This thesis describes the isolation and characterisation of the C. albicans NRG1 gene, which encodes a repressor of filamentous growth in this pathogenic fiingus. A C. albicans SBP1 cDNA was previously isolated in a screen for transacting factors that bind to a STRE-like element (consensus sequence: CCCCT) (Leng, 1999). In S. cerevisiae, STRE is a stress-responsive element that is required for the regulation of many stress-responsive genes (Marchler et al., 1993). In C. albicans, this element had been identified in the promoters of two hypha-specific genes, ALS8 and HYR1. Since many conditions that induce yeast-hypha morphogenesis in C. albicans impose a stress, it was proposed that the STRE- binding protein (Sbpl) might influence yeast-hypha morphogenesis and/or stress responses in this human pathogen. The cDNA was then used to isolate the complete C. albicans SBP1 locus by colony hybridisation. Both the cDNA and gene were sequenced, revealing an ORF capable of encoding a protein of 310 amino acids containing a C2H2-zinc finger motifs near its C-terminus. The zinc finger region of this protein displayed the highest sequence similarity to S. cerevisiae NRG1 (67 % identity), and hence the gene was renamed CaNRGl. To examine the role of CaNrgl, a C. albicans nrgl/nrgl null mutant and a mutant over-expressing the NRG1 gene were created. Overexpression of NRG1 did not reveal any obvious phenotypes, but inactivation of NRG1 caused constitutive filamentous and invasive growth, as well as increased sensitivity to some stresses. Also, the expression of the hypha-specific genes, ALS8, ECE1, HWP1 and HYR1, was derepressed in the nrgl/nrgl mutants. Similar phenotypes were observed for a C. albicans tupl/tupl null mutant. These observations suggest that Nrgl represses filamentous growth in C. albicans, possibly by recruiting Tupl to specific promoters. Unlike the tupl/tupl mutant, nrgl/nrgl cells formed normal hyphae following pH and serum stimulation, they generated chlamydospores at normal rates, and they grew at 42 C. Transcript profiling of 2002 C. albicans genes revealed that Nrgl regulates a subset of Tupl-repressed genes, which includes known hypha- specific genes and some virulence factors. The data also showed that Tupl regulates other genes, which are not regulated by Nrgl, including glucose sensitive genes, amino acid and sterol biosynthesis genes, and genes encoding other virulence determinants. Taken together, this study demonstrates that Nrgl is a transcriptional repressor that regulates a set of functions required for yeast-hypha morphogenesis and virulence in C. albicans.

Recently, through reporter gene studies, the novel BTB/POZ protein, Kaiso, has been identified as a transcriptional repressor. The purpose of this study was to determine if Kaiso recruited the Histone Deacetylase Complex to mediate repression and if the previously identified Kaiso Binding Site (KBS; TCCTGCNA) is a physiological target regulated by Kaiso. The two objectives are complementary because an HDAC interaction identifies the mechanism of transcriptional regulation used by Kaiso and regulation of the KBS element identifies a novel, non-methylation dependent, physiological target under transcriptional regulation by Kaiso. Through coimmunoprecipitation and Western blot analyses, Kaiso does not interact with HDAC1, HDAC2 or mSIN3A. These results were surprising since all three of these proteins are common to a variety of repression complexes. mSIN3A is a common component of SIN3 mediated repression and HDAC1/HDAC2 are part of various repression complexes including SIN3, NuRD and CtBP. Although the remaining HDAC proteins were not assayed for an interaction, Kaiso transcriptional activity was demonstrated to be insensitive to the HDAC inhibiting drug, Trichostatin A (TSA). These results indicate either a non-HDAC mechanism of action or alternatively, transcriptional activation. Complementary to the observations of no Kaiso-HDAC interaction and TSA insensitivity was the findings that Kaiso activates transcription of the KBS cis-element in HCT116, HCA-7 and 293 cells, but not MOCK cells in reporter gene assays. Taken together, these results indicate that Kaiso is a dual functioning protein capable of both transcriptional activation and repression and that the mechanism of repression is not through the direct recruitment of HDAC proteins. / Thesis / Master of Science (MSc)

The Escherichia coli transcription factor NikR is responsible for nickel-mediated repression of the nik operon. The crystal structure of NikR in complex with its operator sequence provided insight into the mechanistic details of nickel-activated NikR-DNA complex formation. The crystal structure revealed that the α3 helix and its preceding loop (residues 63-79) in two of the metal-binding domains—which become structurally ordered after high-affinity nickel binding—make non-specific contacts with the DNA phosphodiester backbone. The proposed mechanism of NikR binding to DNA suggests that the non-specific interactions between the DNA phosphodiester backbone and the positively-charged residues Lys64 and Arg65 anchor NikR to the DNA, thereby allowing the protein to initiate a one-dimensional search for its recognition sequence. The DNA-binding studies presented here strongly support an important role for Lys64 and Arg65 in NikR-DNA complex formation which is in agreement with the proposed model of NikR binding to DNA.

