Prolactin (PRL) is a polypeptide hormone that regulates diverse biological processes such as those involved in mammary gland and reproductive organ development. These biological functions are mediated by interaction of PRL with its membrane-bound receptor, a member of the class I cytokine receptor superfamily. Ligand binding to the PRL receptor (PRLR) induces dimerization of the receptor and activation of Janus kinase-2 (Jak2). These events lead to PRLR tyrosine phosphorylation creating binding sites for cellular signaling molecules containing SH2 domains such as the signal transducer and activator of transcription 5 (Stat5). Jak2/Stat5 pathway has emerged as the main signaling pathway mediating many physiological effects of PRL such as the terminal differentiation of mammary epithelial cells and milk protein production. / Work in this thesis was performed to understand the molecular mechanisms of PRL actions in target tissues by investigating the role of cytoplasmic protein tyrosine phosphatases containing SH2 domain, SHP-1 and SHP-2, in regulation of PRLR intracellular signaling events. These phosphatases participate in signaling as enzymes and/or adapter proteins. Both SHP-1 and SHP-2 contain specific tyrosine residues at their C-terminal parts that undergo phosphorylation in response to PRLR stimulation. The focus of this doctoral work was to investigate the adapter function of these proteins, which is mediated by their C-terminal tyrosine residues. These studies revealed that these tyrosine residues can function to recruit legitimate substrate(s) for the catalytic domain of SHP-2 including a highly phosphorylated p29 protein. Subsequent studies showed that p29 is growth factor receptor-binding protein-2 (Grb2), suggesting that Grb2 is phosphorylated in response to PRL and the level of phosphorylation is regulated by SHP-2. Grb2 phosphorylation by PRLR was further shown to be a new mechanism by which PRLR inhibits EGFR-induced signaling. / In addition, our results indicate that the inhibitory effect of SHP-1 on cytokine-induced Jak/Stat pathway could occur independently of its catalytic activity. Based on our data, SHP-1 C-terminal tyrosine residues contribute to the inhibitory effect of SHP-1 by recruiting inhibitory complex Grb2/SOCS-1 and targeting SOCS-1 to Jak2. Together these findings highlight new mechanisms of PRL/cytokine receptor signaling by the protein tyrosine phosphatases SHP-1 and SHP-2.

In vertebrates, PBX and MEIS/PREP are normally localized to the cytoplasm and are mutually dependant on one another for their translocation to the nucleus. Recently, phosphorylation and cytoskeleton anchoring to myosin heavy chain II B were proposed as regulatory factors for PBX subcellular localization. Moreover, MEIS-independent regulation of PBX1 subcellular localization has also been reported. In this study, we investigated the role of actin and myosin in the control of PBX1 subcellular localization and the PBX1-independent regulation of MEIS1 subcellular localization. Our data suggest that PBX1 interacts with actin via myosin, and the interaction interface was mapped to the PBC-B domain of PBX1. Moreover, there was no nuclear translocation of the PBX ortholog EXD when actin filaments were destroyed with swinholide A in insect S2 cells. We propose that PBX1 interacts with actin indirectly via myosin heavy chain II B, but that association with actin is not required for regulation of subcellular localization. In cultured NIH 3T3 cells, we obtained evidence for a novel MEIS1 isoform which is not able to interact with PBX1 and is therefore constitutively cytoplasmic. We also mapped a Rev-like nuclear export signal in the HR2 domain of MEIS1, revealing common and isoform-specific regulation of MEIS1 subcellular localization. / Chez les vertébrés, les protéines PBX et MEIS/PREP sont normalement localisées dans le cytoplasme et sont réciproquement dépendantes pour leur translocation au noyau. Cependant, deux autres facteurs furent récemment proposés d’être aussi importants pour la localisation sous-cellulaire de PBX, soit la phosphorylation ainsi que l’ancrage au cytosquelette par la chaîne lourde IIB de la myosine. De plus, il a été rapporté que la localisation sous-cellulaire de PBX pouvait être contrôlée de façon indépendante de MEIS. Dans les travaux présentés ici, nous étudions les rôles de l’actine et de la myosine dans la régulation de la distribution sous-cellulaire de PBX1 ainsi que la régulation de la distribution sous-cellulaire de MEIS1 qui ne dépend pas de PBX1. Nos données suggèrent que PBX1 interagit avec l’actine via la myosine et que l’interface d’interaction avec PBX1 se situe dans son domaine PBC-B. Cependant, lorsque les filaments d’actine de cellules de mouches S2 sont détruits, suite au traitement avec la swinholide A, EXD, l’orthologue de PBX, n’est pas relocalisé au noyau. Nous proposons donc que PBX1 interagit avec l’actine via la chaîne lourde IIB de la myosine mais que cette association n’est pas requise pour la régulation de la localisation sous-cellulaire. D’autre part, nous avons identifié, dans des cellules NIH 3T3, un nouvel isoforme de MEIS1 qui est incapable d’interagir avec PBX1 et qui demeure constitutivement cytoplasmique. Finalement, nous avons identifié un signal d’export nucléaire de type Rev dans le domaine HR2 de MEIS1. Nous avons donc identifié un mécanisme isoforme-spécifique de même qu’un mécanisme commun de régulation de la distribution sous-cellulaire de MEIS1.

