There are approximately 20,000 genes in the human genome. Around 2% of these genes code for transcriptional repressors known as KRAB-ZFPs. It is already known that Zinc-Finger Proteins contain two main functional domains at either end of the polypeptide. In today's database, you will find a KRAB (Kruppell-associated Box) domain at one end and a tandem array of Zinc-finger repeats at the other end. The carboxyl terminal tandem Zinc-finger repeats function as sequence-specific DNA-binding domains. The amino terminal KRAB domain serves as a repressor domain, which will recruit a co-repressor termed KAP-1 (KRAB Associated Protein-1). Located in between these two domains is a region of uncharacterized DNA referred to as the "Linker Region". This thesis will explore the DNA-binding domains of 6 known KRAB-ZFPs, as well as utilize the linker regions to derive an evolutionary history for this superfamily. / by Alain Chamoun. / Thesis (M.S.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

L’ARN polymérase III synthétise de nombreux petits ARN non traduits, dont les ARNt et l’ARNr 5S, essentiels à la croissance de toute cellule. Dans ce travail, nous nous sommes intéressés à la régulation de la transcription par l’ARN polymérase III chez la levure Saccharomyces cerevisiae. Nous avons détecté Sub1 sur les gènes de classe III in vivo. Nous avons également observé que Sub1 est capable de stimuler la transcription par l’ARN III reconstituée in vitro avec les facteurs TFIIIB et TFIIIC recombinants et avec l’ARN Pol III purifiée. Sub1 stimule deux étapes de la transcription : l’initiation et la réinitiation facilitée. Des expériences supplémentaires nous montrent que la protéine interagit directement avec TFIIIB et TFIIIC. Enfin, nous avons pu constater que la délétion de Sub1 dans la levure conduit à une diminution de la transcription par l’ARN Pol III en phase exponentielle de croissance. Par la suite, nous avons cherché à déterminer quel lien pouvait exister entre l’activateur Sub1 et le répresseur Maf1 de la transcription par l’ARN Pol III. Enfin, nous avons également souhaité identifier d’autres éléments pouvant interagir avec la protéine Sub1 au cours de sa fonction de régulateur. / RNA polymerase III synthetizes many small untranslated RNA, including tRNA and 5S rRNA which are essential to cell growth. In this work, we took an interest in RNA polymerase III transcription regulation in the baker’s yeast, Saccharomyces cerevisiae. We have detected Sub1 on all class III genes in vivo. We also observed that Sub1 is able to stimulate RNA polymerase III transcription which has been reconstituted in vitro with TFIIIB et TFIIIC recombinants factors and purified RNA polymerase III. Sub1 stimulates two steps of RNA polymerase III transcription : initiation and facilitated reinitiation. Supplementary experiments established that Sub1 directly interacts with TFIIIB and TFIIIC transcription factors. Finally, we showed that Sub1 deletion in yeast leads to a decrease in RNA polymerase III transcription during exponential phase. Then, we tried to determine which link could exist between Sub1, the activator, and Maf1, the repressor of RNA polymerase III transcription. Furthermore, we attempted to identify other elements which could interact with Sub1 during transcription regulation.