The Escherichia coli transcription factor NikR is responsible for nickel-mediated repression of the nik operon. The crystal structure of NikR in complex with its operator sequence provided insight into the mechanistic details of nickel-activated NikR-DNA complex formation. The crystal structure revealed that the α3 helix and its preceding loop (residues 63-79) in two of the metal-binding domains—which become structurally ordered after high-affinity nickel binding—make non-specific contacts with the DNA phosphodiester backbone. The proposed mechanism of NikR binding to DNA suggests that the non-specific interactions between the DNA phosphodiester backbone and the positively-charged residues Lys64 and Arg65 anchor NikR to the DNA, thereby allowing the protein to initiate a one-dimensional search for its recognition sequence. The DNA-binding studies presented here strongly support an important role for Lys64 and Arg65 in NikR-DNA complex formation which is in agreement with the proposed model of NikR binding to DNA.

The LuxR protein family is a class of quorum-sensing regulated bacterial transcription factors that alter gene expression as a function of ligand detection. This coincides with a high population density and/or a low rate of signal ligand diffusion. The majority of LuxR proteins are activated only in the presence of the signal ligand, an acyl-homoserine lactone (AHL). EsaR, from the corn pathogen Pantoea stewartii, represents a subset of LuxR homologues that are active in the absence of AHL and deactivated by its presence. The mechanism by which EsaR responds to AHL in a manner opposite to that of the majority of LuxR homologues remains elusive. Unlike the majority of LuxR homologues, which require AHL for purification, EsaR can be purified and biochemically investigated in the absence and presence of AHL. This work sought to answer questions regarding the structure-function relationship of the LuxR homologue, EsaR. Fluorescence anisotropy was used to determine the relative DNA-binding affinity of wild type EsaR and three AHL-independent EsaR variants in the presence and absence of AHL. This enabled for quantitative analysis of the relative binding affinities of these AHL-independent variants for the EsaR binding site, the esa box. The results demonstrate that one AHL-independent EsaR variant has a slightly higher affinity for the esa box in the presence, rather than the absence of AHL. The affinity of the other two for the DNA is not impacted by AHL, potentially due to an inability to transduce the signal of ligand detection to the DNA binding domain. Constructs containing only the EsaR N-terminal domain (NTD) were also developed. These constructs circumvented solubility issues associated with the full-length protein, allowing for additional biochemical analysis. It was determined that the EsaR NTD alone is sufficient for multimerization and ligand binding. Additionally, preliminary X-ray crystallography efforts have established some of the early parameters required to solve the crystal structure of the EsaR ligand binding domain in both the presence and absence of AHL. If pursued, these structures would be the first solved of a LuxR homologue ligand binding domain in both the presence and absence of the native AHL, potentially demonstrating the conformational change that occurs as a result of ligand binding. Collectively, these findings have established some of the groundwork required to resolve the question of what sort of conformational changes occur in EsaR as a result of ligand binding. / Master of Science

