African swine fever virus (ASFV) is a large double-stranded DNA virus which causes a lethal haemorrhagic fever in domestic pigs. This virus primarily infects cells from the monocyte/macrophage lineage and its ability to manipulate the function of these cells is key to the pathogenesis of this disease. ASFV encodes several proteins involved in immune evasion. One of these proteins, A238L, has been shown to inhibit host macrophage gene transcription. This protein has been shown to interact with several cellular proteins involved in signal transduction: a serine/threonine protein phosphatase, calcinerurin (CaN), the transcription factor NF-кB, and most recently the transcriptional co-activator CREB binding protein (CBP/P300). However its exact mechanism of action is not fully understood. Previous work has been limited to the investigation of individual signaling pathways and/or the expression of individual host genes. The aim of this study was to investigate the global effect of A238L on host macrophage gene transcription and also to carry out further investigation into the mechanism by which this protein functions. To determine the global effect of A238L on host macrophage gene transcription differential gene expression between porcine cells expressing A238L and control cells was examined using a porcine oligonucleotide microarray. These results demonstrated that A238L was a potent inhibitor of host macrophage gene expression. Functional characterisation of the annotated genes showed that a large proportion of A238L down-regulated genes are typically induced in response to cell stress. Significantly, genes regulated by the I kappa B kinase (IKK), mitogen-activated protein kinase (MAPK) and janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathways were all shown to be down regulated by A238L. Genes associated with the MAPK pathways were particularly enriched. The transcription of A238L-regulated genes is controlled by numerous different transcription factors, including NF-кB. All of the transcription factors identified interact with the transcription co-activator CBP/P300. This provides a common link between these factors, and indicates that A238L may target CBP/P300 to inhibit gene transcription. This observation supports recent work demonstrating that A238L interacts with and inhibits CBP/P300 function. To explore the potential mechanisms involved in the nuclear localisation of A238L, ASFV-infected Vero cells, expressing A238L under the control of its own promoter, were examined under a range of conditions using confocal microscopy. The results demonstrated that A238L was actively imported into the nucleus and exported by a CRM 1 mediated pathway, although a pool of A238L protein remained in the cytoplasm. Sequence analysis of A238L identified the presence of two putative nuclear localisation signals (NLS-1 and NLS-2). NLS-2 was located within A238L’s CaN docking motif. Mutation of these motifs indicated that both NLS-1 and NLS-2 are active and exhibit functional redundancy. Mutation of the CaN docking motif alone, in the presence of intact NLS-2, resulted in a dramatic increase in the nuclear localisation of A238L. These results are consistent with a model in which A238L functions within both the nucleus and the cytoplasm and suggest that binding of CaN to A238L masks NLS-2, contributing to the cytoplasmic retention of A238L.

Forkhead box Class O (FOXO), one subfamily of the Forkhead box (Fox) family, which is featured by the Forkhead (FH) DNA-binding domain, includes four human transcription factors: FOXO1, FOXO3a, FOXO4, and FOXO6. The tumor suppressor FOXO3a is involved in multiple physiological and pathological processes, such as breast cancer and acute myeloid leukemia, and is related to human longevity. It plays essential role in metabolism, cell cycle arrest, DNA repair, and apoptosis. Besides the FH domain, FOXO3a contains three other regions (CR1-3), conserved within FOXO subfamily. It specifically binds a consensus Forkhead response element (FRE) DNA sequence through the FH domain, and recruits transcriptional coactivator CBP/p300 to activate gene transcription. FOXO3a functions through interacting with other proteins as well. FOXO3a binds p53 through the FH domain and the CR3 region, which are also engaged in an intramolecular interaction, and the solution structure of the former one was determined. This intramolecular interaction regulates coactivator recruitment and is disrupted by FRE DNA. A novel transactivation domain (TAD) CR2C was identified in addition to the known TAD CR3, both of which promiscuously associate with the KIX domain of CBP/p300 in equilibrium between two conformational states, the structures of which were determined by NMR spectroscopy. These two TADs of FOXO3a form additional multivalent binding to the TAZ1 and TAZ2 domains of CBP/p300, further increasing the promiscuity and complexity of the interaction. The coactivator recruitment is modulated by AMPK phosphorylation, which enhances the multivalent interaction between FOXO3a and CBP/p300, and thus the transactivation. These results indicate the significance of intrinsically disordered regions (IDRs) of FOXO3a in transcriptional activation and protein interaction, provide insight of the role of FOXO3a in gene transcription and apoptosis under various conditions, and potentially contribute to the cancer therapy.

