Le gène CFTR, impliqué lorsqu'il est muté dans la mucoviscidose, est finement régulé au niveau tissulaire (principalement exprimé dans les organes cibles de la mucoviscidose) et au cours du développement. Par exemple, dans les tissus pulmonaires, l'expression du gène CFTR est plus forte chez le fœtus que chez l'adulte (75:1), où seulement deux copies en moyenne par cellule sont détectées.L'objectif de ce travail était de déterminer les mécanismes moléculaires responsables de cette régulation. Nous avons identifié de nombreux motifs cis-régulateurs au niveau de la région promotrice et de la région 3'UTR. Nous avons également caractérisé des facteurs de transcription, dont certains présentent une spécificité tissulaire et développementale. C'est notamment le cas des protéines de la famille FOX, des régulateurs clés dans le développement du système reproducteur et pulmonaire. Cette étude a également permis d'identifier le rôle de certains microARNs dans la déstabilisation des transcrits CFTR. Finalement, nous proposons un rôle combiné de ces différents acteurs dans la régulation transcriptionnelle et post-transcriptionnelle du gène CFTR. L'identification des éléments répresseurs devrait fournir de nouvelles cibles thérapeutiques pour la mucoviscidose. / CFTR gene, involved in cystic fibrosis, displays a tightly regulated spatio-temporal pattern of expression (mainly expressed in taget tissues of cystic fibrosis). In lung, CFTR transcripts are abundant during fetal development compared to the adult stage (75:1), where only two copies per cell are detected. The aim of this work was to determine the molecular mechanisms involved in this regulation. We have identified several cis-regulatory motifs in the 5'UTR and the 3'UTR parts. We have characterized transcription factors with tissue- and temporal-specific activity. Members of FOX family are crucial regulators in reproductive duct and lung formation. We have also identified microRNAs in destabilizing CFTR transcripts. Finally, we propose a coupling role of trans-acting regulators in the transcriptional and post-transcriptional regulation of the CFTR gene. Characterizing the repressors would help to identify novel therapeutic tools in cystic fibrosis.

Le premier objectif a été de sélectionner des promoteurs de gènes cellulaires actifs spécifiquement dans les HCC à l’aide d’une recherche bibliographique puis en utilisant la base de donnée UniGene. Leur activité a été vérifiée par RT-qPCR et CHIP dans des lignées modèles HCC et dans des hépatocytes. Ces promoteurs ont été clonés en amont de la luciférase dans la région intergénique 20 du génome HSV-1 afin d’étudier leur force d’activité, 2 types de cinétiques et leur activité différentielle en fonction du type cellulaire et dans le contexte d’une infection virale. Le deuxième objectif a été de construire des virus oncolytiques ciblés pour l’expression de la protéine Us3, une protéine virale impliquée dans le contrôle de la réponse apoptotique induite par HSV-1. L’expression de la protéine Us3 est placée sous contrôle d’un promoteur cellulaire spécifique d’HCC. L’hypothèse est qu’en l’absence d’activité du promoteur cellulaire dans les cellules non HCC, la protéine Us3 ne sera pas synthétisée et, par conséquent, l’apoptose qui ne sera pas réprimée, inhibera le cycle de réplication et par conséquent, la production virale dans les cellules saines. Dans les cellules HCC, le promoteur actif permettra la réplication virale aboutissant à la destruction de lamasse tumorale. Un virus HSV-1 Us3- a été construit en utilisant la technique de recombinaison en plasmide BAC (Bacterial artificial chromosome), puis 2 virus oncolytiques en réintroduisant le gène Us3 sous contrôle du promoteur ANGPTL3 ou du promoteur HRE (hypoxia responsive element). Leur comportement oncolytique a été étudié en réalisant des courbes de croissance sur lignées cellulaires d’HCC et cellules hepatocyte-like. / Our long-term purpose is to develop transcriptionally targeted oncolytic vectors, derived from herpes simplex virus type 1 (HSV-1), designed to eradicate hepatocellular carcinomas (HCC). We have identified several HCC-specific promoters, as well as other cancer-specific promoters, that maintain their specificity when expressed from the virus genome. More precisely, we have demonstrated that these promoters are able to drive reporter gene (luciferase) expression from the virus genome in HCC-derived cells, both in cultured cells and in nude mice, but not in fresh human hepatocytes or in the WRL38 hepatocyte-like cells. HSV-1 infection induces, but then inhibits, a cellular antiviral apoptotic response, and the early virus protein US3 is a key actor in inhibiting apoptosis. We have hypothesized that inhibition of US3 expression in hepatocytes should led to early apoptotic death of these cells, therefore precluding virus multiplication and spread. In contrast, expression of US3 in cancer cells is expected to block apoptosis, leading to the achievement of the virus life cycle, cell lysis, and virus spread within the tumours. We report in this communication the construction and properties of two different potentially oncolytic HSV-1 vectors. One of them expresses US3 protein under the control of the HCC-specific promoter ANGPTL3, while the second promoter contains 9 repeats of the hypoxia responsive elements of vascular-endothelial growth factor (VEGF) (9xHRE promoter). Growth curves of these viruses were performed on different HCC cell lines to show their oncolytic properties.