BMI1 est une protéine appartenant à la famille des polycombs impliquée dans la régulation épigénétique de la transcription. Il a été montré que cette protéine est essentielle à la régulation de la prolifération, de la sénescence et du métabolisme ainsi qu’à l’auto-Renouvellement des cellules souches hématopoïétiques et cancéreuses. Ce répresseur transcriptionnel au fort potentiel oncogénique est retrouvé surexprimé dans de nombreux types de cancer ; dans le cas de la Leucémie Myéloïde Chronique (LMC) le niveau d’expression de BMI1 augmente avec l’aggravation de la pathologie. Cependant, les voies de signalisation impliquées dans sa surexpression et le rôle qu’il joue au sein de cette maladie demeurent méconnus. En réprimant l’expression de BMI1 par ARN interférence nous avons pu mettre en évidence que ce polycomb était essentiel à la prolifération cellulaire ainsi qu’au potentiel clonogénique des cellules de LMC. Nous avons également démontré pour la première fois que BMI1 soutenait la croissance tumorale à travers la répression d’un processus autophagique délétère pour la cellule cancéreuse. Une approche transcriptomique nous a permis d’identifier la cible transcriptionnelle impliquée dans ce processus, la Cycline G2. Nous avons, pour finir, trouvé une molécule, via une approche bioinformatique, capable de réinduire l’expression de la Cycline G2 dans les cellules de LMC, l’alexidine dihydrochloride. Cette molécule induit une forte autophagie dans les cellules cancéreuses ainsi que de l’apoptose. Elle s’est également montrée capable de resensibiliser à l’imatinib (un inhibiteur de BCR-ABL) une lignée pourtant résistante. / The polycomb protein Bmi1 is a major epigenetic regulator. It has been shown that this protein is essential for the regulation of cell proliferation, senescence and metabolism but also self-Renewal of hematopoïetic and cancer stem cells. This transcriptional repressor, with a strong oncogenic potential, is overexpressed in many types of cancer. In case of Chronic Myeloid Leukemia (CML) the expression level of BMI1 is associated with worsening prognosis. However, the signaling pathways involved in its overexpression and its role in this disease remains unclear. By using RNAi to repress BMI1 expression we highlighted that this polycomb was essential for proliferation and clonogenicity of CML cells. We also demonstrated, for the first time, that BMI1 supported tumor growth through repression of deleterious cancer cell autophagy. A transcriptomic approach allowed us to identify a transcriptional target involved in this process: the Cyclin G2. Through a bioinformatic approach, we finally found a molecule capable of expression re-Induction of Cyclin G2 in CML cells : alexidine dihydrochloride. This molecule induced a high level of autophagy as well as apopotosis in cancer cells. It had also been able to re-Sensitize to imatinib a resistant cell line. In conclusion, our results revealed a new role for the polycomb BMI1 in supporting the CML pathology. Moreover, our work allowed the identification of two new approaches for therapeutically targeting this oncogene functions.

Colorectal cancer is one of the most common cancer types worldwide. Both genetic and epigenetic alterations play a critical role in its initiation and progression. One of the genes frequently epigenetically silenced or lost in many types of human cancer is tumor suppressor gene Hypermethylated in Cancer 1 (HIC1). It encodes for transcriptional repressor regulating its target genes directly or indirectly. Twelve genes whose expression is repressed by HIC1 have been identified to date. These genes encode for transcription factors, cell cycle and apoptosis regulators or proteins involved in angiogenesis as well as cell migration and invasiveness. Employing mouse embryonic fibroblasts upon Hic1-conditional knockout we have revealed six novel genes potentially repressed by Hic1 including Toll-like receptor 2 (Tlr2). Here we show that Tlr2 is one of the Hic1 target genes and that Hic1 inactivation in the intestine leads to increased Tlr2 production. Moreover, enhanced inflammatory response upon chemical-induced colitis as well as increased tumor formation in ApcMin mice was observed in Hic1-deficient mice. Expression profiling in human fibroblast upon HIC1 knockdown revealed increased expression of another potential target gene, transcription factor E2F7. Our study describes a new relationship between HIC1 and...