Forkhead box Class O (FOXO), one subfamily of the Forkhead box (Fox) family, which is featured by the Forkhead (FH) DNA-binding domain, includes four human transcription factors: FOXO1, FOXO3a, FOXO4, and FOXO6. The tumor suppressor FOXO3a is involved in multiple physiological and pathological processes, such as breast cancer and acute myeloid leukemia, and is related to human longevity. It plays essential role in metabolism, cell cycle arrest, DNA repair, and apoptosis. Besides the FH domain, FOXO3a contains three other regions (CR1-3), conserved within FOXO subfamily. It specifically binds a consensus Forkhead response element (FRE) DNA sequence through the FH domain, and recruits transcriptional coactivator CBP/p300 to activate gene transcription. FOXO3a functions through interacting with other proteins as well. FOXO3a binds p53 through the FH domain and the CR3 region, which are also engaged in an intramolecular interaction, and the solution structure of the former one was determined. This intramolecular interaction regulates coactivator recruitment and is disrupted by FRE DNA. A novel transactivation domain (TAD) CR2C was identified in addition to the known TAD CR3, both of which promiscuously associate with the KIX domain of CBP/p300 in equilibrium between two conformational states, the structures of which were determined by NMR spectroscopy. These two TADs of FOXO3a form additional multivalent binding to the TAZ1 and TAZ2 domains of CBP/p300, further increasing the promiscuity and complexity of the interaction. The coactivator recruitment is modulated by AMPK phosphorylation, which enhances the multivalent interaction between FOXO3a and CBP/p300, and thus the transactivation. These results indicate the significance of intrinsically disordered regions (IDRs) of FOXO3a in transcriptional activation and protein interaction, provide insight of the role of FOXO3a in gene transcription and apoptosis under various conditions, and potentially contribute to the cancer therapy.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Il apparait de nos jours évident que les cancers ne peuvent se réduire uniquement à des aberrations génétiques, et qu'un nouveau paramètre est à prendre en considération, l'épigénétique. Au cours de ma thèse je me suis efforcé de caractériser la protéine fusion BRD4-NUT. Cette protéine résulte d'une translocation t(15;19) observée dans les carcinomes de la ligne médiane (NMC), extrêmement agressifs et létaux. La protéine BRD4 possède un double bromodomaine capable de s'associer à la chromatine acétylée, et recrute divers facteurs sur la chromatine. NUT est une protéine de fonction inconnue exprimée exclusivement au cours de la spermatogenèse. J'ai pu démontrer que la protéine fusion BRD4-NUT était suffisante pour induire la tumorigenèse, par un mécanisme de séquestration de la proteine histone acétyltransférase (HAT) CBP/p300. NUT interagit avec CBP/p300 et suractive son activité d'acétylation, créant des foci hyperacétylés de chromatine. BRD4-NUT empêche ainsi CBP/p300 d'aller co-activer la transcription de nombreux gènes, et bloque notamment la réponse apoptotique p53-dépendante. Une inhibition de BRD4-NUT – par siRNA, mutation des bromodomaines ou dérégulation de l'acétylation des foci par des inhibiteurs des histones déacétylases (HDAC) – réenclenche la voie d'apoptose et la mort de ces cellules tumorales. Cette étude est un exemple précis de l'impact qu'une dérégulation épigénétique peut avoir sur l'homéostasie cellulaire et son mécanisme d'induction de la tumorigenèse. Je me suis également interessé à caracteriser la protéine NUT lors de son contexte physiologique, la spermatogenèse, ou lors de son expression illégitime dans des lignées tumorales sans fusion avec BRD4. La protéine NUT est exprimée au niveau des stades de maturation des cellules germinales spermatides, et pourrait participer au remodelage du génome et à l'établissement de l'épigénome final du spermatozoïde. Nut semble également conférer un avantage prolifératif lors de son expression anormale dans au moins trois lignées cellulaires, U2OS, A549 et A7R5. Ainsi, la protéine NUT, seule ou fusionnée avec BRD4, est un facteur Cancer/Testiculaire capable d'influer négativement sur l'homéostasie des cellules somatiques dans lesquelles ses fonctions, normalement restreintes à la spermatogenèse, participent à la tumorigenèse.

