Steroid hormones and receptors play a role in regulating biological events underlying brain development and sexual differentiation. Current evidence indicates that circulating sex steroid hormones are not entirely responsible for development of neural sex differences in song birds such as the zebra finch. p68, as a coactivator specific for estrogen receptor alpha (ERα) and an essential factor in early tissue development and maturation might play a role in sexual differentiation. Zebra finches have a sexually dimorphic song control nuclei in the brain, males have larger song nuclei than females, and are ideal model for investigating the mechanisms controlling sexual differentiation of the brain and behavior. Western blot analysis showed a significant sex difference at post hatch day 10 (P10). Immunohistochemistry showed localization of p68 immunoreactive cells in the ZF brain including nuclei that compose the avian song system. p68 is probably developmentally regulated and may be modulated by endogenous estrogen and estrogen receptors suggesting a role for p68 in sexual differentiation. INDEX WORDS: p68, coactivator, RNA helicase, steroid receptor, song control nuclei, zebra finch (ZF)

Les "protéine arginine méthyltransférases" (PRMT) sont impliquées dans de nombreux processus cellulaires : transcription, maturation et transport des ARN, traduction, transduction du signal, réplication et réparation de l'ADN, et apoptose. Différents travaux ont montré que des dérégulations de ces mécanismes impliquant les PRMT peuvent induire certains cancers, faisant de ces enzymes de nouvelles cibles potentielles en chimiothérapie. Il s’avère donc crucial de comprendre le mode d’action des PRMT à l’échelle atomique, à la fois au niveau fondamental et pour le développement de nouveaux médicaments. Les travaux décrits ici s’intéressent à la protéine PRMT4/CARM1 et s’appuient sur des études structurales par bio-cristallographie, pour comprendre les mécanismes de la réaction de méthylation catalysée par CARM1 et découvrir des inhibiteurs spécifiques, mais aussi sur des études en solution, pour caractériser l’interaction entre CARM1 et ses substrats. / Protein arginine methyltransferases (PRMTs) are involved in several cellular mechanisms: transcription, RNA maturation and transport, translation, signal transduction, DNA replication and repair, and apoptosis. Different studies showed that deregulation of those mechanisms involving PRMTs can induce some cancers, making these enzymes new potential targets for chemotherapy. It is therefore crucial to understand the mode of action of PRMTs at the atomic scale, both at the fundamental level and for the development of new drugs. The studies described here focus on PRMT4/CARM1 and rely on structural studies by bio-crystallography, in order to understand the methylation mechanisms catalyzed by CARM1 and to discover specific inhibitors, but also on in vitro studies, to characterize the interaction between CARM1 and its substrates.

E6-associated protein (E6-AP), which was originally identified as an ubiquitin-protein ligase, also functions as a co-activator that enhances the hormone-dependent transactivation of estrogen (ER) and progesterone (PR) receptors. To investigate the in vivo role of E6-AP in mammary gland development, we generated transgenic mouse lines that specifically overexpress either wild-type human E6-AP (E6-APWT) or the ubiquitin-protein ligase defective mutant E6-AP (E6-APC833S) in the mammary gland. Here we show that overexpression of E6-APWT results in impaired mammary gland development. In contrast, overexpression of E6-APC833S or loss of E6-AP (E6-APKO) increases lateral branching and alveolus-like protuberances in the mammary gland. We also show that the mammary phenotypes observed in the E6-AP transgenic and knockout mice are in large part due to the alteration of PR-B protein levels. RNAi-mediated knockdown of E6-AP in T47D breast cancer cells increased PR-B protein levels and stability. In vitro ubiquitination assay using purified E6-AP and PR-B reinforce these conclusions and demonstrate that E6-AP promotes PR-B turnover in an ubiquitin-dependent manner. Furthermore, we also show that E6-AP regulates progesterone-induced Wnt-4 expression by modulating the steady state level of PR-B in both mice and in human breast cancer cells. This novel mechanism appears to regulate normal physiology of the mammary gland and its dysregulation may prove to contribute importantly to mammary cancer development and progression. To test this hypothesis, we examined the E6-AP transgenic mice for tumor formation over a period of 6, 9, 12, 18 and 24 months. Our data shows that, unlike the E6-APWT mice that show normal phenotype, the E6-APC833S mice develop mammary hyperplasia at high penetrance (80%); with a median latency of 18 months. Our findings indicate that the inactivation of the E3-ligase function of E6-AP is sufficient to initiate the process of mammary tumor development. These findings strongly suggest that E6-AP may act as a tumor suppressor by down regulating the ER-alpha, PR-B and thereby their signaling pathways.

No description available.

No description available.

