Transcriptional deregulation has emerged as one of the leading causes in various human diseases. More than fifty percent of cancers arise due to frequent mutations in genes regulating transcription. Higher-order assembly via protein-protein interactions is one common mechanism of transcriptional regulation. Despite their critical role in regulating gene transcription and therapeutic relevance, detailed mechanistic understanding of these assemblies remains scarce. The primary focus of this thesis is to uncover important structural principles underlying the assembly and stability of multi-domain protein assemblies by characterizing components of the IFNβ enhanceosome and the CtBP-mediated repression complex. Using a combination of biochemical and structural analyses, I showed that the transcriptional activator C-terminal binding protein 2 (CtBP2) is a tetramer by solving the 3.6Å cryoEM structure of CtBP2. I highlighted the types of interactions that stabilize the homo-tetramer and showed the relevance of the tetramer in CtBP2 transcriptional activity. Second, I used an integrative approach to investigate the structural features leading to activation of interferon regulator factor 3 (IRF3) and its interaction with DNA. Although this work mostly focused on components of the CtBP2-mediated complex and IFNβ enhanceosome, the principles described here can be applied to other complexes. Therefore, these studies provide an overall understanding on how other macromolecular complexes regulate gene transcription. Furthermore, our structural-based analyses will provide a basis for designing drugs that can regulate gene transcription in cancer and immunological disorders.

DRM is a conserved transcription factor complex that includes E2F/DP and pRB family proteins and plays important roles in the cell cycle and cancer. Recent work has unveiled a new aspect of DRM function in regulating genes involved in development and differentiation. These studies, however, were performed with cultured cells and a genome-wide study involving intact organisms undergoing active proliferation and differentiation was lacking. Our goal was to extend the knowledge of the role of DRM in gene regulation through development and in multiple tissues. To accomplish this, we employed genomic approaches to determine genome-wide targets of DRM using the nematode Caenorhabditis elegans as a model system. In this dissertation, I focus on the DRM component LIN-54 since it was proposed to exhibit DNA-binding activity. First, we confirmed the DNA-binding activity of C.elegans LIN-54 in vivo, and showed it is essential to recruit the DRM complex to its target genes. Next, chromatin immunoprecipitation and gene expression profiling revealed that LIN-54 controls transcription of genes implicated in cell division, development and reproduction. This work identified an interesting contrast in DRM function in soma vs. germline: DRM promotes transcription of germline-specific genes in the germline, but prevents their ectopic expression in the soma. Furthermore, we discovered a novel characteristic of DRM, sex chromosome-biased binding and function. We demonstrated that C. elegans DRM preferentially binds autosomes, yet regulates X-chromosome silencing by counteracting the H3K36 histone methyltransferase MES-4. By using genomics, cytology, and genetics, we defined DRM as an important player in the regulation of germline X-chromosome gene expression, and addressed molecular mechanisms vii behind the antagonistic interactions between DRM and MES-4. I present a model to explain the interplay of DRM and MES-4, and propose a novel function of DRM and MES-4 in maintaining proper chromosome gene expression dosage. This work extends our knowledge of the conserved roles of DRM in development, and provides a new view of differing DRM functions in soma versus germline. Furthermore, we defined a novel chromosome-specific aspect of DRM-mediated regulation.

Nonsense-mediated mRNA decay (NMD) specifically targets mRNAs with premature translation termination codons for rapid degradation. NMD is a highly conserved translation-dependent mRNA decay pathway, and its core Upf factors are thought to be recruited to prematurely terminating mRNP complexes, possibly through the release factors that orchestrate translation termination. Upf1 is the central regulator of NMD and recent studies have challenged the notion that this protein is specifically targeted to aberrant, nonsense-containing mRNAs. Rather, it has been proposed that Upf1 binds to most mRNAs in a translation-independent manner. In this thesis, I investigated the nature of Upf1 association with its substrates in the yeast Saccharomyces cerevisiae. Using biochemical and genetic approaches, the basis for Upf1 interaction with ribosomes was evaluated to determine the specificity of Upf1 association with ribosomes, and the extent to which such binding is dependent on prior association of Upf1’s interacting partners. I discovered that Upf1 is specifically associated with Rps26 of the 40S ribosomal subunit, and that this association requires the N-terminal Upf1 CH domain. In addition, using selective ribosome profiling, I investigated when during translation Upf1 associates with ribosomes and showed that Upf1 binding was not limited to polyribosomes that were engaged in translating NMD substrate mRNAs. Rather, Upf1 associated with translating ribosomes on most mRNAs, binding preferentially as ribosomes approached the 3’ ends of open reading frames. Collectively, these studies provide new mechanistic insights into NMD and the dynamics of Upf1 during translation.

