Der Signaltransduktor und Aktivator der Transkription 1 (STAT1) ist ein Mitglied der konservierten Proteinfamilie von zytokinregulierten Transkriptionsfaktoren und spielt bei vielen biologischen Prozessen, wie beispielsweise der Steuerung der Immunantwort und der Zellproliferation, eine entscheidende Rolle. Bei Stimulation der Zellen mit Interferon-gamma (IFN-gamma) phosphorylieren rezeptorassoziierte JAK-Kinasen einen kritischen Tyrosinrest im Carboxyterminus von STAT1. Die Tyrosin-phosphorylierten STAT1-Homodimere akkumulieren daraufhin im Zellkern, wo sie spezifische Bindestellen in den Promotoren von STAT1-abhängigen Zielgenen erkennen. In der vorliegenden Arbeit wurde untersucht, ob drei oberflächenexponierte und konservierte Aminosäurereste in der DNA-Bindedomäne (Lys 359, Lys 361 und Asp 367) an der Weiterleitung der Signalintensität beteiligt sind. Zu diesem Zweck führte ich einen Mutageneseansatz durch und substituierte die drei Aminosäurerreste zu Alaninen, doch keiner der drei erhaltenen Punktmutanten zeigte eine gegenüber dem Wildtyp-Molekül geänderte Kinetik der Tyrosin-Phosphorylierung, der induzierbaren Kernakkumulation sowie der Dissoziation von hochaffinen DNA-Bindestellen. Auch blieben die transkriptionelle Aktivierung eines Reportergenkonstrukts und die Expression von sämtlichen der untersuchten IFN-gamma-abhängigen endogenen Zielgene durch die Mutationen unverändert. Zusammendfassend fand ich keine Unterschiede im detektier-baren Verhalten der Mutanten gegenüber dem Wildtyp-Molekül. Doch kann nicht ausgeschlossen werden, dass sich Änderungen in den hier nicht erfassten nicht-kanonischen STAT1-Funktionen ergeben könnten, die kausal für die Homologie dieser Reste verantwortlich sind.

Transcriptional activation of Major Histocompatability Complex (MHC) I and II molecules by the cytokine interferon gamma (IFN-g) is a key step in cell-mediated immunity against pathogens and tumors. Following IFN-g induction, JAK/STAT signaling triggers activation of MHC genes. Recent evidence suggests suppression of MHC I and II expression on multiple tumor types plays important roles in tumor immunoevasion. One such tumor is malignant melanoma, the leading cause of skin cancer related deaths. Despite awareness of MHC expression defects, the molecular mechanisms by which melanoma cells suppress MHC and escape from immune-mediated destruction remain unknown. Here we analyze dysregulation of the JAK/STAT pathway and its role in suppression of MHC II in melanoma cell lines at the Radial Growth Phase (RGP), the Vertical Growth Phase (VGP) and the Metastatic Phase (MET). RGP and VGP cells express both MHC II and the MHC master regulator, the Class II Transactivator (CIITA). MET cells lack not only MHC II and CIITA, but also both STAT 1 and the STAT 1 coactivator, the Interferon Response Factor (IRF) 1. Our studies have implicated that the suppression of MHCII on the cell surface of metastatic melanoma is due to silencing at the level of STAT1 transcription. Furthermore, we determined that silencing of STAT1 is, in part, due to hemi-methylation of the STAT1 promoter.

Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality amongst immuno-compromised individuals and is the leading cause of congenital diseases amongst newborn infants. Mouse CMV (MCMV) infection of inbred mice has been extensively used as a model for HCMV pathogenesis and host-virus interaction. Macrophages are a key target cell type in the pathogenesis of human and mouse CMV infections. Macrophages are semi-permissive to CMV infection, however, the nature of this restrictive mechanism of infection is open for investigation. In this thesis, I hypothesized that macrophage permissivity is determined by the dynamic interplay of the innate response during the immediate-early (IE) period of infection. To test this hypothesis, I first developed and validated a flow cytometry based assay. In MCMV infected macrophages, I found heterogeneous expression from the major IE promoter (MIEP) leading to the development of a refractory subpopulation for IE expression. I further developed a computational modelling approach to help elucidate the dynamics of infection during this period. Modelling work revealed that the occurrence of refractory subpopulation could be caused by either 1) pre-existence of heterogeneous permissivity of macrophages prior to infection or 2) through an emergent process. Experimental testing of the models shows that the heterogeneous IE expression of homogeneously infected macrophages is caused by an emergent process. MCMV infection using type I interferon receptor and signal transducers and activator of transcription 1 (Stat1) knockout macrophages reveals that the emergence of refractory subpopulation is predominantly mediated by type I interferon through Stat1. Comparative molecular analysis between progressively infected and refractory subpopulations reveals that MCMV MIEP activation in the refractory subpopulation is stochastically inhibited by high expression of type I interferon induced antiviral components.

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STAT1 (Signaltransduktor und Aktivator der Transkription 1) gehört zu einer evolutionär hochkonservierten Proteinfamilie, die eine essentielle Rolle bei der Weiterleitung von Zytokinsignalen spielt. Das STAT1-Protein ist bei zahlreichen biologischen Prozessen, wie etwa der Zellproliferation oder der Steuerung der Immunantwort, von Bedeutung und gehört zu einer der am besten untersuchten humanen Trans-kriptionsfaktoren. Bekannt ist, dass die über STAT1 vermittelte Signaltransduktion durch eine Rezeptor-assoziierte Phosphorylierung eines kritischen Tyrosinrestes im carboxyterminalen Molekülbereich von STAT1 initiiert wird. Nach der Ausbildung von transkriptionell aktiven Dimeren translozieren diese in den Zellkern und binden auf den Promotoren von Interferon-gesteuerten Zielgenen. In dieser Arbeit wurden Punktmutanten in der Linker-Domäne des STAT1-Moleküls generiert, um die Bedeutung der reziproken SH2-Phosphotyrosin-Interaktion bei der Bildung von tyrosinphosphorylierten Dimeren in funktioneller Hinsicht studieren zu können. Die Substitution eines kritischen Lysinrestes in Position 550 im STAT1-Protein zu Alanin oder Glutamat war mit einer im Vergleich zum Wildtyp-Molekül verminderten Tyrosinphosphorylierung und einer verkürzten nukleären Akkumulationsphase der mutierten Proteine nach Stimulation von transient exprimierenden Zellen mit Interferon-gamma (IFN-gamma) verbunden. Bei normaler Dissoziationskinetik von STAT1-spezifischen DNA-Sequenzen fanden sich für die K550-Mutanten erhöhte In-vitro- und In-vivo-Dephosphosphorylierungsraten und identifizierten diese damit als gegenüber dem Wildtyp-Protein bevorzugte Substrate der inaktivierenden TC45-Phosphtase. Die mutationsbedingte Destabilisierung in der Architektur der Linker-Domäne resultierte in einer supprimierten Aktivierung von IFN-gamma-gesteuerten Zielgenen, wobei die Signalamplitude von der Ladung der eingeführten Seitengruppe bestimmt wurde. Zusammenfassend zeigen diese Beobachtungen, dass der Linker-Domäne eine wichtige Bedeutung bei der Regulation der Tyrosindephosphorylierung zukommt und unterstreichen überdies deren Rolle bei dem vermuteten Austausch zwischen einer parallelen und antiparallelen Dimerkonformation, die für eine optimale Genexpression essentiell ist.