Alors que le complexe snRNP est bien décrit chez les vertébrés, il nécessite plus d’études chez les invertébrés. Le snARN 7SK sert de maintient structural pour la fixation d’HEXIM à P-TEFb. En retour, HEXIM inhibe l’activité kinase de CDK9 via une fixation directe avec la Cycline T. En conséquence, les interactions entre le snARN 7SK et HEXIM va piéger le complexe P-TEFb sous une forme inactive qui conduit à inhiber l’élongation transcriptionnelle. Dans notre étude, nous montrons qu’un contrôle de l’activité P-TEFb existe aussi chez la Drosophile. Et la dynamique d’équilibre entre les deux formes de P-TEFb dépend également du snARN 7SK. Ce modèle est donc utilisé pour étudier le rôle biologique de la snRNP, et plus spécialement d’HEXIM, dans un contexte intégré. Nous avons donc analysé le profile d’expression d’HEXM durant le cycle de vie de la Drosophile et plus particulièrement pendant l’embryogenèse et l’organogenèse. L’expression permanente et ubiquitaire d’HEXIM suggère qu’elle est nécessaire au développement. Le fait que la perte de fonction d’HEXIM mène à de nombreux et sévères défauts confirme cette hypothèse. En utilisant le modèle des disques imaginaux de l’aile et de l’œil, nous avons étudié plus en profondeur le rôle d’HEXIM et nous avons montré qu’elle est essentielle pour la viabilité cellulaire. De plus, la perte de fonction d’HEXIM conduit à des changements du destin cellulaire et à des modifications des profiles d’expression de plusieurs gènes sélecteurs ou de morphogènes. De façon surprenante, la diminution d’HEXIM induit l’accumulation de Ci155 qui est requise pour activer l’expression de Ptc, ainsi que l’activation ectopique de la voie Hh. Cette accumulation notable de Ci155 est également détectée dans les cellules “immortelles” et dans les tissus en cours de régénération à la suite d’une ablation par voie génétique. Sur la base de ces données, nous proposons un rôle possible de l’accumulation de Ci155 dans le phénomène de prolifération compensatrice. Finalement, nous avons caractérisé un nouvel analogue du snARN 7SK chez la Drosophile, qui a été nommé dm7SK-like snARN. Ce dernier a une structure secondaire très similaire à celle de ces homologues vertébrés, alors que la séquence primaire est assez différente. De plus, presque tous les domaines structuraux importants pour les interactions avec HEXIM et les autres partenaires sont conservés chez cet ARN. Des interactions directes ont été démontrées entre HEXIM et cet ARN suggérant qu’il est un analogue structural du snARN 7SK. Ainsi, la présence de deux analogues du snARN 7SK suggère un autre niveau de régulation de l’expression des gènes, au moins chez la Drosophile. / Whereas 7SK snRNP complex has been well characterized in vertebrates, its activities still remain to be further elucidated in invertebrates. 7SK snRNA serves as a structural scaffold for the efficient binding of HEXIM to P-TEFb. HEXIM in turn inhibits the kinase activity of CDK9 via its direct binding to CyclinT. Consequently, the interaction between 7SK snRNA and HEXIM sequesters the active P-TEFb complex into the inactive form, thereby suppressing the transcription elongation. In this study, we first show that a similar P-TEFb control system exists in Drosophila. In addition, the dynamic equilibrium of the two complexes of P-TEFb in Drosophila also depends on 7SK snRNA. Thank to this similarity, we are able to examine the biological role of 7SK snRNP complex, especially HEXIM protein, in an integrative organism as Drosophila model. We next document the expression profile of HEXIM throughout the life cycle of Drosophila, especially during embryogenesis and organogenesis. The continuous and ubiquitous expression of HEXIM suggests its necessity during development. We demonstrate that HEXIM is indeed essential for the proper development of Drosophila, since its down-regulation results in numerous severe defects. By using wing and eye imaginal discs as study models, we further examine biological roles of HEXIM, and reveal that it is required for cell viability. Moreover, HEXIM knockdown leads to changes in cell fate commitments, and modifications in expression patterns of several selector genes and morphogens. Strikingly, down-regulation of HEXIM significantly induces the accumulation of Ci155, which is required for Ptc expression, and the ectopic activation of Hh signaling. This remarkable accumulation of Ci155 is also detected in “undead cells” and regenerated tissue upon genetic ablation. Given these findings, we thus propose a putative role of Ci155 accumulation in compensatory proliferation. Finally, we characterize a novel analog of 7SK snRNA in Drosophila, which is named dm7SK-like snRNA. This snRNA displays a very similar secondary structure with its vertebrate homologs, although the primary sequence is relatively different. More importantly, almost all of the structural elements crucial for the interaction with HEXIM and other partners are found conserved in this novel dm7SK-like snRNA. A direct interaction between dHEXIM and this snRNA also suggests that it is a functional analog of 7SK snRNA in Drosophila. Thus, the intriguing finding of the two analogs of 7SK snRNA would propose another regulation level of gene expression, at least in Drosophila.

Abstract not available

<p> <p>&nbsp / </p> </p> <p align="left">This study utilized large-scale gene expression data to define the regulatory networks of genes expressing in the hippocampus to which multiple disease pathologies may be associated. Specific aims were: ident i fy key regulatory transcription factors (TFs) responsible for observed gene expression patterns, reconstruct transcription regulatory networks, and prioritize likely TFs responsible for anatomically restricted gene expression. Most of the analysis was restricted to the CA3 sub-region of Ammon&rsquo / s horn within the hippocampus. We identified 155 core genes expressing throughout the CA3 sub-region and predicted corresponding TF binding site (TFBS) distributions. Our analysis shows plausible transcription regulatory networks for twelve clusters of co-expressed genes. We demonstrate the validity of the predictions by re-clustering genes based on TFBS distributions and found that genes tend to be correctly assigned to groups of previously identified co-expressing genes with sensitivity of 67.74% and positive predictive value of 100%. Taken together, this study represents one of the first to merge anatomical architecture, expression profiles and transcription regulatory potential on such a large scale in hippocampal sub-anatomy.</p>