The mammalian cytosolic chaperone Hsc70 functions in protein folding and sorting into the mitochondria. Two of its main regulators are the J domain proteins and the nucleotide exchange factors (NEFs) .The Hsp40-like DJA1, DJA2 and DJA4 proteins have been found to be the primary J domain functional partners of Hsc70. They are involved in protein targeting to the mitochondria, they activate the Hsc70 ATPase and DJA2 is highly efficient at supporting protein refolding whereas DJA1 inhibits it. The NEFs Bag1, HspBP1 and Hsp110 all function in conjunction with the DJAs to stimulate the Hsc70 ATPase but the low required concentrations and an ability to increase refolding yields suggest that Hsp110 might be the general NEF in the mammalian cytosol. Additionally, the J domain protein MPP11 was found to associate with ribosomes and to be important for fully efficient folding of nascent polypeptide, thus linking the translation and chaperone systems. / Dans le cytosol des cellules mammifère le chaperon Hsc70 est impliqué dans le repliement général de protéines et leur importation vers la mitochondrie. Il est principalement régulé par les protéines contenant un domaine J et les facteurs d'échange de nucléotides (NEFs). Les protéines similaires à Hsp40 DJA1, DJA2 et DJA4 sont les principaux partenaires à domaine J fonctionnels de Hsc70. Ces protéines sont impliquées dans l'importation de preproteines vers la mitochondrie, elles activent l'activité d'hydrolyse d'ATP de Hsc70 et DJA2 permet très efficacement à Hsc70 de replier des protéines dénaturées. Les NEFs Bag1, HspBP1 et Hsp110 arrivent tous, en conjonction avec les DJA, à stimuler l'hydrolyse d'ATP par Hsc70 mais la faible concentration requise et son habileté à augmenter l'activité de repliement de protéines de Hsc70 nous permet de supposer que Hsp110 est le facteur d'échange de nucléotide principal. Nous avons de plus trouvé que la protéine à domaine J MPP11 interagit avec les ribosomes et qu'elle est importante pour obtenir un repliement efficace des protéines nouvellement synthétisées.

The morphological and cellular changes observed during embryonic development are mainly caused by a reorganisation of gene expression programmes at the cellular level. These reorganisations are a consequence of extracellular and intracellular signals that converge in the nucleus to alter gene expression and thus give rise to cells with entirely novel properties. The mechanisms governing the regulation of gene expression in a given cell type during embryonic development are still ill-defined. In order to understand more how the cardiac genetic programme is governed, we undertook to clone the rat cDNA of theGATA-6 transcription factor. The GATA family of transcription factors encodes zinc-finger DNA binding proteins that recognise the consensus WGATAR sequence that constitutesa regulatory region for numerous genes. In the haematopoiesis system, GATA-1, -2 and -3 have an expression restricted to subsets of heamotopoietic cells and they all have unique, essential non-redundant roles in the differentiation and survival of these lineages. Interestingly, our laboratory and others have characterised a fourth member of the vertebrate GATA family, GATA-4, whose expression is highly enriched in the developing myocardium. Furthermore, GATA-4 expression is essential for cardiogenesis as tested in an in vitro system of cardiac differentiation or by gene-targeted mutation in the mouse. Here we describe the cloning of the rat GATA-6 cDNA and describe that GATA-6 is also highly enriched in the myocardium both at the RNA and the protein levels implying that a cardiac GATA subfamily may, during cardiogenesis, play analogous roles to the haematopoietic GATA subfamily. The characterisation of the GATA-6 protein revealed that it binds to consensus GATA sites with high affinity and that the GATA-6 gene encodes for a transcriptional activator with properties very similar to those of GATA-4. Finally we provide some evidence that GATA-6 expression might be under the control of GATA-4 and

