The hsFLJ22386 gene, an unknown gene, was located on chromosome 16p13.3. Its protein product contains 287 amino acids with the molecular weight of 32.2 kDa approximately. Predicted by bioinformatics, hsFLJ22386 might be a protein containing a leucine zipper domain. Based on the results of reverse transcriptase polymerase chain reaction, it revealed that FLJ22386 was expressed in several nervous system tissues and several organ tissues (liver, spleen, small intestine and kidney). In human cancer cell lines, the RT-PCR results showed that FLJ22386 was expressed in brain tumor cell lines (T98G, U87MG, U251, GBM8401), nasopharyngeal epithelial cell line (NNE-3)and carcinoma cell lines (NPCTW01, NPCTW04), hepatoma cell lines (J5, Hep3B, SK-Hep-1) and lymphoma cell lines (RPMI, P3HR1, Raji, U937). Human FLJ22386 coding sequence was inserted into pEGFP-C2 plasmid, and the tag-fused gene was transfected into NIH3T3 cells to see if it has the ability to promote cell proliferation. To further investigate the protein level expression and biological functions of the gene, glutathione S-transferase-hsFLJ22386 fusion protein was expressed and used to generate anti-FLJ22386 polyclonal antibody. According to the results of RT-PCR and anchorage dependent growth assay, it is presumed that FLJ22386 may play a role in cell proliferation.

Jun and Fos are components of the AP1 family of transcription factors that bind to the promoters of a diverse multitude of genes involved in critical cellular responses such as cell growth and proliferation, cell cycle regulation, embryonic development and cancer. The specific protein-DNA interactions are driven by the binding of basic zipper (bZIP) domains of Jun and Fos to TPA response element (TRE) and cAMP response element (CRE) within the promoters of target genes. Here, using a diverse array of biophysical techniques, including in particular isothermal titration calorimetry in conjunction with molecular modeling and semi-empirical analysis, I characterize AP1-DNA interactions in thermodynamic and structural terms. My data show that the binding of bZIP domains of Jun-Fos heterodimer to TRE and CRE are under enthalpic control accompanied by entropic penalty at physiological temperatures. This is in agreement with the notion that protein-DNA interactions are largely driven by electrostatic interactions and intermolecular hydrogen bonding. A larger than expected heat capacity change suggests that the basic regions within the bZIP domains are unstructured in the absence of DNA and interact in a coupled folding and binding manner. Further analysis demonstrates that Jun-Fos heterodimer can tolerate single nucleotide variants of the TRE consensus sequence and binds in the biologically relevant micromolar to submicromolar range. Of particular interest is the observation that the Jun-Fos heterodimer binds to specific variants in a preferred orientation. 3D atomic models reveal that such preference in orientation results from asymmetric binding and may in part be attributable to chemically distinct but structurally equivalent residues within the basic regions of Jun and Fos. I further demonstrate that binding of the biologically relevant Jun-Jun homodimer to TRE and CRE occurs with favorable enthalpic contributions accompanied by entropic penalty at physiological temperatures in a manner akin to the binding of Jun-Fos heterodimer. However, anomalously large negative heat capacity changes provoke a model whereby Jun loads onto DNA as unfolded monomers coupled with subsequent folding and homodimerization upon association. The data also reveal that the heterodimerization of leucine zippers is modulated by the basic regions and these regions may undergo at least partial folding upon heterodimerization. Large negative heat capacity changes accompanying the heterodimerization of leucine zippers are consistent with the view that leucine zippers do not retain a-helical conformation in isolation and the formation of the native coiled coil a-helical dimer is attained through a coupled folding-dimerization mechanism. Taken together, this dissertation marks the first comprehensive thermodynamic analysis of an otherwise well-studied and vitally important transcription factor. My studies shed new light on the forces driving the AP1-DNA interaction in thermodynamic and structural terms. The implications of these novel findings on the development of novel therapies for the treatment of disease with greater efficacy coupled with low toxicity cannot be overemphasized.