Human Immunodeficiency Virus Type 1 (HIV-1) is the causative agent of Acquired Immunodeficiency Syndrome (AIDS), currently the leading cause of death from infectious diseases. Since HIV-1 co-opts the host cellular machinery, the study of cellular factors involved is a rational approach in discovering novel therapeutic targets for AIDS drug development. In this thesis, we present studies on two such proteins. APOBEC3G is from the family of cytidine deaminases known to keep endogenous retroviruses and retrotransposons at bay to maintain stability of the human genome. APOBEC3G targets Vif-deficient HIV-1 particles and renders them noninfectious, partially through deaminase-dependent hypermutation of the provirus during reverse transcription. APOBEC3G largely localizes in mRNA processing (P) bodies, cytoplasmic structures involved in RNA metabolism. Here we explore the significance of APOBEC3G localization in P bodies. We found that disrupting P bodies does not affect virion incorporation of endogenous APOBEC3G, implying that the APOBEC3G fraction in P bodies is not directly involved in the production of nascent, non-infectious particles. We also study UPF1, another host protein encapsidated by HIV-1. It is an essential protein mainly studied for its role in nonsense-mediated decay (NMD) pathway and belongs to the same helicase superfamily as MOV10, a recently identified antiviral factor. We found that UPF1 is incorporated in HIV-1 virions in a nucleocapsid-dependent manner and is required for single-cycle infectivity at an early, post-entry step of the viral life cycle. This novel function of UPF1 most likely does not involve NMD since depletion of UPF2 does not affect viral infectivity.

No description available.

No / During development, multipotent progenitor cells establish tissue-specific programs of gene expression. In this paper, we show that p63 transcription factor, a master regulator of epidermal morphogenesis, executes its function in part by directly regulating expression of the genome organizer Satb1 in progenitor cells. p63 binds to a proximal regulatory region of the Satb1 gene, and p63 ablation results in marked reduction in the Satb1 expression levels in the epidermis. Satb1(-/-) mice show impaired epidermal morphology. In Satb1-null epidermis, chromatin architecture of the epidermal differentiation complex locus containing genes associated with epidermal differentiation is altered primarily at its central domain, where Satb1 binding was confirmed by chromatin immunoprecipitation-on-chip analysis. Furthermore, genes within this domain fail to be properly activated upon terminal differentiation. Satb1 expression in p63(+/-) skin explants treated with p63 small interfering ribonucleic acid partially restored the epidermal phenotype of p63-deficient mice. These data provide a novel mechanism by which Satb1, a direct downstream target of p63, contributes in epidermal morphogenesis via establishing tissue-specific chromatin organization and gene expression in epidermal progenitor cells.

Thymic epithelial cells (TECs) are the main component of the thymic stroma, which supports T-cell proliferation and repertoire selection. Here, we demonstrate that Cbx4, a Polycomb protein that is highly expressed in the thymic epithelium, has an essential and non-redundant role in thymic organogenesis. Targeted disruption of Cbx4 causes severe hypoplasia of the fetal thymus as a result of reduced thymocyte proliferation. Cell-specific deletion of Cbx4 shows that the compromised thymopoiesis is rooted in a defective epithelial compartment. Cbx4-deficient TECs exhibit impaired proliferative capacity, and the limited thymic epithelial architecture quickly deteriorates in postnatal mutant mice, leading to an almost complete blockade of T-cell development shortly after birth and markedly reduced peripheral T-cell populations in adult mice. Furthermore, we show that Cbx4 physically interacts and functionally correlates with p63, which is a transcriptional regulator that is proposed to be important for the maintenance of the stemness of epithelial progenitors. Together, these data establish Cbx4 as a crucial regulator for the generation and maintenance of the thymic epithelium and, hence, for thymocyte development.