During cell specification, temporal and spatially restricted gene expression programs are set up, forming different cell types and ultimately a multicellular organism. In this thesis, we have studied the molecular mechanisms by which sequence specific transcription factors and coactivators regulate RNA polymerase II (Pol II) transcription to establish specific gene expression programs and what epigenetic patterns that follows. We found that the transcription factor Dorsal is responsible for establishing discrete epigenetic patterns in the presumptive mesoderm, neuroectoderm and dorsal ectoderm, during early Drosophila embryo development. In addition, these different chromatin states can be linked to distinct modes of Pol II regulation. Our results provide novel insights into how gene regulatory networks form an epigenetic landscape and how their coordinated actions specify cell identity. CBP/p300 is a widely used co-activator and histone acetyltransferase (HAT) involved in transcriptional activation. We discovered that CBP occupies the genome preferentially together with Dorsal, and has a specific role during development in coordinating the dorsal-ventral axis of the Drosophila embryo. While CBP generally correlates with gene activation we also found CBP in H3K27me3 repressed chromatin. Previous studies have shown that CBP has an important role at transcriptional enhancers. We provide evidence that the regulatory role of CBP does not stop at enhancers, but is extended to many genomic regions. CBP binds to insulators and regulates their activity by acetylating histones to prevent spreading of H3K27me3. We further discovered that CBP has a direct regulatory role at promoters. Using a highly potent CBP inhibitor in combination with ChIP and PRO-seq we found that CBP regulates promoter proximal pausing of Pol II. CBP promotes Pol II recruitment to promoters via a direct interaction with TFIIB, and promotes transcriptional elongation by acetylating the first nucleosome. CBP is regulating Pol II activity of nearly all expressed genes, however, either recruitment or release of Pol II is the rate-limiting step affected by CBP. Taken together, these results reveal mechanistic insights into cell specification and transcriptional control during development. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p><p> </p>

La signalisation par l’estrogène a longtemps été considérée comme jouant un rôle critique dans le développement et la progression des cancers hormono-dépendants tel que le cancer du sein. Deux tiers des cancers du sein expriment le récepteur des estrogènes (ER) qui constitue un élément indiscutable dans cette pathologie. L’acquisition d’une résistance endocrinienne est cependant un obstacle majeur au traitement de cette forme de cancer. L’émergence de cancers hormono-indépendants peut est produite par l’activation de ER en absence d’estrogène, l’hypersensibilité du récepteur aux faibles concentrations plasmique d’estrogène ainsi que l’activation de ER par des modulateurs sélectifs. L’activité du ER est fortement influencée par l’environnement cellulaire tel que l’activation de voie de signalisation des facteurs de croissances, la disponibilité de protéines co-régulatrices et des séquences promotrices ciblées. Présentement, les études ont principalement considérées le rôle de ERα, cependant avec la découverte de ERβ, notre compréhension de la diversité des mécanismes potentiels impliquant des réponses ER-dépendantes s’est améliorée. L’activation des voies des kinases par les facteurs de croissance entraîne le développement d’un phénotype tumoral résistant aux traitements actuels. Nos connaissances des voies impliquées dans l’activation de ER sont restreintes. ERα est considéré comme le sous-type dominant et corrèle avec la plupart des facteurs de pronostic dans le cancer du sein. Le rôle de ERβ reste imprécis. Les résultats présentés dans cette thèse ont pour objectif de mieux comprendre l’implication de ERβ dans la prolifération cellulaire par l’étude du comportement de ERβ et ERα suite à l’activation des voies de signalisation par les facteurs de croissance. Nous démontrons que l’activation des récepteurs de surfaces de la