A fim de contornar a problemática imposta pela recalcitrância da parede celular à hidrólise, o processo de produção de etanol a partir de biomassa vegetal requer um passo denominado de pré-tratamento, que consiste em tornar a biomassa mais acessível à ação de glicosil hidrolases que atacam a parede celular. O melhor conhecimento de processos de degradação de parede operados pela própria planta tem o potencial de direcionar as pesquisas em bioernegia, indicando quais os mecanismos mais eficientes para desmontar o complexo de polímeros da parede celular. Cunhado pré-tratamento biológico, este consiste na hidrólise de polissacarídeos estruturais empregando mecanismos e elementos chave de processos de degradação de parede celular que ocorrem na própria planta. Um exemplo de evento com esta característica é a formação de aerênquima lisígeno, que consiste na abertura de espaços de gás em tecidos parenquimáticos. A formação do aerênquima em cana-de-açúcar se dá de forma modular, sendo o processo constituído por seis etapas: 1) percepção do sinal inicial; 2) separação celular: 3) expansão celular; 4) morte celular programada; 5) hidrólise de hemiceluloses e 6) hidrólise de celulose. O presente trabalho teve como principal foco estudar a regulação das duas etapas iniciais, visando obter conhecimentos que possibilitem para tornar a biomassa de cana de açúcar menos resistente à penetração de enzimas. O aerênquima ocorre na raiz de cana-de-açúcar por um processo constitutivo. Sua independência de um indutor externo foi corroborada mediante tratamento com nutrientes e inibidor da percepção por etileno (1-MCP) pela análise dos cinco centímetros mais apicais da raiz. O atraso na formação do aerênquima após tratamento com nutrientes levou à expressão diferencial de genes relacionados à degradação de parede celular, morte celular programada e sinalização por etileno. Por outro lado, o 1-MCP não afetou visivelmente a formação do aerênquima, porém alterou o balanço hormonal na raiz. O padrão transcricional das raízes tratadas com 1-MCP revelou aumento da expressão de genes relacionados à expansão celular e estresse oxidativo. Tais padrões são discutidos à luz da regulação hormonal da formação do aerênquima através do estabelecimento de um balanço hormonal definido entre auxina e etileno. Ambos os experimentos levaram à seleção de quatro genes candidatos: dois fatores de transcrição (ScRAV1 e ScERF1) e duas glicosil hidrolases (ScEPG1 e ScARA1), sequenciados e analisados quanto à diversidade hom(e)óloga, sequências promotoras e estrutura gênica em comparação a S. bicolor. A topologia das reconstruções filogenéticas e a distribuição diferencial de sítios para RAV e ERF nos promotores de ScEPG1 e ScARA1 não parece refletir padrões de expressão distintos, visto que os diferentes hom(e)ólogos demonstraram ser igualmente expressos. Em sistema heterólogo, o fator de transcrição RAV apresentou atividade repressora sobre o promotor de ScEPG1. Tal papel de RAV sugere regulação negativa sobre a degradação da lamela média e sobre o processo de perda de adesão e separação celular. A identificação de reguladores-chave da formação do aerênquima consiste em importante elo entre a sinalização hormonal e a degradação de parede celular, possibilitando a melhor compreensão dos mecanismos de controle da degradação endógena de parede celular. Os dados produzidos possibilitam a aplicação biotecnológica dos genes sequenciados, além de consistirem em significativo avanço no conhecimento sobre a fisiologia do processo estudado e na dinâmica da regulação da expressão gênica acoplada a aspectos filogenéticos das sequências alvo. / In order to circumvent the problems imposed by the cell wall recalcitrance to hydrolysis, the process