Luman (LZIP/CREB3) is a basic leucine zipper (bZIP) transcription factor that has been linked to the endoplasmic reticulum (ER) stress response. In the event of ER stress, Luman is proteolytically cleaved, or ‘activated’, through regulated intramembrane proteolysis (RIP), resulting in an amino-terminal fragment that translocates to the nucleus to activate transcription of downstream unfolded protein response (UPR)-related genes. The general mode of activation of the key signal transducers of the UPR appears to be an alteration of their oligomeric states. Structural and functional similarities to these proteins suggest that Luman may be activated in a similar manner. In this thesis, we demonstrate through in vitro and in vivo studies that Luman can form homodimers in the cell. Through the use of mutagenesis, we show that Luman dimerization is mediated through the leucine zipper and we provide evidence that Luman dimerization is required for its transcription activation function. However, we found that Luman dimerization is not required for its activation by proteolytic cleavage.

Une cellule dendritique (CD) qui exprime le facteur de transcription GILZ durant l’apprêtement de l’antigène et sa présentation aux cellules effectrices, génère des lymphocytes T régulateurs (LTregs) CD25high Foxp3+ sécréteurs d’IL-10. La production de GILZ est dépendante de l’action des glucocorticoïdes, de l’IL-10 et du TGF-.Nous avons mis en évidence chez l’homme qu’une corticothérapie orale de 48h induit l’expression de GILZ dans les cellules présentatrices de l’antigène circulantes (CPAs) de sujets allergiques. Les CPAs isolées après la corticothérapie génèrent des LTregs CD25high Foxp3+ IL-10+ spécifiques de l’allergène.Nous également constaté in vitro que les mastocytes participent à l’activation des CDs au cours des réactions allergiques en régulant l’expression de GILZ. Les médiateurs d’origine mastocytaire, dont l’histamine, diminuent l’expression de GILZ dans les CDs et altèrent ainsi leur capacité à activer des LTregs. Nous avons identifié le mécanisme par lequel l’histamine diminue l’expression de GILZ dans les CDs humaines. L’histamine inhibe l’activité transcriptionnelle de Foxo3, un facteur de transcription régulant l’expression de GILZ. Enfin, nous avons démontré que des souris transgéniques qui surexpriment GILZ constitutivement dans les CDs sont protégées contre le développement de l’asthme allergique. L’ensemble de ces résultats permet d’envisager de nouvelles stratégies d’immunomodulation dans l’allergie, centrée sur la régulation de l’expression de GILZ dans les CDs. / We previously showed in vitro that DCs with a high level of GILZ activate regulatory T cells (Tregs) whereas DCs with low level of GILZ trigger effector T lymphocytes. Glucocorticoids (GCs), IL-10 and TGF- are potent inducers of GILZ expression. The aim of this thesis is to extend the above findings to induction of tolerance to allergens. Modulation of GILZ expression by DCs should induce allergen-specific Tregs able to inhibit the activation and proliferation of allergen specific T cell clones. In order to validate this concept we demonstrated that:- allergen-specific tolerance can be achieved in allergic patients treated with oral GC through the induction of GILZ expression in their antigen-presenting cells, and the role of allergen-specific Tregs in this effect,- mast cells play a role in the activation of DCs by inhibiting their expression of GILZ and thus their ability to stimulate Tregs against harmless environmental allergens,- GILZ-expressing DCs protect against allergic asthma in a model of transgenic mice over-expressing GILZ in their DCs.The present study supports the concept of an immune regulation of allergic responses through the modulation of GILZ expression by DCs and opens new perspectives in the development of innovative immunotherapies in the treatment of allergic diseases.