La régulation de l’expression des gènes joue un rôle fondamental dans la réponse et l’adaptation des cellules à leur environnement. L'expression des gènes peut être régulée à plusieurs étapes distinctes, mais un niveau de contrôle critique est l’initiation de la transcription. Celle-ci implique le recrutement séquentiel de nombreux régulateurs différents, dont les complexes co-activateurs. De nombreuses études ont démontré et caractérisé leurs fonctions dans la transcription. Cependant, il est moins bien compris comment les co-activateurs sont directement régulés par les conditions environnementales. Des travaux précédents de mon laboratoire de thèse ont montré, dans la levure fissipare Schizosaccharomyces pombe, que le complexe co-activateur SAGA contrôle l’expression des gènes en réponse aux nutriments et contribue ainsi à l’équilibre entre la prolifération cellulaire et la différenciation sexuelle. L’objectif de mon travail de thèse a été de comprendre comment le complexe SAGA répond à la disponibilité en nutriments et régule l’expression des gènes de différenciation. Pour cela, j’ai combiné des approches de génétique, de biochimie et de protéomique quantitative. Des analyses d’interactions génétiques m’ont permis de montrer que SAGA, par l’intermédiaire de sa sous-unité acétyltransférase Gcn5, contrôle l’équilibre entre prolifération et différenciation en aval des voies de signalisation TORC1 et TORC2. Puis, des études biochimiques ont établi que les voies de signalisation TORC1 et TORC2 contrôlent SAGA via la phosphorylation différentielle d’une sous-unité architecturale du complexe, nommée Taf12. En effet, lorsque les nutriments sont présents, TORC1 active la phosphatase PP2A, via la kinase Greatwall, pour déphosphoryler Taf12. Au contraire, la carence en nutriments active la voie de signalisation TORC2-AKT, qui permet la phosphorylation de Taf12, afin de moduler l’intensité de la réponse de différenciation. Nous avons également identifié d’autres sous-unités de SAGA qui sont différentiellement phosphorylées en fonction du niveau en nutriments et qui pourraient donc également contribuer à la régulation de SAGA. Notamment, nous avons observé que les sous-unités Ada3 et Sgf29, impliquées dans la régulation de l’activité de Gcn5, sont également phosphorylées dans les conditions carencées en nutriments. Enfin, j’ai observé que TORC2 et Gcn5 contrôlent la transition G2/M de façon synergique, suggérant que SAGA et les voies de signalisation des kinases TOR interagissent fonctionnellement dans le contrôle d’autres processus. Mon travail révèle que SAGA est une cible directe des voies de signalisation qui détectent les nutriments et établit un nouveau mécanisme par lequel TORC1 et TORC2 convergent pour contrôler l’expression génique et le destin cellulaire / The regulation of gene expression plays a fundamental role in the ability of cells to respond to external changes. One critical level of regulation is transcription, which is controlled by large complexes with many distinct activities. Little is known about how these activities integrate developmental or environmental signals to regulate transcription. We are using S. pombe as a model system to address this issue, in the context of cell fate control by nutrient availability. Previous work in the lab has established that, in this yeast, the SAGA co-activator complex controls whether cells proliferate or not in response to nutrients. Following up on these observations, we determined which nutrient-sensing signaling pathways regulate SAGA activities. A comprehensive genetic approach demonstrated that SAGA functions downstream of the TOR kinase-containing complexes, TORC1 and TORC2. In parallel, quantitative mass spectrometry analysis of the SAGA complex revealed that the Taf12 subunit is differentially phosphorylated, depending on nutrient levels. In agreement with our genetic analyses, Taf12 phosphorylation depends on the PP2A phosphatase, which we found is activated by TORC1 when nutrients are present. Conversely, upon nutrient starvation, TORC2 is activated allowing the AKT kinase to phosphorylate Taf12. We are now testing the in vivo roles of these modifications as well as their impact on SAGA functions at nutrient-regulated promoters. Altogether, our results contribute to a better understanding of the control of transcription by signal transduction pathways.