Avec plus d’un million et demi de morts chaque année, la tuberculose reste aujourd’hui la seconde cause de mortalité liée à un agent infectieux. De plus l’organisation mondiale de la santé (OMS) a estimé en 2011 qu’un tiers de la population mondiale était porteuse du bacille Mycobacterium tuberculosis responsable de la maladie. Depuis la fin des années 1980, une recrudescence du nombre de cas de tuberculose est observée à l’échelle mondiale. Cette recrudescence est due à la fois à l’apparition de souches résistantes, mais également à l’épidémie de VIH qui est un facteur de prédisposition au déclenchement de la maladie.En 2000, le répresseur transcriptionnel mycobactérien EthR a été identifié comme étant un régulateur clé dans la bioactivation de l’éthionamide (ETH), un antituberculeux utilisé pour le traitement de seconde intention. En 2009, l’inhibition de ce répresseur par le développement de molécules « drug-like » a permis de potentialiser l’activité de l’éthionamide d’un facteur 3 chez la souris infectée et a permis de valider cette cible pour une future approche thérapeutique.Ce travail repose sur la découverte et l’optimisation de nouveaux inhibiteurs de ce répresseur transcriptionnel mycobactérien, à partir d’une petite molécule appelée « fragment » qui a été cocristallisée avec la protéine. Par la combinaison d’un criblage in silico, d’un criblage in vitro des touches identifiées, de l’étude des structures radiocristallographiques des complexes ligands/protéines et de la chimie médicinale, le développement de trois approches complémentaires dites « fragmentgrowing », « fragment-merging » et « fragment-linking » a permis de développer des composés présentant de fortes activités. Ces résultats permettront très prochainement de sélectionner une nouvelle molécule issue de ce travail dans la perspective de nouveaux essais sur le modèle murin. / Tuberculosis (TB) remains the leading cause of death due to a single infective agent with more than 1.5 million people killed each year. In 2011, the world health organization (WHO) estimated that one third of the world’s population is infected with Mycobacterium tuberculosis, the pathogen responsible for the disease. This phenomenon may be due to an explosive escalation of TB incidence that occurred in the 1980s due to the emergence of both resistant strains and HIV epidemic.In 2000, EthR, a mycobacterial transcriptional repressor, was identified as a key modulator of ethionamide (ETH) bioactivation. ETH is one of the main second-line drugs used to treat drug resistant strains. In 2009, it was shown that co-administration of ETH and drug-like inhibitors of EthR was able to boost ETH activity threefold in a mouse-model of TB-infection, thus validating the target for a new therapeutic strategy.This work deals with the discovery and optimisation of new EthR inhibitors, based on a small molecule, called a “fragment”, co-crystallized with the protein. We combined in silico screening, in vitro evaluation of the hit compounds, study of co-crystal structures and medicinal chemistry to develop three complementary approaches called “fragment growing”, “fragment merging” and “fragment linking” that led to the discovery of very potent inhibitors. Based on these results, we are currently selecting a potential candidate for new in vivo experiments.