underlying biofuel production requires a step called pretreatment, aimed at making biomass more accessible to the action of glycosil hydrolases that attack the cell wall. The increased knowledge of wall degradation processes operated by the plant itself has the potential to influence research in the bioernergy field, pointing out the most efficient mechanisms to disassemble the cell wall complex polymeric structure. The term coined biological pretreatment means taking advantage of key elements and mechanisms of cell wall degradation processes occurring in the plant itself in order to disassemble the entangled polysaccharide structure. One example of endogenous cell wall degradation process is the lysigenous aerenchyma formation, which consists in the opening of gas spaces in parenchyma tissue. The aerenchyma formation in sugarcane is thought to be a modular process that occurs in six steps: 1) target cells perception; 2) cell separation; 3) cell expansion; 4) programmed cell death; 5) hemicellulose hydrolysis and 6) cellulose hydrolysis. This work has as the main objective to study the regulation of the two initial steps, aiming at acquiring knowledge that would afford to turn biomass less resistant to enzyme penetration. The aerenchyma develops within the roots of sugarcane as a constitutive process. Its independence from an external inducer was corroborated in this work by subjecting sugarcane plants to treatment with nutrients and one inhibitor of ethylene perception (1-MCP) when the five more apical centimeters of the root were analyzed. After treatment with nutrients, the delayed aerenchyma formation led to differential expression of cell wall degradation-, programmed cell death- and ethylene signalling-related genes. Under visual inspection, 1-MCP did not show effect on aerenchyma development. Instead, it changed the hormone balance mainly in the two most apical root segments. The transcriptional pattern of 1-MCP treated roots revealed increased expression of cell expansion- and oxidative stress-related genes. Such patterns are discussed in the light of the hormone regulation of the aerenchyma development through the establishment of a balance between auxin and ethylene within root segments. Both experiments led to the selection of four two candidate genes, two transcription factors (ScRAV1 and ScERF1) and two glycosil hydrolases (ScEPG1 and ScARA1), that were sequenced and analyzed regarding homEURologous diversity, promoter sequences and gene structure compared to S. bicolor. The topology of the phylogenetic reconstructions and the differential distribution of sites for RAV and ERF within ScEPG1 and ScARA1 promoters did not reflect distinct expression patterns. Different hom(e)ologous of each target gene were equally expressed. In an heterologous system, RAV transcription factor interacted with ScEPG1 promoter, reducing the activity encoded by the reporter gene. This suggests the repressive role of RAV on pectin degradation within the middle lamella, leading to reduced cell adhesion and cell separation, possibly regulated by this glycosyl hydrolase. The identification of key regulators of the aerenchyma formation is an important link between hormone signaling and the wall degradation within the sugarcane roots. It enables a better understanding of control mechanisms underlying wall modifications when performed by the plant itself. Altogether, the data produced in this work allows biotechnological application of sequenced genes. Moreover, the produced data unveil key aspects regarding physiologycal aspects of aerenchyma development and highlights features related to the dynamics of gene expression in sugarcane coupled to the phylogenetic aspects of the four target sequences.