Ácido ajulêmico (AJA) é um análogo sintético do ácido THC-11-óico, um metabólito do composto tetrahidrocanabinol (THC), principal ingrediente ativo da maconha, uma droga derivada da planta cannabis sativa. A principal característica do composto AJA é que este apresenta potentes efeitos analgésico e antiinflamatório, sem a ação psicotrópica do THC. AJA não é ulcerogênico em doses terapêuticas e encontra-se atualmente na fase I de testes clínicos, apesar de seu mecanismo de ação não ser completamente entendido. Vários estudos têm reportado que AJA se liga de maneira direta a isoforma da família PPAR de receptores nucleares, induzindo sua atividade transcricional em modelos humanos e em ratos, quando administrado em concentrações farmacológicas. Atualmente AJA se encontra em fase I de testes clínicos, sob aprovação do FDA (Food and Drug Adminstration, E.U.A.). Neste trabalho, é determinada e analisada a estrutura cristalográfica do complexo PPAR -LBD:AJA, mostrando que de fato este receptor pode ser o (ou um) mediador da ação terapêutica de AJA in vivo. Na segunda parte da tese é apresentada da estrutura cristalográfica com complexo entre o domínio catalítico da integrase de HIV e parte do coativador transcricional p75 (também conhecido como LEDGF), mostrando as bases estruturais do reconhecimento molecular no hospedeiro por enzimas retrovirais, passo esse crucial para a replicação viral. Tal importância tem sido explorada no desenvolvimento de fármacos anti-retrovirais, que possam inibir o passo de integração do cDNA viral no genoma humano, atacando o sítio ativo da enzima Devido a características da interface observada no modelo cristolográfico, sugerimos tal região pode vir a ser um novo alvo no desenho de pequenas moléculas que interfiram no reconhecimento molecular / Ajulemic acid (AJA) is a synthetic analog of the tetrahydrocannabinol (THC) metabolite THC-11-oic acid. THC is a major active ingredient of the drug marijuana derived from the plant cannabis sativa. It has been shown that AJA has potent analgesic and anti-inflammatory activity without the psychotropic action of THC. At therapeutic doses AJA is not ulcerogenic, making it a promising anti-inflarnatory drug. Furthermore, AJA is currently under phase I of clinical tests by Indevus Phmaceuticals (USA). However, the mechanism of AJA action remains unknown. It has been shown by biochemical assays that AJA binds directly and specifically to the peroxisome proliferators-activated receptor (PPAR) indicating that this may be a potential target for drug-development in the treatment of pain and inflammation. In this work we describe the crystal structure of the ligand binding region of this receptor in complex with ajulêmico acid, showing that in fact they may be partners in vivo, also providing structure-based answers for current questions, for example, the specificity for the isoform . The binding mode of AJA gives clues about modifications on its structure that might lead to development of more specific and potent molecules. In the second half, it is presented the crystal structure of macromolecular complex between the catalytic core domain of HIV-1 Integrase and the integrase-binding domain of LEDGF (also known as p75). Inspection of the crystallographic model suggests the presence of a specific interface, sharing a high number of tight contacts, apparent lentiviral tropism of LEDGF. Also, our results, along with in vitro assays previously reported, encourage efforts to exploit vim-host protein interactions for the development of novel antiretroviral drugs

L'énergie indispensable au fonctionnement de la cellule est produite principalement par la mitochondrie grâce au mécanisme de phosphorylation oxydative impliquant des protéines codées par le génome nucléaire et celui de la mitochondrie. La coordination transcriptionnelle de ces génomes est nécessaire à la biogenèse de mitochondries fonctionnelles, et est assurée par divers facteurs de transcription, tels que les NRFs (Nuclear Respiratory Factors) et les ERRs (Estrogen-Related Receptors). Leur efficacité transcriptionnelle est contrôlée par les coactivateurs de la famille PGC-1 (Peroxisome proliferator-activated receptor γ Coactivator-1) – PGC-1α, PGC-1β et PRC (PGC-1-Related Coactivator) – dont l'expression dépend de signaux endogènes ou environnementaux. Afin de préciser le rôle de PRC dans le dialogue nucléo-mitochondrial, nous avons utilisé plusieurs modèles cellulaires de carcinomes folliculaires thyroïdiens humains (RO82 W-1, FTC-133 et XTC.UC1) présentant une richesse en mitochondries, une orientation métabolique et des niveaux d'expression de PRC et de PGC-1α différents. Ce travail a mis en évidence le rôle clef du complexe ERRα–PRC dans la biogenèse de mitochondries fonctionnelles. PRC semble par ailleurs coordonner les phases du cycle cellulaire selon l'efficacité du métabolisme énergétique mitochondrial et le statut redox de la cellule. Dans ces modèles, notre travail a mis en évidence un rôle du monoxyde d'azote et du calcium comme régulateurs de la biogenèse et de la fonction mitochondriales PRC-dépendantes. L'ensemble de ces données fait du coactivateur PRC et des voies qui régulent sa fonction des cibles thérapeutiques potentielles dans les tumeurs.