Les cancers, qu’il s’agisse de leucémies ou de tumeurs solides, sont le résultat de proliférations cellulaires anormales et non contrôlées au sein des tissus. Ces proliférations anarchiques sont le reflet d’une surexpression et/ou sur-activation de protéines intracellulaires engendrées par un événement oncogénique. Aujourd’hui encore il est donc nécessaire de trouver de nouvelles molécules à usage thérapeutique ciblant spécifiquement ces protéines. C’est dans ce contexte que les facteurs de transcription STAT5 constituent de véritables cibles de choix puisque ces protéines participent activement à la leucogénèse. L’implication directe des protéines STAT5 dans la génèse des leucémies a été démontrée par l’utilisation de formes mutées constitutivement active de STAT5. Les facteurs de transcription STAT5 jouent un rôle essentiel dans la voie de signalisation JAK/STAT. Cette voie aboutit à la régulation de grandes fonctions biologiques telles que la prolifération cellulaire, la différenciation cellulaire ou encore l’apoptose. L’objectif de ce projet consiste donc à cibler spécifiquement les protéines STAT5 dans le but de rétablir le processus de mort cellulaire et empêcher la prolifération des cellules cancéreuses. Les inhibiteurs spécifiques des protéines STAT5 sont sélectionnés selon la méthode SELEX qui permet d’isoler des ligands structurés de forte affinité pour la protéine. L’affinité et la spécificité de ces inhibiteurs, appelés aptamères, sont caractérisées à partir de modèles cellulaires de leucémies dépendant de l’activité des facteurs de transcription STAT5. Les aptamères sont aujourd’hui de véritables outils thérapeutiques en pleine évolution. / Leukemias are due to abnormal cell proliferation, which is the result of intracellular over-expression or excessive activation of protein due to oncogenic event. Still today, it is necessary to find new therapeutic molecules, which specifically target these proteins. STAT5, via the JAK/STAT signaling pathway, controls fundamental cellular processes, including .cell survival, proliferation and differentiation. To struggle against tumorigenesis, JAK/STAT signaling pathway has to be inhibited. The aim of this project is to target specifically STAT5 factors to restore healthy signal transduction. We generated aptamers by an iterative in vitro selection. Aptamers are short-structured single strand DNAs or RNAs that bind with high affinity and specificity to their target. Once STAT5B recombinant proteins are produced, they are subjected to SELEX process. The number of rounds depends on various parameters. After seven rounds, two sequences are retrieved. The specificity and affinity of these aptamers are assessed by fluorescent immunoassays. Binding affinity and kinetics of interaction are characterized by SPR. Aptamer anti proliferative effects are determined by evaluation of the growth of cells depending on STAT5. Finally, we developed several .assays aiming at understanding the mechanism of an aptamer action on STAT5B such as phosphorylation measurement and EMSA. Aptamers are now emerging therapeutic tools; they exhibit significant advantages relative to protein therapeutics.

No description available.

Dans l'introduction, diverses voies de trafic intracellulaire et endocytose seront discutées. Je familiarise le lecteur avec des protéines inactivant les ribosomes, en mettant l'accent sur la structure, l'endocytose, et le trafic intracellulaire de la toxine bactérienne Shiga toxin (STX). STx et la ricine suivent la voie rétrograde pour exercer leur effet toxique sur les cellules. Ils sont respectivement, une menace maladie infectieuse pour la santé humaine et des outils potentiels pour le bioterrorisme pour lequel aucun antidote n’existe actuellement. D'un criblage à haut débit, Retro-1 et Retro-2 avaient déjà été identifiés comme de puissants inhibiteurs de la voie rétrograde à l'interface des endosomes précoces-TGN, et Retro-2 a été démontré pour protéger les souris contre la ricine. Parmi les facteurs de trafic analysés, seule la protéine SNARE syntaxine-5 a été ré- localisée dans les cellules traitées avec Rétro - 2. / In the introduction, various endocytic and intracellular trafficking pathways will be discussed. I acquaint the reader with ribosome-inactivating proteins, with emphasis on the structure, endocytosis, and intracellular trafficking of the bacterial toxin Shiga toxin (STx). STx and ricin follow the retrograde route to exert their toxic effect on cells. They are respectively, an infectious disease threat to human health and potential tools for bioterrorism for which no antidote currently exists. From a high throughput screening, Retro-1 and Retro-2 had previously been identified as potent inhibitors of the retrograde route at the early endosomes-TGN interface, and Retro-2 was demonstrated to protect mice against ricin. Of the trafficking factors analyzed, only the SNARE protein syntaxin-5 was re-localized in Retro-2 treated cells. Yet, whether syntaxin-5 is the direct target of Retro-2 and whether its re-localization was directly responsible for retrograde transport inhibition remained to be established.