Hearing loss or deafness, in its most serious form, affects an estimated 28 million people in America. One of the forms of hearing loss, known as ototoxicity, refers to damage to the ear (-oto) due to xenobiotics. Cisplatin is the most widely used antineoplastic agent in the treatment of various solid tumors. Cisplatin toxicity can lead to severe effects on the kidneys, nervous system and auditory system which significantly reduce the quality of life of cancer patients. While nephrotoxicity could be alleviated by hydration and diuresis, cisplatin-induced ototoxicity is permanent and there is currently no approved treatment for this condition. Previous studies have shown that the generation of reactive oxygen species (ROS) is a critical event that initiates damage to the outer hair cells (OHCs), stria vascularis (SVA) and spiral ganglion cells (SG) of the cochlea, leading to hearing loss after cisplatin treatment. However, the mechanism(s) underlying the transition from ROS generation to the manifestation of ototoxicity is (are) not clearly defined. Recent studies have also implicated inflammatory pathways in cisplatin-induced cell death. Various transcription factors have been linked to the induction of inflammation mediated by cisplatin in the cochlea. Recent study demonstrate that cisplatin activates signal transducer and activator of transcription 1 (STAT1) a transcription factor implicated in inflammation, which mediates damage to utricular hair cell and could also confer cisplatin-induced hearing loss. The aim of this study is to further define a role of STAT1 in cochlear inflammation and in cisplatin-mediated ototoxicity. Based on preliminary data, we hypothesize that STAT1 plays an integral role in cisplatin-mediated inflammation and hearing loss. Our data show that STAT1 couples ROS to the inflammatory process in the cochlea. The major source of ROS appears to be the NOX3 NADPH oxidase system, knockdown of which by short interfering (si)RNA reduces STAT1 activation by cisplatin and alleviates hearing loss. Activation of STAT1 by cisplatin involves phosphorylation of Serine 727 by mitogen activated protein kinases such as extracellular signal regulated kinase (ERK) 1/2 and p38. Knockdown of STAT1 by trans-tympanic administration of siRNA reduces damage to OHCs and protects against cisplatin-induced hearing loss in rats. STAT1 siRNA attenuates the production of inflammatory mediators, such as tumor necrosis factor- α (TNF-α), and reduces the recruitment of inflammatory cells to the cochlea. Furthermore, inhibition of TNF-α by trans-tympanic administration of etanercept, a clinically used TNF-α antagonist, protects against OHC damage and cisplatin-induced hearing loss. These data suggest that targeting STAT1 or the inflammatory genes it regulates could serve as useful strategies for preventing cisplatin-induced hearing loss and improve the overall quality of life of cancer patients.

Signal Transducer and Activator of Transcription (STAT) 2 is one of seven members of the STAT family of transcription factors with dual roles in signal transduction and gene activation. STAT2 is a central transcription factor that regulates the antiviral, apoptotic and cell growth inhibitory effects of type I interferons (IFN-α/β), a small family of secreted glycoproteins induced by the host after sensing the presence of tumor cells and pathogens. The creation of Stat2-/- mice established the pivotal role of STAT2 in type I IFN signaling and in antiviral immunity. In vitro studies conducted in STAT2 deficient tumor cell lines suggested a role in suppressing tumor cell growth in response to IFN treatment. Based on these properties STAT2 is presumed to have tumor suppressor functions but data to support this notion in animal models of cancer are limited. To address the role of STAT2 in cancer, I used the murine B16-F1 tumor transplantation model of human melanoma. The B16-F1 melanoma cell line was established from a spontaneous tumor that arose in mice. I discovered that tumor cells transplanted subcutaneously in Stat2-/- mice grew more aggressively than in the counterpart wild type mice. Closer examination of B16-F1 tumors harvested from wild type and Stat2-/- mice revealed an unexpected dramatic similar reduction of STAT2 and STAT1 proteins. Yet soluble factors secreted by B16-F1 tumors established in Stat2-/- mice alone were sufficient to enhance proliferation of B16-F1 tumor cells. I further showed that tumor-bearing wild type mice treated with IFN-β developed smaller tumors compared to Stat2-/- mice, whose tumors continued to grow and hence were unresponsive to IFN intervention. Lastly, to elucidate a mechanism that leads to enhanced tumor growth in Stat2-/- mice, I questioned the involvement of the host immune response in restricting tumor growth. I