famille ErbB, spécifiquement ErbB2/ErbB3, inhibe l’activité transcriptionnelle de ERβ, malgré la présence du coactivateur CBP, tout en activant ERα. De plus, l’inhibition de ERβ est attribuée à un résidu sérine (Ser-255) situé dans la région charnière, absente dans ERα. Des études supplémentaires de ErbB2/ErbB3 ont révélé qu’ils activent la voie PI3K/Akt ciblant à son tour la Ser-255. En effet, cette phosphorylation de ERβ par PI3K/Akt induit une augmentation de l’ubiquitination du récepteur qui promeut sa dégradation par le système ubiquitine-protéasome. Cette dégradation est spécifique pour ERβ. De façon intéressante, la dégradation par le protéasome requiert la présence du coactivateur CBP normalement requis pour l’activité transcriptionnelle des récepteurs nucléaires. Malgré le fait que l’activation de la voie PI3K/Akt corrèle avec une diminution de l’expression des gènes sous le contrôle de ERβ, on observe une augmentation de la prolifération des cellules cancéreuses. L’inhibition de la dégradation de ERβ réduit cette prolifération excessive causée par le traitement avec Hrgβ1, un ligand de ErbB3. Un nombre croissant d’évidences indique que les voies de signalisations des facteurs de croissance peuvent sélectivement réguler l’activité transcriptionnelle de sous-types de ER. De plus, le ratio ERα/ERβ dans les cancers du sein devient un outil de diagnostique populaire afin de déterminer la sévérité d’une tumeur. En conclusion, la caractérisation moléculaire du couplage entre la signalisation des facteurs de croissance et la fonction des ERs permettra le développement de nouveaux traitements afin de limiter l’apparition de cellules tumorales résistantes aux thérapies endocriniennes actuelles. / It has long been appreciated that estrogenic signaling plays a critical role in the development of hormone-dependent cancers such as breast cancer. Two-thirds of breast cancers express estrogen receptor (ER) which has been demonstrated to play an irrefutable role in tumour development and progression. However the acquisition of endocrine resistance has become a major obstacle in the treatment of hormone-dependent cancers that have acquired a hormone-independent state. Hormone-independent cancers emerge from an array of pathways involving ER activation in the absence of estrogen, hypersensitivity of ER to low serum levels of estrogen and activation by estrogen antagonists. The activity of ER is critically influenced by the cellular environment such as growth factor signaling pathways, availability of coregulatory proteins and the promoter sequence of target genes. The mechanisms studied have mostly considered the role of ERα, however with the discovery of the second subtype, ERβ, the understanding on the diversity of potential mechanisms involving ER-dependent responses have improved. Hormonal-independent activation of ER can occur in estrogen-dependent breast tumours, with concomitant rise in kinase signaling pathways, resulting in the acquisition of a therapeutic resistant phenotype in treated women. Our knowledge is relatively limited on which pathways trigger ER signaling and how these phosphorylation-coupled events affect ER activity. ERα is considered the dominant subtype and correlates with most of the prognostic factors in breast cancers. Conversely the role of ERβ remains unclear. The results presented in this thesis were carried out with the objective of gaining a better understanding of ERβ’s role in cellular proliferation by examining the behavior of ERβ and ERα during the activation of growth factor signaling pathways by cell-surface receptor-tyrosine kinases. We demonstrate here that the activation of cell surface receptors of the ErbB family, specifically ErbB2/ErbB3, inhibits the transcriptional activity of ERβ despite the presence of the coactivator CBP, yet activated ERα. Furthermore the inhibition of ERβ was attributed to a specific serine residue located within the hinge region, not present in ERα. Additional studies of ErbB2/ErbB3-initiated signaling revealed that it triggered the activation of the PI3K/Akt pathway which targeted the serine residue within the hinge region of ERβ. In fact, phosphorylation of ERβ by the