Pichia pastoris, a methylotrophic yeast is widely used for recombinant protein production. It has a well characterized methanol utilization (MUT) pathway, the enzymes of which are induced when cells are cultured in the presence of methanol. In this study, we have identified an unannotated zinc finger protein, which was subsequently named ROP (repressor of phosphoenolpyruvate carboxykinase, PEPCK) and characterized its function. ROP expression is induced in P. pastoris cells cultured in biotin depleted glucose ammonium medium as well as a medium containing methanol as the sole source of carbon. In glucose-abundant, biotin depleted cultures, ROP induces the expression of a number of genes including that encoding PEPCK. Interestingly, a strain in which the gene encoding ROP is deleted (ΔROP) exhibits biotin-independent growth. Based on a number of studies, it was proposed that the ability of ΔROP to grow in the absence of biotin is due to the activation of a pyruvate carboxylase-independent pathway of oxaloacetate biosynthesis. It was also proposed that PEPCK, which normally functions as a gluconeogenic enzyme, may act as an anaplerotic enzyme involved in the synthesis of oxaloacetate. ROP was shown to be a key regulator of methanol metabolism when P. pastoris cells are cultured in YPM medium containing yeast extract, peptone and methanol but not YNBM medium containing yeast nitrogen base and methanol. In P. pastoris cells cultured in YPM, ROP functions as a transcriptional repressor of genes encoding key enzymes of the methanol metabolism such as the alcohol oxidase I. (AOXI). Deletion of the gene encoding ROP results in enhanced expression of AOXI and growth promotion while overexpression of ROP results in repression of AOXI and retardation of growth of P. pastoris cultured in YPM medium. Subcellular localization studies indicate that ROP translocates from cytosol to nucleus in cells cultured in YPM but not YNBM. To understand the mechanism of action of ROP, we examined its DNA-binding specificity. The DNA-binding domain of ROP shares 57% amino acid identity with that of Mxr1p, a master regulator of genes of methanol metabolism. We demonstrate that the DNA-binding specificity of ROP is similar to that of Mxr1p and both proteins compete with each other for binding to AOXI promoter sequences. Thus, transcriptional interference due to competition between Mxr1p and ROP for binding to the same promoter sequences is likely to be the mechanism by which ROP represses AOXI expression in vivo. Mxr1p and ROP are examples of transcription factors which exhibit the same DNA-binding specificity but regulate gene expression in an antagonistic fashion.

La découverte du système des peptides natriurétiques (NP), au début des années 80, fut une découverte majeure qui révéla le rôle endocrinien du cœur. Les connaissances sur la relaxation vasculaire, la diurèse et la natriurèse provoquées par ce système ont évolué vers un niveau de complexité insoupçonné à cette époque. Nous savons à présent que les NP sont impliqués dans plusieurs autres mécanismes dont la prolifération cellulaire, l’apoptose, l’inhibition du système rénine-angiotensine-aldostérone (RAAS) et le métabolisme des adipocytes. Le métabolisme des lipides est maintenant devenu une cible de choix dans la lutte contre l’obésité. Cette condition aux proportions pandémiques est un facteur de risque majeur dans l’apparition de l’hypertension et du syndrome métabolique (MetS). La compréhension des mécanismes et des défauts de la voie des NP pourrait avoir un impact positif sur le contrôle du MetS et de l’hypertension. L’expression du récepteur des peptides natriuretiques de type 1 (NPR1/GCA) est contrôlée par plusieurs agents incluant son propre ligand, le peptide natriurétique de l’oreillette (ANP). La découverte d’une boucle de retro-inhibition, dans les années 90, a été un événement majeur dans le domaine des NP. En effet, suite à une stimulation à l’ANP, le NPR1/GCA peut inhiber l’activité transcriptionnelle de son propre gène par un mécanisme dépendant du cGMP. Notre groupe a identifié un élément cis-régulateur responsable de cette sensibilité au cGMP et mon projet consistait à identifier la ou les protéine(s) liant cet élément de réponse au cGMP (cGMP-RE). Nous avons identifié un clone liant le cGMP-RE en utilisant la technique du simple hybride chez la levure et une banque d’ADN complémentaire (ADNc) de rein humain. Ce clone provient d’un