found that tumor specific T cell priming by Stat2-/- dendritic cells (DCs) was defective since generated cytotoxic T cells (CD8+ T lymphocytes) produced low levels of IFN-γ and IL-2 and adoptive transfer of these B16-F1 tumor specific CD8+ T cells in B16-F1 bearing Stat2-/- mice did not cause tumor regression with IFN-β intervention. Collectively, my findings reveal that host STAT2 restricts the establishment of melanoma tumors. More importantly, type I IFN/STAT2 signaling on DCs plays a pivotal role in tumor antigen cross-presentation to CD8+ T cells and in the development of a protective antitumor response resulting in tumor rejection. To now address whether STAT2 expression in cancer cells could influence tumor establishment and the antitumor effects of type I IFNs, STAT2 expression was silenced in B16-F1 tumor cells. Contrary to my expectation, silencing STAT2 augmented the growth inhibitory effects of IFN-β both in vitro and in vivo. However, loss of STAT2 expression in the tumor did not cause B16-F1 tumor cells to grow more aggressively compared to control B16-F1 cells. Furthermore, compared to B16-F1 control cells, STAT2-silenced B16-F1 cells showed an initial delay but later persistent STAT activation and formation of the ISGF3 transcriptional complex (consisting of STAT1, STAT2 and IRF9). This observation paralleled with an initial delay and then later an increase in the expression of IFN regulated genes. In addition, reduced activation of STAT5 induced by IFN-β was observed in STAT2-silenced B16-F1 cells. This may partially explain the enhanced growth inhibitory effects of type I IFNs. Together these results shed light on the unexpected role of tumor STAT2 expression in diminishing the efficacy of type I IFN treatment of melanoma. / Biochemistry

Au cours de la gestation précoce, la régulation de la physiologie endométriale est cruciale au bon déroulement de l’implantation. Chez les mammifères, une famille de facteurs de transcription est fortement impliquée dans la régulation de la physiologie endométriale, les facteurs STAT. Chez la vache, des analyses haut-débit ont révélé que l’expression endométriale de STAT1 est régulée au cours de la période préimplantatoire. Le but de cette thèse est donc d’apporter de nouvelles données sur l’expression et la régulation endométriales de STAT1 mais également sur ses fonctions biologiques au cours de la gestation précoce chez la vache.Grâce à différents modèles physiologiques et expérimentaux, l’impact de la progestérone, de l’IFNT (signal majeur de reconnaissance maternelle de la gestation chez les ruminants) et de la gestation sur l’expression et la régulation de STAT1 (y compris sa phosphorylation) a été analysé dans l’endomètre bovin et sur des cultures primaires de cellules endométriales. Ainsi, l’expression de STAT1 (transcrit et protéine) ainsi que sa phosphorylation sont augmentés en présence du conceptus et de l’IFNT, indépendamment du taux circulant de progestérone à l’implantation chez la vache. Pour avoir une meilleure connaissance des rôles de STAT1, l’identification de ses gènes cibles a été entreprise : d’abord avec une approche gènes candidats (avec la famille des gènes SOCS), puis par une approche exploratoire.Les facteurs SOCS sont connus pour être des régulateurs négatifs de la voie de signalisation des cytokines. L’utilisation des différents modèles physiologiques et expérimentaux évoqués plus haut a permis l’analyse de l’expression et de la régulation des huit membres de la famille des gènes SOCS au cours de la gestation précoce chez la vache. L’application d’un protocole d’immunoprécipitation de la chromatine sur des cultures primaires de cellules stromales bovines montre le recrutement rapide de STAT1 par l’IFNT sur les promoteurs des gènes SOCS IFNT-dépendants. D’autre part, l’identification systématique des gènes cibles de STAT1 a été entreprise via l’élaboration d’un protocole d’immunoprécipitation de la chromatine suivit de séquençage haut-débit, appliqué à des échantillons d’endomètre bovin. L’ensemble de ces travaux suggèrent l’implication de STAT1 dans la signalisation endométriale de l’IFNT, dans la régulation du système immunitaire maternel et également dans le contôle des phénomènes d’apposition et d’adhérence, fonctions cruciales à l’implantation chez la vache. / During the early stage of the pregnancy, the regulation of the endometrial physiology is crucial to the right establishment of the implantation. In mammals, a transcription factor family is highly involved in the regulation of endometrial physiology, the STAT family. In cattle, high-throughput analyses light up the regulation of endometrial STAT1 expression during the pre-implantation period. Thus, the aim of this work is to bring new insights about endometrial STAT1 expression and regulation but also on