Abstract One of the major tasks in cancer research is to understand the regulation of transcription factors that are crucial in controlling cell proliferation, differentiation and apoptosis. To achieve this, transcriptional co-regulators and signaling pathways that control transcription factors have been extensively studied. One good example is the Runx family of proteins and two of their co-regulators, transcription co-activator with PDZ domain-binding motif (TAZ) and Yes-associated protein (YAP). TAZ and YAP are paralogous and serve as either co-activators or co-repressors depending on the cellular context. In an attempt to understand the underlying molecular mechanisms for this dual role in transcription regulation, and especially considering that Yorkie (a Drosophila homologue of YAP) is under control of the hippo kinase pathway, we considered the possibility that TAZ and YAP are the target of this pathway. As such, we sought to test the signaling pathways, including the hippo-like pathway, that regulate the function of TAZ and YAP in mammalian cells. In this thesis research, I first set out to characterize how TAZ, in synergy with the histone acetyltranserase monocytic leukemia zinc finger protein (MOZ), activates Runx-dependent transcription in a signal-responsive manner. Then I used Runx-dependent transcription as a model system to study the regulation of TAZ. I found that both class I and II histone deacetylases interacted with TAZ and repressed its co-activator activity. In addition, TAZ is acetylated by and synergizes with CBP and p300, two paralogous acetyltransferases, to activate transcription. These results suggest that the activity of TAZ is subjected to regulation by acetylation and deacetylation. These results also provide a molecular basis for the hypothesis that TAZ and YAP recruit or release co-repressors in response to cellular stimuli. I then investigated the hippo-like pathway in mammalian cells. Excitingly, I found that the ki / Résumé Un des défis majeurs de la recherche sur le cancer est de comprendre la régulation de certains facteurs de transcription qui jouent un rôle clé dans le contrôle de la prolifération, de la différenciation et de l'apoptose. Pour cette raison, de nombreuses études approfondies sur les corégulateurs et les voies de signalisation ont été menées. Parmi celles-ci, la famille des protéines Runx et deux de leurs corégulateurs, TAZ (transcription co-activator with PDZ domain-binding motif) et YAP (Yes-associated protein) en sont un bon exemple. TAZ et YAP sont des protéines paralogues et agissent comme des coactivateurs ou des corepresseurs en fonction du contexte cellulaire. Puisque Yorkie, l'orthologue de YAP chez la drosophile, est contrôlé par la voie de signalisation de hippo, l'identification de celle qui régule les fonctions de TAZ et de YAP dans les cellules de Mammifères a été menée afin de comprendre les mécanismes moléculaires du double rôle de ces corégulateurs dans la régulation transcriptionnelle. Dans ce travail de thèse, la manière dont TAZ active la transcription dépendante de Runx, en synergie avec l'histone acétyltransférase MOZ (monocytic leukemia zinc finger protein) a été étudiée. Par la suite, la transcription médiée par Runx a été utilisée comme modèle afin d'étudier la régulation de TAZ. Il a ainsi été démontré que les histones deacétylases de classe I et II interagissent avec TAZ et répriment son activité de coactivateur dans la transcription dépendante de Runx. En outre, TAZ est acétylé par CBP/p300, deux acétyltransférases paralogues, et synergise avec ces deux protéines pour activer la transcription. Ces résultats suggèrent donc que l'activité de TAZ est régulée par un mécanisme d'acétylation et de déacétylation et que TAZ et YAP recrutent et relâchent des corépresseurs en réponse à différents stimuli cellulaires. À la lumière de ces informations, la voie de sig