Les cellules souches embryonnaires (cellules ES) constituent un excellent système modèle pour étudier les mécanismes épigénétiques contrôlant la transcription du génome mammifère. Un nombre important de membres de la famille des facteurs de remodelage de chromatine ATP-dépendants ont une fonction essentielle pour l’auto-renouvellement des cellules ES, ou au cours de la différentiation. On pense que ces facteurs exercent ces rôles essentiels en régulant l’accessibilité de la chromatine au niveau des éléments régulateurs de la transcription, en modulant la stabilité et le positionnement des nucléosome.Dans ce projet, nous avons conduit une étude génomique à grande échelle du rôle d’une dizaine des remodeleurs (Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Ep400, Brg1, Smarca3, Smarcad1, Smarca5, ATRX et Chd1l) dans les cellules ES. Une double stratégie expérimentale a été utilisée : Des expériences d’immunoprécipitation de la chromatine suivi par un séquençage à haute-débit (ChIP-seq) sur des cellules ES étiquetées pour les différents remodeleurs, pour étudier leur distribution sur le génome, et un approche transcriptomique sur des cellules déplétées de chaque remodeleur par traitement avec des vecteurs shRNA (knockdown). Nous avons établi les profils de liaison des remodeleurs sur des éléments régulateurs (promoteurs, enhancers et sites CTCF) sur le génome, et montré que ces facteurs occupent toutes les catégories d’éléments régulateurs du génome. La corrélation entre les données ChIP-seq et les données transcriptomiques nous a permis d’analyser le rôle des remodeleurs dans les réseaux de transcription essentiels des cellules ES. Nous avons notamment démontré l’importance particulière de certains remodeleurs comme Brg1, Chd4, Ep400 et Smarcad1 dans la régulation de la transcription chez les cellules ES. / The characteristics of embryonic stem cells (ES cells) make them one of the best models to study the epigenetic regulation exerted by different actors in order to control the transcription of the mammalian genome. Members of the Snf2 family of ATP-dependent chromatin remodeling factors were shown to be of specific importance for ES cell self-renewal and during differentiation. These factors are believed to play essential roles in modifying the chromatin landscape through their capacity to position nucleosomes and determine their occupancy throughout the genome, making the chromatin more or less accessible to DNA binding factors.In this project, a genome-wide analysis of the function of a number of ATP-dependent chromatin remodelers (Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1, Ep400, ATRX, Smarca3, Smarca5, Smarcad1 and Alc1) in mouse embryonic stem (ES) cells was conducted. This was done using a double experimental strategy. First, a ChIP-seq (Chromatin Immunoprecipitation followed by deep sequencing) strategy was done on ES cells tagged for each factor in the goal of revealing the genomic binding profiles of the remodeling factors. Second, loss-of-function studies followed by transcriptome analysis in ES cells were performed in order to understand the functional role of remodelers. Data from both studies were correlated to acquire a better understanding of the role of remodelers in the transcriptional network of ES cells. Specific binding profiles of remodelers on promoters, enhancers and CTCF binding sites were revealed by our study. Transcriptomic data analysis of the deregulated genes upon remodeler factor knockdown, revealed the essential role of Chd4, Ep400, Smarcad1 and Brg1 in the control of transcription of ES cell genes. Altogether, our data highlight how the distinct chromatin remodeling factors cooperate to control the ES cell state.

Le récepteur minéralocorticoïde (MR), activé par l’aldostérone, exerce de nombreuses fonctions pléïotropes, notamment au niveau rénal où il régule l’homéostasie hydrosodée. Des dysfonctionnements de la signalisation minéralocorticoïde sont impliqués dans des pathologies majeures chez l’Homme. Dans ce travail, nous avons identifié par ChIP sequencing le premier cistrome du MR dans une lignée cellulaire rénale humaine. La caractérisation des cibles génomiques a permis de décrire l’élément de réponse spécifique du MR, et de démontrer l’existence de deux modes d’action pour le MR : par liaison directe à l’ADN, ou indirecte via la liaison à d’autres facteurs de transcription. Le MR est physiologiquement confronté à une dualité face au récepteur glucocorticoïde (GR) avec lequel il partage un ligand, le cortisol, et des cibles génomiques, dont le gène PER1. Sur ce dernier, les deux récepteurs se distinguent par des recrutements dynamiques et cycliques différents, variants selon l’hormone, et contemporains de celui de partenaires transcriptionnels, régulant ainsi des effets à court ou à long-terme. Enfin, par ChIP en série et en tandem, nous avons montré que le MR et le GR agissent sous forme d’homodimères ou d’hétérodimères.L’identification du cistrome du MR, et la caractérisation de ses mécanismes d’action moléculaires, améliore notre compréhension de la physiopathologie de la signalisation minéralocorticoïde, et pourrait aboutir, notamment par le développement d’antagonistes sélectifs du MR comme la Finérénone, à de nouvelles stratégies thérapeutiques. / The mineralocorticoid receptor (MR), activated by aldosterone, exhibits numerous pleiotropic functions, most notably at the renal level where it regulates electrolytic homeostasis. Dysfunctions in the mineralocorticoid signaling pathway are involved in major diseases in Human. During this work, we have identified by ChIP sequencing the first MR cistrome in a human renal cell lineage. The characterization of the identified genomic targets allowed us to define a specific MR responsive element, and to demonstrate the existence of two transactivation processes for MR: through direct binding to DNA or through indirect interaction via binding to other transcription factors. MR is physiologically confronted with a duality with the glucocorticoid receptor (GR), since they share a common ligand, cortisol, and some of their genomic targets, whose PER1 gene. On the latter, MR and GR are distinguished by different dynamic and cyclical recruitment, varying according to hormone, and coordinated with the one of transcriptional partners, translating into the regulation of short-term and long-term effects. Finally, by serial and tandem ChIP experiment, we have demonstrated that MR and GR act as homodimer and as heterodimer.Identification of new MR genomic targets and characterization of its molecular mechanisms of action, improve our understanding of the pathophysiology of the mineralocorticoid signaling pathway. This could ultimately, notably through the development of selective MR antagonists like Finerenone, lead to new therapeutic strategies.