ADNc de 1083-bp dont le gène est localisé sur le chromosome 1 humain (1p33.36) et codant pour une protéine dont la fonction était inconnue jusqu’ici. Nous avons nommé cette nouvelle protéine GREBP en raison de sa fonction de cGMP Response Element Binding Protein. Des essais de liaison à l’ADN ont montré que cette protéine possède une affinité 18 fois plus élevée pour le cGMP-RE que le contrôle, tandis que des expériences de retard sur gel (EMSA) ont confirmé la spécificité des interactions protéine-ADN. De plus, l’immuno-précipitation de la chromatine (ChIP) a prouvé que GREBP lie le cGMP-RE dans des conditions physiologiques. La liaison de GREBP au cGMP-RE inhibe l’expression du gène rapporteur luciférase sous contrôle du promoteur de npr1/gca. L’inhibition de GREBP à l’aide d’ARN interférant active le promoteur de npr1/gca. Dans les cellules NCI-H295R, l’ANP stimule l’expression de grebp de 60% après seulement 3 heures et inhibe l’expression de npr1/gca de 30%. GREBP est une protéine nucléaire surtout exprimée dans le cœur et ayant le facteur eIF3F comme partenaire. Les variations nucléotidiques du gène sont plus fréquentes chez les patients hypertendus que chez des patients normotendus ou hypertendus souffrant de MetS. Nous rapportons ici l’existence d’un gène spécifique à l’humain qui agit comme répresseur transcriptionnel de npr1/gca et potentiellement impliqué dans le développement de l’hypertension. / The natriuretic peptide (NP) system was a milestone discovery that revealed the endocrine role of the heart for the first time in the early 1980s. From its vasodilatory, natriuretic and diuretic actions, knowledge about this system has evolved to a degree of complexity unsuspected at that time. Now, through cGMP generation, NPs are involved in several other mechanisms, such as cell proliferation, apoptosis, renin-angiotensine-aldosterone system (RAAS) inhibition, and fat cell function. The latter point is of growing interest in lipid metabolism and has become an important issue in the fight against obesity. This pandemic condition is one of the main risk factors leading to hypertension development and metabolic syndrome (MetS) progression. Thus, understanding, at least in part, the lipid mobilization pathways controlled by NPs could have a positive impact in MetS management. As with hypertension, identifying defects in signaling pathways will certainly help to identify mechanisms implicated in lost sensitivity of the NP system. Natriuretic peptide receptor 1 (npr1/gca) expression is controlled by several agents including its own ligand, the atrial natriuretic peptide (ANP). A major finding in NPs field occured in the mid-90s when a mechanism involving a retro-inhibition loop was described. Indeed, after ANP stimulation, NPR1/GCA down-regulates the transcriptional activity of its gene via a cGMP-dependent mechanism. Since our group previously identified a cis-acting element responsible for this cGMP sensitivity, I proceeded to explore novel putative protein binding to the cGMP-response element (cGMP-RE). Using the yeast-one-hybrid technique with a human kidney cDNA library, we identified a strongly positive clone able to bind cGMP-RE. The clone was derived from a 1083-bp long cDNA of a gene of yet unknown function localized on human chromosome 1 (1p33.36). We named this new protein GREBP for cGMP-Response Element-Binding Protein. DNA-binding assays showed 18-fold higher cGMP-RE-binding capacity than the controls while electromobility shift assay (EMSA) indicated a specific binding for the cGMP-RE and chromatin immuno-precipitation (ChIP) confirmed the binding of GREBP to the element under physiological conditions. By acting on cGMP-RE, GREBP inhibited the activity of a luciferase-coupled NPR1 promoter construct. In H295R cells, ANP heightened GREBP expression by 60% after just 3 hours of treatment while inhibiting npr1/gca expression by 30%. Silencing GREBP with specific small interfering RNA increased the activity of the luciferase-coupled NPR1/GCA promoter and NPR1/GCA mRNA levels. GREBP is a nuclear protein mainly expressed in the heart and has the eIF3F factor as partner. Its nucleotide variations are more frequent in non-obese hypertensive patients than normotensive subjects or hypertensive patients suffering from MetS. We report here the existence of a human specific gene acting as a transcriptional repressor of npr1/gca gene that could be implicated in hypertension development.