its biological functions during the early pregnancy in cattle. Using physiological and experimental models, the impact of progesterone, IFNT (major signal of the maternal recognition of pregnancy in ruminants) and pregnancy on the expression and the regulation of STAT1 transcript and protein (including its phosphorylation status) have been analyzed in the bovine endometrium and endometrial cells. Thus, STAT1 (transcript, protein and phosphorylation) is up-regulated by the presence of the conceptus and by IFNT but independent of progesterone level at implantation in cattle. To better understand endometrial STAT1 functions, the identification of STAT1 target genes has been initiated: first, on a candidate genes family, SOCS genes, and secondly, with an explorative approach.The proteins SOCS are known to be negative regulator of cytokine signalling pathway. Using physiological and experimental models previously quoted, the eight members of SOCS genes expression and regulation were analyzed during the early pregnancy in cattle. Chromatin immunoprecipitation protocol applied on stromal cells show the recruitment of STAT1 on SOCS promoters by a rapid treatment of IFNT. Moreover, the exhaustive identification of STAT1 target gene has been initiated, using a chromatin immunoprecipitation followed by high-throughput sequencing on bovine endometrium samples. Collectively, this data suggests the involvement of STAT1 in IFNT signalling pathway but also in the regulation of maternal immune system and the apposition/adhesion process, all that being crucial for the implantation in cattle.

Le facteur de transcription STAT1 est un effecteur majeur de la réponse à l’interféron exerçant ainsi un rôle clé dans l’immunité innée. Il est également un suppresseur de tumeur et régule des effets antiprolifératifs et pro-apoptotiques. Afin de mettre en évidence de nouvelles implications anti-tumorales de STAT1, ce projet de doctorat a porté sur son implication fonctionnelle i) dans l’expression des immunoglobulines (Ig), processus essentiel de l’immunité adaptative et ii) dans la réponse cellulaire aux traitements par les agents génotoxiquesNotre étude cinétique, après induction de l’expression de STAT1 dans des cellules initialement totalement déficientes, a montré son implication dans l’expression des IgG membranaires. Le mécanisme régulateur est indirect : il impliquerait une inhibition de l’activation de STAT3 conduisant à l’inhibition de l’expression de BLIMP1, un acteur essentiel de la différenciation plasmocytaire. La capacité de STAT1 à pouvoir se fixer sur le promoteur de BLIMP1, de même que STAT3 n’exclut pas une implication transcriptionnelle.Au cours d’un traitement génotoxique par le MNNG, nous avons décrit la participation de STAT1 à un complexe de réparation de l’ADN. Seule la présence de STAT1 permet l’intégration au complexe MLH1/p53 de la kinase c-Abl. Dans ce contexte, on observe une cytotoxicité sous le contrôle de l’activité kinase de c-Abl, une réparation rapide et efficace de l’ADN, un arrêt seulement transitoire du cycle et une orientation vers la survie cellulaire équivalent à une résistance à ce traitement.Ces résultats mettent en évidence deux nouveaux rôles anti-tumoraux de STAT1 par sa contribution à des complexes régulateurs essentiels et la désigne comme une potentielle cible thérapeutique. / The transcription factor STAT1, as a major effector of interferon, plays a key role in innate immunity. Through its strong anti-proliferative and pro-apoptotic properties STAT1 is also considered as a tumor suppressor. The aim of this project was to delineate new potential tumor suppressor properties for STAT1 in two signaling mechanisms: i) Ig expression in plasmacytoid cells and ii) cellular response to genotoxic stress.Our kinetic experiments, upon “de novo” expression of STAT1 in a rare STAT1-deficient cell line showed its modulation of membrane IgG expression. The underlying mechanism involves a STAT1-dependent inactivation of STAT3 and subsequently a decreased expression of BLIMP1, a major contributor to plasma cell differentiation. Since STAT1, like STAT3, is able to bind to the BLIMP1 promoter elements a transcriptional interference cannot be excluded.During alkylating agent treatment with MNNG we have observed the presence of STAT1 in DNA repair complex. STAT1 expression allows the recruitment into a MLH1/p53 complex of the kinase c-Abl. This complex leads to cytotoxic dependence on c-Abl kinase activity, an efficient DNA repair with a transient cell cycle arrest and to signaling mechanisms toward cell survival. At longer term of exposure STAT1 also lead to cellular resistance to treatment.These results provide evidences for new anti-tumor roles of STAT1 in two major regulatory systems and indicate STAT1 as a potential therapeutic target.