The Australian zebra finch (Taeniopygia guttata) serves as an excellent model organism for studying the mechanisms that influence brain sexual differentiation. The brain and behavior of the zebra finch are sexually dimorphic. The regions of the brain that control the learning and production of song (song control nuclei) are significantly larger in the male brain than in the female brain and only males sing courtship songs, thus the majority of past research has focused on the development of these sex differences. In the majority of mammals, brain sexual differentiation occurs because hormones secreted from the gonads act to initiate male or female brain development. In zebra finches, estradiol is sufficient to masculinize the male brain, however manipulations of developmental hormone exposure fail to fully reverse the sex differences in song nuclei size. Furthermore, genetic females induced to develop functional testicular tissue do not develop a completely masculinized song system and castration has no effect on development of the song system in males. The source of the increased estrogenic signal in male zebra finch brain has yet to be identified, but data suggest that other neuronal factors play a role in development of the song control nuclei. Coregulators, such as coactivators and corepressors, are proteins and RNA activators that work by enhancing or depressing transcriptional activity of the nuclear steroid receptor with which they associate. Coregulators also modulate the development of sex-specific brain morphology and behavior in rodents and birds and may help to explain the difficulties observed in altering song nuclei development via castration and gonadal hormone replacement. As an estrogen receptor-α coactivator, ribosomal protein L7 (RPL7) is able to make the brain more sensitive to estradiol by enhancing the effects of steroid receptor action. Therefore, this dissertation addressed the following questions regarding RPL7: (1) is RPL7 expression sexually dimorphic in the song nuclei of the zebra finch brain?; (2) is RPL7 protein expression regulated by steroid hormones?; and (3) does decreasing RPL7 protein expression with antisense oligonucleotides alter neuronal survival in vivo and song nuclei size and neuron number in vitro? Collectively, these studies will provide valuable information about the role of steroid receptor coactivators in development of the zebra finch song system and on the role of coactivators on sexual differentiation of the brain.

Ácido ajulêmico (AJA) é um análogo sintético do ácido THC-11-óico, um metabólito do composto tetrahidrocanabinol (THC), principal ingrediente ativo da maconha, uma droga derivada da planta cannabis sativa. A principal característica do composto AJA é que este apresenta potentes efeitos analgésico e antiinflamatório, sem a ação psicotrópica do THC. AJA não é ulcerogênico em doses terapêuticas e encontra-se atualmente na fase I de testes clínicos, apesar de seu mecanismo de ação não ser completamente entendido. Vários estudos têm reportado que AJA se liga de maneira direta a isoforma da família PPAR de receptores nucleares, induzindo sua atividade transcricional em modelos humanos e em ratos, quando administrado em concentrações farmacológicas. Atualmente AJA se encontra em fase I de testes clínicos, sob aprovação do FDA (Food and Drug Adminstration, E.U.A.). Neste trabalho, é determinada e analisada a estrutura cristalográfica do complexo PPAR -LBD:AJA, mostrando que de fato este receptor pode ser o (ou um) mediador da ação terapêutica de AJA in vivo. Na segunda parte da tese é apresentada da estrutura cristalográfica com complexo entre o domínio catalítico da integrase de HIV e parte do coativador transcricional p75 (também conhecido como LEDGF), mostrando as bases estruturais do reconhecimento molecular no hospedeiro por enzimas retrovirais, passo esse crucial para a replicação viral. Tal importância tem sido explorada no desenvolvimento de fármacos anti-retrovirais, que possam inibir o passo de integração do cDNA viral no genoma humano, atacando o sítio ativo da enzima Devido a características da interface observada no modelo cristolográfico, sugerimos tal região pode vir a ser um novo alvo no desenho de pequenas moléculas que interfiram no reconhecimento molecular / Ajulemic acid (AJA) is a synthetic analog of the tetrahydrocannabinol (THC) metabolite THC-11-oic acid. THC is a major active ingredient of the drug marijuana derived from the plant cannabis sativa. It has been shown that AJA has potent analgesic and anti-inflammatory activity without the psychotropic action of THC. At therapeutic doses AJA is not ulcerogenic, making it a promising anti-inflarnatory drug. Furthermore, AJA is currently under phase I of clinical tests by Indevus Phmaceuticals (USA). However, the mechanism of AJA action remains unknown. It has been shown by biochemical assays that AJA binds directly and specifically to the peroxisome proliferators-activated receptor (PPAR) indicating that this may be a potential target for drug-development in the treatment of pain and inflammation. In this work we describe the crystal structure of the ligand binding region of this receptor in complex with ajulêmico acid, showing that in fact they may be partners in vivo, also providing structure-based answers for current questions, for example, the specificity for the isoform . The binding mode of AJA gives clues about modifications on its structure that might lead to development of more specific and potent molecules. In the second half, it is presented the crystal structure of macromolecular complex between the catalytic core domain of HIV-1 Integrase and the integrase-binding domain of LEDGF (also known as p75). Inspection of the crystallographic model suggests the presence of a specific interface, sharing a high number of tight contacts, apparent lentiviral tropism of LEDGF. Also, our results, along with in vitro assays previously reported, encourage efforts to exploit vim-host protein interactions for the development of novel antiretroviral drugs