No description available.

IKAROS est un facteur de transcription majeur dans l’hématopoïèse qui agit en recrutant à la chromatine de nombreux partenaires décisifs dans le renouvellement cellulaire et l’engagement vers des lignages spécifiques. Il est notamment requis dans les cellules lymphoïdes pour réprimer les gènes cibles de la voie de signalisation NOTCH. IKAROS est aussi important dans le développement des cellules érythroïdes dans lesquelles il facilite le passage d’une globine fœtale à adulte chez l’embryon grâce au recrutement des complexes remodeleurs de la chromatine NuRD et BAF. En condition normale, la voie de signalisation NOTCH réprime la différenciation en cellules érythroïdes. Il est donc important que les gènes cibles de la voie NOTCH soient finement régulés afin d’amener une cellule progénitrice à se différencier en érythrocyte énucléé. Dans les cellules hématopoïétiques, incluant les cellules érythroïdes, IKAROS est un régulateur important du gène Hes1, cible effectrice majeure de la voie NOTCH. En effet, IKAROS participe activement à la répression du gène Hes1, permettant le développement des cellules érythroïdes. Nous avons donc émis l’hypothèse que dans ces cellules, IKAROS pourrait avoir une action plus généralisée sur le contrôle des gènes ciblés par NOTCH, comme observé dans les cellules lymphoïdes. Il pourrait ainsi agir en recrutant les complexes enzymatiques nécessaires à la régulation de ces gènes comme NuRD et BAF afin d’assurer le développement des cellules érythroïdes. Étant donné que la régulation des gènes est aussi dépendante du motif de méthylation de l’ADN, nous avons étendu notre questionnement à cet autre aspect de la régulation qu’IKAROS pourrait utiliser pour contrôler les gènes de la voie NOTCH. Pour ce faire, nous avons procédé à l’analyse bio-informatique d’un séquençage d’ARN de cellules érythroïdes murines préalablement réalisé au laboratoire afin d’en extraire les gènes régulés par IKAROS, mais aussi par NOTCH. L’analyse nous a permis d’extraire deux motifs d’expression intéressants observés dans les cellules érythroïdes pour lesquels IKAROS réprime ou active des gènes qui sont normalement réceptifs à l’activation de la voie NOTCH. Parmi les gènes réprimés par IKAROS en sont ressortis les gènes cibles de NOTCH Cdkn1a (P21WAF1/CIP1) et Trp53 (TP53), dont l’expression augmente fortement quand IKAROS est muté et que NOTCH est actif. Parmi les gènes activés par IKAROS en sont ressortis les gènes cibles de NOTCH Prdm16 et Nrarp, dont l’expression diminue fortement quand IKAROS est muté et que NOTCH est actif. IKAROS est donc un régulateur pouvant être répresseur, mais aussi activateur d’une multitude de gènes ciblés par NOTCH dans les cellules érythroïdes. Par des expériences d’immunoprécipitation de la chromatine, nous avons pu observer qu’IKAROS semblait toujours agir en partenariat avec le complexe NuRD et que la présence du complexe BAF était plutôt dépendante de la voie NOTCH. L’association IKAROS-NuRD semble servir de plateforme pour imposer un état de chromatine bivalente (avec co-présence de H3K4me3 et de H3K27me3) associée à une pause transcriptionnelle. Dans ce contexte, les éléments nécessaires à l’initiation de la transcription (présence de la marque H3K4me3) des gènes ciblés par NOTCH sont recrutés mais, l’élongation transcriptionnelle est affectée. L’état de chromatine bivalente peut être associé à l’activité des déméthylases de l’ADN Ten-Eleven-Translocation (TET) qui empêchent alors l’hyperméthylation de ces régions. Nos résultats démontrent qu’IKAROS peut utiliser la protéine TET1 pour réguler des gènes cibles de la voie NOTCH, en y formant l’hydroxyméthylcytosine (5-hmC). Celle-ci peut aussi marquer les régions de régulation génique caractérisées par une chromatine bivalente et une pause transcriptionnelle. Ces travaux décrivent IKAROS comme un facteur agissant de façons multiples dans la régulation des gènes cibles de NOTCH dans les cellules érythroïdes. Nous proposons qu’IKAROS et son partenaire NuRD soient requis pour mettre en place un état de chromatine bivalente et de pause transcriptionnelle pour faciliter l’activation physiologique des gènes cibles de NOTCH lors de la signalisation. IKAROS peut ainsi prendre part à l’activation ou la répression de gènes cibles de NOTCH, tout en facilitant la déméthylation