PI3K/Akt pathway led to an increase in receptor ubiquitination which promoted its degradation by the ubiquitin-proteasome system which was subtype specific. Interestingly, proteasomal degradation required the presence of the coactivator CBP, which is normally involved in assisting nuclear receptor transcriptional activity. Although the activation of the PI3K/Akt pathway correlated with a decrease in the expression of ERβ target genes it led to an increase in the proliferation of breast cancer cells. Inhibiting the degradation of ERβ reduced the enhanced proliferation of breast cancer cells brought about by the treatment of ErbB3’s ligand, Hrgβ1. Increasing evidence indicates that growth factor signaling pathways can selectively regulate the transcriptional activity of ER subtypes, and the ratio of ERα/ERβ expression in breast tumours is becoming a popular prognostic factor to evaluate the severity of the tumour. Therefore the molecular characterization of the coupling between growth factor signaling and ER function should provide improved therapeutical approaches to overcome or delay the onset of resistance to endocrine therapy in hormone-dependent cancers.

La signalisation par l’estrogène a longtemps été considérée comme jouant un rôle critique dans le développement et la progression des cancers hormono-dépendants tel que le cancer du sein. Deux tiers des cancers du sein expriment le récepteur des estrogènes (ER) qui constitue un élément indiscutable dans cette pathologie. L’acquisition d’une résistance endocrinienne est cependant un obstacle majeur au traitement de cette forme de cancer. L’émergence de cancers hormono-indépendants peut est produite par l’activation de ER en absence d’estrogène, l’hypersensibilité du récepteur aux faibles concentrations plasmique d’estrogène ainsi que l’activation de ER par des modulateurs sélectifs. L’activité du ER est fortement influencée par l’environnement cellulaire tel que l’activation de voie de signalisation des facteurs de croissances, la disponibilité de protéines co-régulatrices et des séquences promotrices ciblées. Présentement, les études ont principalement considérées le rôle de ERα, cependant avec la découverte de ERβ, notre compréhension de la diversité des mécanismes potentiels impliquant des réponses ER-dépendantes s’est améliorée. L’activation des voies des kinases par les facteurs de croissance entraîne le développement d’un phénotype tumoral résistant aux traitements actuels. Nos connaissances des voies impliquées dans l’activation de ER sont restreintes. ERα est considéré comme le sous-type dominant et corrèle avec la plupart des facteurs de pronostic dans le cancer du sein. Le rôle de ERβ reste imprécis. Les résultats présentés dans cette thèse ont pour objectif de mieux comprendre l’implication de ERβ dans la prolifération cellulaire par l’étude du comportement de ERβ et ERα suite à l’activation des voies de signalisation par les facteurs de croissance. Nous démontrons que l’activation des récepteurs de surfaces de la famille ErbB, spécifiquement ErbB2/ErbB3, inhibe l’activité transcriptionnelle de ERβ, malgré la présence du coactivateur CBP, tout en activant ERα. De plus, l’inhibition de ERβ est attribuée à un résidu sérine (Ser-255) situé dans la région charnière, absente dans ERα. Des études supplémentaires de ErbB2/ErbB3 ont révélé qu’ils activent la voie PI3K/Akt ciblant à son tour la Ser-255. En effet, cette phosphorylation de ERβ par PI3K/Akt induit une augmentation de l’ubiquitination du récepteur qui promeut sa dégradation par le système ubiquitine-protéasome. Cette dégradation est spécifique pour ERβ. De façon intéressante, la dégradation par le protéasome requiert la présence du coactivateur CBP normalement requis pour l’activité transcriptionnelle des récepteurs nucléaires. Malgré le fait que l’activation de la voie PI3K/Akt corrèle avec une diminution de l’expression des gènes sous le contrôle de ERβ, on observe une augmentation de la prolifération des cellules cancéreuses. L’inhibition de la dégradation de ERβ réduit cette prolifération excessive causée par le traitement avec Hrgβ1, un ligand de ErbB3. Un nombre croissant d’évidences indique que les voies