conduzida para determinar miRNAs candidatos a regularem a expressão de Slc2a4, Hk2 e Gys1. Os animais diabéticos apresentaram perda de peso, poliúria, glicosúria, hiperglicemia e aumento de frutosamina plasmática; a insulinoterapia melhorou estas variáveis. O diabetes reduziu a expressão dos mRNAs Slc2a4 (~55%), Hk2 (~47%) e Gys1 (~45%), e das proteínas GLUT4 (~77%), HK2(~52%) e GYS1 (~49%); a insulinoterapia restaurou essas variáveis. A expressão de 20 miRNAs foi avaliada neste estudo; 8 foram modulados pelo diabetes, sendo três supra-regulados, miR-1 (~28%), miR-29b (~118%) e miR-29c (~51%); e cinco infra-regulados, miR-93 (~39%), miR-199a (~30%), miR-345-3p (~23%), miR-532-3p (~26%) e miR-150 (~32%). Exceto pelo miR-1 e miR-150, a insulinoterapia reverteu as demais alterações. Além disso, miR-29b e miR-29c correlacionaram-se negativamente com GLUT4 e HK2, e positivamente com glicemia, glicosúria e frutosamina, sugerindo uma possível relação causal; enquanto que miR-199a e miR-532-3p correlacionaram-se positivamente com GLUT4 e HK2, e também com as variáveis metabólicas, sugerindo uma regulação indireta sobre os mRNAs dessas proteínas. No último caso, demonstrou-se que o miR-199a tem como alvo o NFKB1, um repressor do gene Slc2a4, o qual diminuiu no diabetes, explicando, pelo menos parcialmente, o efeito indireto sobre o GLUT4. Em suma, o diabetes aumenta a expressão de miR-29b e miR-29c, e reduz a expressão de miR-199a e miR-532-3p; o primeiro efeito, potencialmente age diretamente na tradução do mRNA Slc2a4 e Hk2, e o segundo, potencialmente age indiretamente, via NFKB, na transcrição dos genes. Como consequência, as proteínas GLUT4 e HK2 diminuem, o que reduziria a utilização de glicose pelo músculo, contribuindo para a hiperglicemia do diabetes. / Diabetes is a metabolic disease characterized by hyperglycemia associated with impaired glucose metabolism and uptake, in which reductions of GLUT4, hexokinase 2 (HK2) and glycogen synthase 1 (GYS1) proteins, encoded respectively by SLC2A4, HK2 and GYS1 genes, play an important role. Recently, a new element have been related to etiopathogeny and pathophysiology of diabetes, the microRNAs (miRNAs), which are small RNAs involved in the regulation of gene expression, usually by affecting the degradation of mRNAs. However, the participation of miRNAs diabetes-induced reduction of expression of genes related to glucose uptake and metabolism in skeletal muscle remains unknown. Thus, the objective of this study was to investigate the expression of miRNAs potentially regulators of the Slc2a4/GLUT4, Hk2/HK2 and Gys1/GYS1 in skeletal muscle of diabetic rats. Male Wistar rats were rendered diabetic by receiving streptozotocin. After 13 days, 3 groups were formed: non-diabetic (ND), and diabetic treated with placebo (DP) or insulin (DI) (NPH insulin, 6U/day). Treatment was conducted for 7 days, totalizing 21 days of diabetes. At the end of the experimental period, metabolic variables were evaluated and the soleus muscle was removed for evaluation of mRNA, miRNA and protein expression. A broad in silico analysis was performed to determine candidate miRNAs as potential regulators of Slc2a4, Hk2 and Gys1. Diabetic rats shown weight loss, polyuria, glycosuria, hyperglycemia and increased plasma fructosamine; insulin treatment improved these variables. Diabetes reduced Slc2a4 (~55%), Hk2 (~47%) and Gys1 (~45%) mRNAs, as well as GLUT4 (77%), HK2 (52%) and GYS1 (49%) proteins; insulin treatment restored these variables. Twenty miRNAs were assessed in this study. Eight miRNAs were modulated by diabetes in skeletal muscle; three were upregulated: miR-1 (28%), miR-29b (118%) and miR-29c (51%), whereas five were downregulated: miR-93 (39%), miR-150 (32%), miR-199a (30%), miR-345-3p (23%) and miR-532-3p (26%). Except for miR-1 and miR-150, all regulations were reverted by insulin treatment. Besides, miR-29b and miR-29c were negatively correlated with GLUT4 and HK2 proteins, and positively with glucose, glycosuria and plasma fructosamine suggesting a direct causal relationship; while miR-199a and miR-532-3p were positively correlated with GLUT4 and HK2 proteins, and also with the metabolic variables, suggesting an indirect causal relationship. In the last case, it was demonstrated that miR-199a has the Slc2a4 repressor Nfkb1 as target, which was reduced in muscle from diabetic rats, explaining, at least partially, the indirect effect upon GLUT4. In conclusion, diabetes increase the expression of miR-29b and miR-29c, and reduce the expression of miR-199a e miR-532-3p; the first effect, potentially acts directly in the translation of Slc2a4 and Hk2 mRNAs, and the second one, potentially acts indirectly, via NFKB, in the transcription of these genes. As a result, the expression of GLUT4 and HK2 decreases, which would reduce the muscle glucose uptake and metabolization, contributing to the hyperglycemia of the diabetes.