de l’ADN ainsi que le recrutement d’éléments transcriptionnels qui favorisent un état de pause transcriptionnelle. NOTCH ainsi que d’autres éléments de régulation sont alors requis pour induire l’activation ou la répression des gènes cibles. / IKAROS is a critical transcription factor in hematopoiesis. It facilitates the chromatin binding of many important co-factors required for chromatin organization during cell renewal and lineage commitment. IKAROS is particularly important in lymphoid cells whereby it is involved in the repression of target genes of the NOTCH signaling pathway. IKAROS is also important in the development of other hematopoietic lineages, including the erythroid cells, in which it facilitates the passage of a fetal to adult globin in the embryo through the recruitment of the chromatin remodeling complexes NuRD and BAF. Under normal conditions, the NOTCH signaling pathway represses development of erythroid cells. It is therefore important to precisely understand how the NOTCH target genes are regulated during passage from hematopoietic progenitor to the enucleated circulating erythrocyte. IKAROS has been demonstrated to be an important regulator of Hes1 gene expression in hematopoietic cells of different lineages. Hes1 is the major effector target of the NOTCH pathway and IKAROS actively participates in its repression. In erythroid cells, the regulation of Hes1 imposed by IKAROS is required for terminal differentiation. We therefore investigated the importance of IKAROS in the regulation of NOTCH-targeted genes in erythroid cells. The combined effect of the mutation of IKAROS with NOTCH signaling was particularly investigated in these cells. To define how IKAROS influences the regulation of NOTCH target genes, we performed the bioinformatics analysis of a RNA-sequencing performed in murine erythroid cells activated or not for NOTCH signaling and whereby IKAROS is absent. We identified genes influenced by IKAROS expression and by NOTCH, and defined the effect of the combination of the absence of IKAROS expression and NOTCH pathway activation. Two particular expression patterns were identified and characterized the combined effect of the absence of IKAROS and NOTCH pathway activation in erythroid cells. Indeed, the absence of IKAROS either favors the overexpression of NOTCH target genes or prevents their response to NOTCH pathway activation. To understand how IKAROS could have an opposite effect on different NOTCH target genes we analysed the effect of IKAROS on their regulation. Among the genes repressed by IKAROS are the target genes of NOTCH Cdkn1a (encoding the P21WAF1/CIP1 protein) and Trp53 (encoding the TP53 protein), whose expression increases strongly when IKAROS is mutated and the NOTCH pathway is activated. Prdm16 and Nrarp are, instead, requiring IKAROS expression for their activation by NOTCH. The characterization of these NOTCH target genes suggests that IKAROS can work in partnership with the NuRD complex to influence the expression. The chromatin characterization of these genes led us to posit that the IKAROS-NuRD could act as a ‘platform’ to impose a bivalent chromatin organization associated with poised transcription. Then, the regulation imposed by IKAROS-NuRD would be required for the physiological activation of NOTCH targets upon external signaling. Finally, since in embryonic stem cells the Ten-Eleven Translocation (TET) enzymes are reported to be frequently associated to bivalent chromatin in order to prevent DNA hypermethylation, we assessed whether IKAROS could interact and ‘use’ TET enzymes to regulate NOTCH target genes. We determined that IKAROS can co-immunoprecipitate with the TET1 enzymes. We show that IKAROS influences both recruitment and activity of TET1 to different NOTCH target genes and favors the accumulation of hydroxymethylcytosine (5-hmC) to these genes. 5-hmC can be considered as a mark of transcriptional pausing/bivalence. Thus, these studies bring new information on the mechanism used by IKAROS to influence gene regulation in hematopoietic cells. Our results suggest that IKAROS primary function is to organize a bivalent chromatin and to promote transcriptional pausing to multiple NOTCH target genes. IKAROS is required to set the epigenetic and promoter organization for rapid activation upon NOTCH signaling.