de signalisations des facteurs de croissance peuvent sélectivement réguler l’activité transcriptionnelle de sous-types de ER. De plus, le ratio ERα/ERβ dans les cancers du sein devient un outil de diagnostique populaire afin de déterminer la sévérité d’une tumeur. En conclusion, la caractérisation moléculaire du couplage entre la signalisation des facteurs de croissance et la fonction des ERs permettra le développement de nouveaux traitements afin de limiter l’apparition de cellules tumorales résistantes aux thérapies endocriniennes actuelles. / It has long been appreciated that estrogenic signaling plays a critical role in the development of hormone-dependent cancers such as breast cancer. Two-thirds of breast cancers express estrogen receptor (ER) which has been demonstrated to play an irrefutable role in tumour development and progression. However the acquisition of endocrine resistance has become a major obstacle in the treatment of hormone-dependent cancers that have acquired a hormone-independent state. Hormone-independent cancers emerge from an array of pathways involving ER activation in the absence of estrogen, hypersensitivity of ER to low serum levels of estrogen and activation by estrogen antagonists. The activity of ER is critically influenced by the cellular environment such as growth factor signaling pathways, availability of coregulatory proteins and the promoter sequence of target genes. The mechanisms studied have mostly considered the role of ERα, however with the discovery of the second subtype, ERβ, the understanding on the diversity of potential mechanisms involving ER-dependent responses have improved. Hormonal-independent activation of ER can occur in estrogen-dependent breast tumours, with concomitant rise in kinase signaling pathways, resulting in the acquisition of a therapeutic resistant phenotype in treated women. Our knowledge is relatively limited on which pathways trigger ER signaling and how these phosphorylation-coupled events affect ER activity. ERα is considered the dominant subtype and correlates with most of the prognostic factors in breast cancers. Conversely the role of ERβ remains unclear. The results presented in this thesis were carried out with the objective of gaining a better understanding of ERβ’s role in cellular proliferation by examining the behavior of ERβ and ERα during the activation of growth factor signaling pathways by cell-surface receptor-tyrosine kinases. We demonstrate here that the activation of cell surface receptors of the ErbB family, specifically ErbB2/ErbB3, inhibits the transcriptional activity of ERβ despite the presence of the coactivator CBP, yet activated ERα. Furthermore the inhibition of ERβ was attributed to a specific serine residue located within the hinge region, not present in ERα. Additional studies of ErbB2/ErbB3-initiated signaling revealed that it triggered the activation of the PI3K/Akt pathway which targeted the serine residue within the hinge region of ERβ. In fact, phosphorylation of ERβ by the PI3K/Akt pathway led to an increase in receptor ubiquitination which promoted its degradation by the ubiquitin-proteasome system which was subtype specific. Interestingly, proteasomal degradation required the presence of the coactivator CBP, which is normally involved in assisting nuclear receptor transcriptional activity. Although the activation of the PI3K/Akt pathway correlated with a decrease in the expression of ERβ target genes it led to an increase in the proliferation of breast cancer cells. Inhibiting the degradation of ERβ reduced the enhanced proliferation of breast cancer cells brought about by the treatment of ErbB3’s ligand, Hrgβ1. Increasing evidence indicates that growth factor signaling pathways can selectively regulate the transcriptional activity of ER subtypes, and the ratio of ERα/ERβ expression in breast tumours is becoming a popular prognostic factor to evaluate the severity of the tumour. Therefore the molecular characterization of the coupling between growth factor signaling and ER function should provide improved therapeutical approaches to overcome or delay the onset of resistance to endocrine therapy in hormone-dependent cancers.