De la synthèse des ARNm jusqu'à leur dégradation, le complexe CCR4-NOT est un régulateur essentiel de l'expression des gènes eucaryotes. CAF1 est une sous-unité catalytique qui joue un rôle central dans la fonction de ce complexe. La protéine humaine hCAF1 possède une activité déadénylase, régule la méthylation des arginines dépendante de PRMT1 et est un régulateur transcriptionnel des récepteurs nucléaires. Bien que l'ensemble des travaux publiés sur hCAF1 lui confère une place importante dans la régulation de l'expression des gènes, son mécanisme d'action et surtout les voies de signalisation qu'elle régule restent encore mal compris dans les cellules humaines. Lors de ce travail de thèse, nous avons mis en évidence une nouvelle fonction de la protéine hCAF1 comme régulateur de la voie des interférons via le contrôle du facteur de transcription STAT1 et la dégradation de ses ARNm cibles. L'identification de hCAF1 comme régulateur de l'activité de STAT1 et de la réponse aux interférons est très importante car des activations anormales de ces voies sont associées à de nombreuses pathologies telles que le cancer ou des maladies immunitaires. En parallèle, nous avons caractérisé un nouvel isoforme nommé hCAF1v2 produit par le gène humain Caf1 suite à un évènement d'épissage alternatif. Nos résultats indiquent que hCAF1v2 présente une divergence fonctionnelle vis-à-vis de hCAF1 puisqu'elle ne possède pas d'activité déadénylase intrinsèque et s'avère requise pour la régulation de la méthylation des arginines via son interaction avec l'enzyme PRMT1. L'ensemble des résultats obtenus identifient une nouvelle voie de signalisation régulée par la protéine hCAF1 dans les cellules humaines et permettent de mieux comprendre l'implication du complexe CCR4-NOT dans les mécanismes de régulation de l'expression des gènes / The multi-subunit CCR4-NOT complex has been implicated in all aspects of the mRNA life cycle, from synthesis of mRNAs in the nucleus to their degradation in the cytoplasm. The CAF1 protein is a catalytic subunit which plays a central role inside the complex. Human CAF1 is a deadenylase, modulates arginine methylation, and is a transcriptional cofactor of several nuclear receptors. The main objective of the thesis was to elucidate the molecular mechanism of hCAF1- mediated gene expression. We reported that hCAF1 is an important negative regulator of the interferon pathway and that hCAF1 is associated in the cytoplasm of resting cells with STAT1, a crucial transcription factor of this pathway. We found that hCAF1 participates in the extinction of the IFN signal via its deadenylase activity, by speeding up the degradation of some STAT1-induced mRNAs. Our findings are important because abnormal activations of this pathway are frequently associated with cancer and auto-immune diseases. In parallel, we characterized a novel isoform called hCAF1v2 produced by alternative splicing of the Caf1 gene. We reported that hCAF1v2 displays divergent functions compared with hCAF1. In fact hCAF1v2 does not have a deadenylase activity and is preferentially associated with PRMT1 to modulate arginine methylation. Altogether, our findings identify a new signalling pathway which is regulated by hCAF1, and reveal novel mechanisms utilized by the CCR4-NOT complex to control gene expression