Transcriptional regulation refers to the molecular systems that control the concentration of mRNA species within the cell. Variation in these controlling systems is not only responsible for many diseases, but also contributes to the vast phenotypic diversity in the biological world. There are powerful experimental approaches to probe these regulatory systems, and the focus of my doctoral research has been to develop and apply effective computational methods that exploit these rich data sets more completely. First, I present a method for mapping genetic regulators of gene expression (expression quantitative trait loci, or eQTL) using Random Forests. This approach allows for flexible modeling and feature selection, and results in eQTL that are more biologically supportable than those mapped with competing methods. Next, I present a method that finds interactions between genes that in turn regulate the expression of other genes. This is accomplished by finding recurring decision motifs in the forest structure that represent dependencies between genetic loci. Third, I present a method to use distributional differences in eQTL data to establish the regulatory roles of genes relative to other disease-associated genes. Using this method, we found that genes that are master regulators of other disease genes are more likely to be consistently associated with the disease in genetic association studies. Finally, I present a novel application of Random Forests to determine the mode of regulation of toxin-perturbed genes, using time-resolved gene expression. The results demonstrate a novel approach to supervised weighted clustering of gene expression data.

The development of methods and techniques is the driving force of scientific research. In this work, we described two large-scale screens in studying transcriptional activation and tumor suppression. In Part I, we studied transcriptional activation mechanisms by deriving and characterizing activation defective mutants. Promoter-specific transcriptional activators stimulate transcription through direct interactions with one or more components of the transcription machinery, termed the “target.” The identification of direct in vivo targets of activators has been a major challenge. We perform a large-scale genetic screen to derive and characterize tra1 alleles that are selectively defective for interaction with Gal4 in vivo. Utilizing these mutants, we demonstrated that Tra is an essential target for Gal4 activation, Gal4 and Tra1 bind cooperatively at the promoter and the Gal4–Tra1 interaction occurs predominantly on the promoter. In addition, we demonstrated that the Gal4-interaction site on Tra1 is highly selective. In Part II, we described a functional genomics approach to discover new tumor suppressor genes. A goal of contemporary cancer research is to identify the genes responsible for neoplastic transformation. Cells that are immortalized but non-tumorigenic were stably transduced with pools of short hairpin RNAs (shRNAs) and tested for their ability to form tumors in mice. ShRNAs in any resulting tumors were identified by sequencing to reveal candidate TSGs, which were then validated both experimentally and clinically by analysis of human tumor samples. Using this approach, we identified and validated 33 candidate TSGs. We found that most candidate TSGs were down-regulated in >70% of human lung squamous cell carcinoma (hLSCC) samples, and 17 candidate TSGs negatively regulate FGFR signalling pathway, and their ectopic expression inhibited growth of hLSCC xenografts. Furthermore, we suggest that by examining at the expression level of TSGs in lung cancer patients, we can predict their drug responsiveness to FGFR inhibitors. In conclusion, we have identified many new lung squamous cell cancer TSGs, using an experimental strategy that can be broadly applied to find TSGs in other tumor types.

Dbp2p, a member of the large family of DEAD-box proteins and a yeast homolog of human p68, was shown to interact with Upf1p, an essential component of the nonsense-mediated mRNA decay pathway. Dbp2p:Upf1p interaction occurs within a large conserved region in the middle of Upf1p that is largely distinct from its Nmd2p and Sup35/45p interaction domains. Deletion of DBP2, or point mutations within its highly conserved DEAD-box motifs, increased the abundance of nonsense-containing transcripts, leading us to conclude that Dbp2p also functions in the nonsense-mediated mRNA decay pathway. Dbp2p, like Upf1p, acts before or at decapping, is predominantly cytoplasmic, and associates with polyribosomes. Interestingly, Dbp2p also plays an important role in rRNA processing. In dbp2Δ cells, polyribosome profiles are deficient in free 60S subunits and the mature 25S rRNA is greatly reduced. The ribosome biogenesis phenotype, but not the mRNA decay function, of dbp2Δ cells can be complemented by the human p68 gene. We propose a unifying model in which Dbp2p affects both nonsense-mediated mRNA decay and rRNA processing by altering rRNA structure, allowing specific processing events in one instance and facilitating dissociation of the translation termination complex in the other.