La leucine zipper de la rétine neurale (Nrl) joue un rôle central dans le développement et l'homéostasie des bâtonnets en activant I'expression de gènes tels que le photopigment Rhodopsine. Nrl est aussi associé à la Rétinite Pigmentaire, faisant ainsi de ce gène un modèle intéressant pour la compréhension des programmes contrôlant le développement et I'homéostasie des photorécepteurs.Ce travail de thèse vise à caractériser les mécanismes régulateurs de I'expression de Nr/ au cours du développement rétinien. L'électroporation in vivo de vecteurs rapporteurs dans des rétines de souris en développement, a révélé des séquences minimales de promoteur Nr/ nécessaires à une expression spécifique dans les photorécepteurs. Nous avons identifié RORI3 comme facteur requis pour cette expression, et montré que les facteurs OTX2, CRX et CREB s'accrochent aussi directement à des régions régulatrices particulières du promoteur. Nous avons construit un virus adéno-associé (AAV) contenant un promoteur minimal Nrl de 0.3 kb, et montré qu'il est adapté à la délivrance de gène spécifiquement dans les photorécepteurs.Nous avons montré que NRL, CRX et NR2E3, les régulateurs principaux de la Rhodopsine, ont une expression rythmique au cours de 24 h, et que l'expression cyclique de Nr/ peut être due à l'activation par RORp, un composant l'horloge circadienne. Enfin, nous avons identifié un nouveau facteur de transcription, NonO, au niveau de la région du promoteur proximal de la Rhodopsine, qui en combinaison avec NRL et CRX, active le promoteur de la Rhodopsine. L'invalidation de NonO au cours du développement rétinien a prouvé son implication pour le développement et I'homéostasie des bâtonnets. / The Neural Retina Leucine zipper transcription factor (Nrl) plays a central role in rod photoreceptor development and homeostasis, by activating the expression of rod-specific genes such as the visual photopigment, Rhodopsin. Nrlhave been also associated with Retinitis Pigmentosa, making this gene an interesting model for understanding genetic programs controlling photoreceptors development and homeostasis.This thesis work aimed at characterizing regulatory mechanisms of Nr/ expression during retinal development. Using in vivo electroporation of reporter vectors carrying distinct portions of Nrlpromoter into neonatal mouse retina, we identified minimal sequences required for expression photoreceptors-specific expression. We identified RORI3 as being required for this expression and showed that OTX2, CRX and CREB transcription factors also directly bind to the defined regulatory regions.We designed a novel adeno-associated virus (AAV) vector containing a minimal Nrl promoter fragment of 0.3 kb, and showed that it is well-suited for gene delivery specifically into photoreceptors.We also showed that NRL, CRX, and NR2E3, the main transcriptional regulators of Rhodopsin, display rhythmic expression over 24 h. and that Nrl might undergo cyclic activation by RORB which is part of the photoreceptor circadian clock. Finally, we investigated the role of a novel Rhodopsin transcriptional regulator, NonO, identified in theRhodopsin proximal promoter region. We demonstrated that NonO co-activates Rhodopsin promoter along with NRL and CRX. By knocking down this gene during retinal development we provided evidence for its role in rod development and homeostasis.