The objective of the research in this thesis is to investigate, at the molecular level, the sequences and/or structures involved in the initiation of mammalian DNA replication and to investigate the protein interactions with DNA cruciforms which have been implicated in the process of replication initiation. Four plasmids containing monkey (CV-1) early replicating nascent origin enriched sequences (ors), which had been shown previously to replicate autonomously in transfected CV-1, COS-7 and HeLa cells, were used in the establishment of an in vitro DNA replication system that uses HeLa cell extracts. The in vitro replication system is dependent upon the presence of an ors sequence, and HeLa cell extracts. Mapping experiments indicate that there is preferential nucleotide incorporation in the ors sequences, suggesting site-specific initiation, and that replication is bidirectional and semiconservative. Electron microscopy confirmed that in vitro initiation occurs within the ors sequence. / Prokaryotic and eukaryotic viral replication origins, mammalian origin enriched sequences (ors) and other mammalian early replicating sequences contain AT-rich sequences and inverted repeats, which have the potential to form bent and cruciform (stem-loop) DNA structures, respectively. Cruciforms have been postulated to form transiently at or near origins to serve as recognition structures for initiator proteins. Using a stable-DNA cruciform as a binding substrate in a band-shift assay, a novel DNA binding activity with specificity for the cruciform-containing DNA and no apparent sequence-specificity was identified in HeLa cell extracts. The activity is protein-dependent and is void of detectable nuclease activity. Cruciform-specific binding was observed to be maximal in early-S phase extracts. A novel cruciform binding protein (CBP) with an apparent molecular weight of 66 kDa was enriched from HeLa cell extracts. Footprinting experiments localized the CBP-DNA cruciform interaction to the four-way junction at the base of the cruciform. CBP appears to interact with the elbow junctions in an asymmetric fashion. Upon CBP binding, structural distortions were observed at the cruciform stems and at a DNA region distal to the junction.

The Hairy enhancer of split 6 (Hes6) protein modulates myogenesis and neurogenesis by repressing inhibitors of differentiation. We demonstrate that Hes6 dependent transcriptional repression in myoblasts is mediated by recruitment to the DNA N-box. Identification of putative targets of Hes6 was attempted by creating inducible clones. Alternately, a target was sought by comparing cells with constitutive Hes6 expression to wildtype cells using DNA microarray technology. Candidate gene analysis of microarray data resulted in the identification of a novel potential target, cardiac helix-loop-helix factor 2 (CHF2), which was confirmed by semi-quantitative RT-PCR analysis. Future studies are required to determine whether Hes6 binds N-boxes within the untranslated regions of the CHF2 gene, and the possibility of negative autoregulation of CHF2 via the formation of Hes6-CHF2 heterodimers in vivo, and its implications in somitogenesis.

Tumor necrosis factor (TNF) is a key inflammatory cytokine that plays a major role in combating infections. Its dysregulation, however, can lead to uncontrolled inflammation which may cause deleterious conditions such as septic shock. Therefore, the expression of TNF is tightly regulated at both the transcriptional and post-transcriptional level. AU-rich elements, present in the 3'-untranslated region of TNF mRNA, are primarily known for their ability to stimulate rapid mRNA decay. ARE-binding proteins such as TTP and AUF-1 mediate this process. We have identified a new protein, Fragile X-related protein 1 (FXR1P), which binds to the ARE of TNF mRNA. The generation of a macrophage cell line, in which the Fxr1 gene is ablated, resulted in higher expression of TNF protein without affecting TNF mRNA steady state level or stability. Analysis of polysomes showed that TNF mRNA was associated preferentially with heavy polysome fractions in macrophages cell lines derived from FXR1P knockout mice, indicating a translational repressor role for FXR1P. We next defined the role of FXR1P in TNF induction by Toll-like receptor (TLR) ligands other than LPS (a TLR4 ligand). The effects of S28463, a synthetic TLR7 ligand, and CpG, a TLR9 ligand, were tested as they have potential clinical therapeutic properties. FXR1P ablation in macrophages did not alter TNF expression compared with wildtype macrophages stimulated with S28463. However, FXR1P-deficient macrophages stimulated with CpG had significantly higher TNF secretion than wildtype cells. Although CpG-stimulated FXR1P-KO cells had higher TNF mRNA steady state levels, no difference in TNF mRNA stability was observed compared with wil / Le facteur onconécrosant de tumeur (TNF) est une cytokine inflammatoire qui joue un rôle important pour combattre diverses infections. La dérégulation de TNF peut cependant entraîner une inflammation non-contrôlée qui est à l'origine de diverses conditions pathologiques. L'expression de TNF est donc fortement régulé aux niveaux transcriptionnels et post-transcriptionnels. Les AREs (éléments riches en adénine\uridine) qui sont présents dans la région 3' non-traduite de l'ARNm de TNF sont reconnus pour leur capacité à stimuler rapidement la dégradation de ces ARNm. Plusieurs protéines se liant aux AREs ont la capacité de médier ce processus. Nous avons identifié une nouvelle protéine, FXR1P (Fragile X-related protein 1) capable de se lier à l'ARE de l'ARNm de TNF. L'utilisation d'une lignée cellulaire de type macrophage provenant de souris où le gène Fxr1 est inactivé a résulté en une expression plus élevée de la protéine TNF sans affecter les niveaux d'ARNm ni la stabilité de ceux-ci. L'analyse de polyribosomes a démontré que l'ARNm de TNF est associé de façon préférentielle avec les fractions lourdes des polyribosomes dans les macrophages FXR1-KO. Ceci indique un rôle de répression de la traduction de l'ARNm de TNF pour FXR1P. Nous avons par la suite définie le rôle de FXR1P lors de l'induction de TNF par les ligands de TLR (récepteur ressemblant à Toll) autres que LPS. Les effets de S28463 (TLR7-L) et de CpG (TLR9-L) ont été testé parce qu'ils possèdent des propriétés thérapeutiques potentielles. La stimulation avec S28463 n'a pas entrainé de différence d'expression de TNF entre les macrophages FXR1-KO et FXR1-WT.