Ce projet de maitrise s’inscrit dans le cadre de l’étude du rôle de DLK dans les neurones. Depuis quelques années seulement, plusieurs auteurs ont démontré l’implication de DLK dans les mécanismes de dégénérescence et de régénérescence neuronale, des processus pouvant survenir lors du développement ou dans le cas de certaines pathologies reliées au système nerveux, comme l’Alzheimer et le Parkinson. Jusqu’à maintenant, peu de travaux sur la régulation de l’activité de DLK dans les cellules nerveuses ont été rapportés dans la littérature. Au cours de ce travail, nous avons démontré que la voie de signalisation DLK/JNK est activée suite à la dépolymérisation des microtubules, des composants majeurs du cytosquelette souvent affectés lors de stress infligés aux cellules. Cette démonstration s’est fait grâce aux traitements des cellules Neuro-2a avec la colcémide, suivis par des immunobuvardages de type Western dont l’anticorps était dirigé contre la forme phosphorylée de JNK (p-JNK), une cible majeure de DLK. Nous avons par la suite été en mesure de confirmer que l’activation de JNK était bel et bien DLK-dépendante à l’aide de cellules déplétées en DLK par des ARN interférents. Dans ces cellules, les niveaux de p-JNK étaient significativement inférieurs, comparés aux cellules contrôles, et ce même lorsqu’on active la voie de signalisation. Nous avons par la suite identifié, à l’aide d’une méthode de séquençage de nouvelle génération, le «RNA-seq», des gènes étant différentiellement exprimés dans les cellules déplétées en DLK. Nous avons été en mesure d’établir un lien entre certains des gènes régulés par DLK et le guidage axonal, un processus essentiel au développement neuronal. Donc, en plus d’avoir démontré un mécanisme possible de régulation de DLK, nous avons, pour la première fois, identifié des gènes régulés par la présence/absence de cette dernière dans les cellules neuronales de mammifères.

Glucocorticoids (GCs) are frequently prescribed pharmacological agents most notably for their immunosuppressant effects. Endogenous GCs mediate biological processes such as energy metabolism and tissue development. At the cellular level, GCs bind to the Glucocorticoid Receptor (GR), a cytosolic receptor that translocates to the nuclei upon ligand binding and alters gene transcription. Among a long list of genes activated by GCs is the Glucocorticoid Induced Leucine Zipper (GILZ). Although GC induced GILZ expression has been well established in lymphocytes, little is known whether cardiomyocytes respond to GCs by inducing GILZ. Unlike lymphocytes, in which GCs induce apoptosis and GILZ mediates GC induced apoptosis, cardiomyocytes respond to GCs by gaining resistance against apoptosis. We determined GILZ expression pattern in cardiomyocytes in vivo and in vitro. Our data demonstrate GILZ induction in cardiomyocytes both in vivo and in vitro by GCs and point to H9C2 cell line as a valid model for studying the biological function of GILZ in cardiomyocytes. I have also determined GILZ functions as GC induced cytoprotective protein against the known cardiac toxicant Doxorubicin. Finally I have determined GILZ stabilizes Bcl-xL pro-survival protein, providing a possible mechanism of cytoprotection in cardiomyocytes.

The homeodomain leucine zipper (HDZip) proteins constitute a plant-specific family of transcription factors, that based on sequence criteria have been grouped into four classes, HDZip I-IV. This thesis describes the phylogeny, function, expression patterns and regulation of the HDZip class I genes in the model species Arabidopsis thaliana. The phylogenetic analyses, traced duplication history and exon/intron organisation of the 17 class I genes in Arabidopsis show that the genes form six monophyletic groups, clades, with an origin in early plant evolution. All genes are expressed in broad tissue distribution patterns and the majority are responsive to water availability and/or light conditions. The expression of the genes show different patterns and dependence on environmental stress conditions, indicating evolutionary changes within and between clades. Ectopic expression of the genes suggest that they regulate genes in part by conserved mechanisms. Therefore, different functional roles seem to have evolved by a divergence mainly in the regulatory properties of the genes. Detailed expression analyses of the paralogous HDZip I genes ATHB7 and ATHB12 show that they have essentially overlapping patterns of activity in response to abscisic acid, ABA, or water deficit in leaves, stems and roots. The water deficit response of ATHB7 and -12 is mediated by ABA and depends on the protein phosphateses ABI1 and ABI2. Transgenic plants with ectopic expression of ATHB7 and/or -12, and athb7 and athb12 mutants, reveal that the genes in roots mediate the growth inhibitory effects of ABA. In this aspect of their function they do not overlap. In leaves and stems, the genes might act as growth regulators redundantly with other factors. Taken together these data suggest that ATHB7 and -12 regulate growth in response water deficit and that other HDZip I genes have related functions in response to environmental stress conditions.

This thesis explores how basic region/leucine zipper (bZIP) transcription factors target gene regulatory sequences. The GCN4 bZIP binds to more than one target site [CRE (TGACGTCA) and cognate AP-1 (TGACTCA)] and exhibits flexibility in -helical structure. These observations suggest that the GCN4 bZIP can establish sequence-selective DNA binding at noncognate target sites. Studies on such noncognate but sequence-selective binding can provide insights into how bZIP proteins search for and localize to their cognate target sites. This thesis investigates DNA binding by the GCN4 bZIP and its structural and functional mimic, the wild-type (wt) bZIP, at noncognate gene regulatory sequences C/EBP (TTGCGCAA), E-box (CACGTG), HRE (GCACGTAG), XRE1 (TTGCGTGA), and related DNA sequences. These DNA-binding activities are sequence-selective, as confirmed by DNase I footprinting and electrophoretic mobility shift assay (EMSA). Full- and half-site DNA-binding affinities, determined by EMSA titrations, decrease from cognate to noncognate binding. At noncognate target sites, the bZIP proteins form a dimer of -helices, as indicated by circular dichroism (CD) spectroscopy and EMSA. These results demonstrate that the bZIP proteins can establish noncognate but sequence-selective DNA binding, and suggest such DNA binding potentially contributes to structure preorganization and rapid translocation of the bZIP proteins when they search for their cognate target sites, to which they then bind with high affinity. This thesis also indicates a highly dynamic DNA-binding model for the bZIP proteins to establish strong and sequence-selective DNA binding. The C/EBP site includes two 5H-LR (TTGCG) half-sites, each of which comprises two 4-bp subsites. The in vitro and in silico results together demonstrate that the basic region at 5H-LR recognizes the 4-bp subsites alternately as distinct units, which requires it to translocate between the subsites, potentially by sliding or hopping. Taken as a whole, this thesis provides further insights into how bZIP transcription factors accomplish sequence-selective DNA binding.

This thesis explores how basic region/leucine zipper (bZIP) transcription factors target gene regulatory sequences. The GCN4 bZIP binds to more than one target site [CRE (TGACGTCA) and cognate AP-1 (TGACTCA)] and exhibits flexibility in -helical structure. These observations suggest that the GCN4 bZIP can establish sequence-selective DNA binding at noncognate target sites. Studies on such noncognate but sequence-selective binding can provide insights into how bZIP proteins search for and localize to their cognate target sites. This thesis investigates DNA binding by the GCN4 bZIP and its structural and functional mimic, the wild-type (wt) bZIP, at noncognate gene regulatory sequences C/EBP (TTGCGCAA), E-box (CACGTG), HRE (GCACGTAG), XRE1 (TTGCGTGA), and related DNA sequences. These DNA-binding activities are sequence-selective, as confirmed by DNase I footprinting and electrophoretic mobility shift assay (EMSA). Full- and half-site DNA-binding affinities, determined by EMSA titrations, decrease from cognate to noncognate binding. At noncognate target sites, the bZIP proteins form a dimer of -helices, as indicated by circular dichroism (CD) spectroscopy and EMSA. These results demonstrate that the bZIP proteins can establish noncognate but sequence-selective DNA binding, and suggest such DNA binding potentially contributes to structure preorganization and rapid translocation of the bZIP proteins when they search for their cognate target sites, to which they then bind with high affinity. This thesis also indicates a highly dynamic DNA-binding model for the bZIP proteins to establish strong and sequence-selective DNA binding. The C/EBP site includes two 5H-LR (TTGCG) half-sites, each of which comprises two 4-bp subsites. The in vitro and in silico results together demonstrate that the basic region at 5H-LR recognizes the 4-bp subsites alternately as distinct units, which requires it to translocate between the subsites, potentially by sliding or hopping. Taken as a whole, this thesis provides further insights into how bZIP transcription factors accomplish sequence-selective DNA binding.