The WT1 gene encodes a transcription factor that is involved in the etiology of the nephroblastoma, Wilms' Tumor, and its associated diseases. Protein-protein interactions have been shown to alter the transcription regulatory functions of WT1 and implicate WT1 in the post-transcriptional event, splicing. Modulation of WT1 activity by its interacting partner and vice versa can extend the functional capacity of both proteins. A new potential WT1-interacting protein, Bone Marrow Zinc Finger 2 (BMZF2 or ZNF255), was isolated by affinity chromatography from HeLa cell nuclear extracts and subsequently identified by mass spectrometry analysis. Amino acid sequence analysis of BMZF2 suggests that it is a potential transcription factor with DNA-binding abilities. GST pulldown experiments indicate that BMZF2 binds the zinc finger region of the WT1 (-KTS) isoform in vitro. Because BMZF2 targets the WT1 (-KTS) zinc finger domain, it may be capable of influencing WT1's transcriptional regulatory functions. The expression patterns of BMZF2 and WT1 suggest that they may be important to the regulation of mammalian development and hematopoiesis.

NCoR and SMRT are corepressors that interact with the ligand-binding domain (LBD) of retinoic acid receptors (RARs) in the absence of ligand to repress transcription. This interaction is allowed through the presence of small alpha-helical motifs found in corepressors that interact with residues lining the hydrophobic pocket of the RARs. Unlike RARalpha and RARgamma, RARbeta weakly interacts with NCoR and SMRT. Here we show that the determinants in RARalpha present in helix 1 (H1) (I188), H7 (A311), H9 (D346), and H10 (M377) are important for corepressor binding. Substitution of these amino acids with RARbeta corresponding residues decreased interaction with SMRT. Moreover, gain of function experiments showed that introduction of some of these determinants in RARbeta resulted in an improved ability to interact with SMRT. Furthermore, the hinge region of RARalpha is very important for the stability of the LBD and corepressor binding. In a mammalian two-hybrid assay, we show that in the presence of NCoR, RARbeta hinge region could not stabilize as much its LBD as RARalpha. However, RARalpha hinge efficiently stabilized the LBD of RARbeta in the presence of the corepressor. We also show that the RARbeta H12 inhibits interaction with corepressors. Deletion of H12 rescued these interactions both in solution and on DNA, and rendered RARbeta a potent repressor in vivo. These results suggest that RARbeta, although having a high degree of identity with RARalpha, has major conformational differences that affect its ability to interact with corepressors. All together, these modified receptors will be useful tools to identify determinants in RARs important for corepressor interaction and help elucidate the precise molecular mechanism by which RARbeta acts as a tumor suppressor.