<p>This thesis reports on the development of a novel comprehensive database (Dragon Database of Genes Implicated in Esophageal Cancer, DDEC) as an integrated knowledge database aimed at representing a gateway to esophageal cancer related data. More importantly, it illustrates how the biocurated genes in the database may represent a reliable starting point for divulging transcriptional regulation, diagnostic markers and the biology related to esophageal cancer.</p>

<p>This thesis reports on the development of a novel comprehensive database (Dragon Database of Genes Implicated in Esophageal Cancer, DDEC) as an integrated knowledge database aimed at representing a gateway to esophageal cancer related data. More importantly, it illustrates how the biocurated genes in the database may represent a reliable starting point for divulging transcriptional regulation, diagnostic markers and the biology related to esophageal cancer.</p>

Today, mankind faces a new challenge in energetic terms: a new Industrial Revolution is imperative, already called by some as an Energetic Revolution. This corresponds to a conversion to clean, environmentally friendly and renewable energy sources. In this context, hydrogen arises as a valid alternative, since its combustion produces a considerable amount of energy and releases solely water as a by-product. In the present thesis, two model cyanobacteria, namely Synechocystis sp. strain PCC 6803 and Anabaena/Nostoc sp. strain PCC 7120, were used to examine the hydrogen metabolism. The efforts were focused on to understand the transcription regulation of the hox genes, encoding the structural elements of the bidirectional hydrogenase enzyme. Here, it is shown that such regulation is operated in a very distinct and intricate way, with different factors contributing to its delicate tuning. While in Synechocystis sp. strain PCC 6803 the hox genes were shown to be transcribed as a single operon, in Anabaena/Nostoc sp. strain PCC 7120 they were shown to be transcribed as two independent operons (possibly three). Two transcription factors, LexA and AbrB-like protein, were identified and further characterized in relation to the hydrogen metabolism. Furthermore, different environmental conditions were demonstrated to operate changes on the transcription of the bidirectional hydrogenase genes. In addition, functional studies of three open reading frames found within the hox operon of Synechocystis sp. strain PCC 6803 suggest that this may be a stress responsive operon. However, based on the gained knowledge, it is still not possible to connect the signal transduction pathways, from the environmental signal, through the response regulator, to the final regulation of the hox genes. Nevertheless, the crucial importance of studying the transcription regulation of the different players involved in the hydrogen metabolism is now established and a new era seems to be rising.

The CCAAT/Enhancer Binding Protein Beta (C/EBPβ) is part of the leucine zipper family of transcription factors and is involved in a myriad of processes including cellular proliferation and differentiation. C/EBPβ is expressed as three isoforms (LAP*, LAP, LIP), translated from a single mRNA by a leaky ribosomal scanning mechanism. While LAP* and LAP have activating functions, LIP is recognized as being a repressor of transcription due to its lack of activation domains. Numerous studies have shown that C/EBPβ acetylation state modulates its activity in a promoter-specific manner. For instance, the acetyltransferases GCN5/PCAF and the deacetylase complex mSin3A/HDAC1 regulate C/EBPβ activity on the C/EBPa promoter. GCN5/PCAF-mediated acetylation of C/EBPβ was shown to positively affect its transcriptional activity in a steroid-dependent mechanism via the glucocorticoid receptor (GR). GR relieves HDAC1 association from C/EBPβ by targeting the deacetylase for proteasomal degradation, hence favouring GCN5-mediated acetylation of C/EBPβ and allowing maximum activation capacity to be reached. In order to further elucidate C/EBPβ activation, I sought to characterize the interplay between GCN5 and HDAC1 in regulating C/EBPβ LAP/LIP activity during murine adipogenesis by identifying their binding domain in C/EBPβ. I identified a minimal domain located within regulatory domain 1 (RD1) of C/EBPβ that is required for both GCN5 and HDAC1 binding. Furthermore, the loss of the identified domain in C/EBPβ appears to partially mimic the GR effect, thus giving C/EBPβ a higher basal transcriptional activity that accelerates NIH 3T3 and 3T3 L1 adipogenesis. Moreover, I also showed that the LIP isoform inhibitory mode of action is partially mediated through the mSin3A/HDAC1 repressor complex, which gives LIP an active repressor function. In addition to LIP inhibitory function, I also showed that a cysteine residue located in LAP* negatively regulates its transactivating function during murine adipogenesis. Although RD1 of C/EBPβ has been suggested to act as a negative regulatory domain, I showed that only five residues are responsible for most of its inhibitory effect. Hence, in an attempt to further define sub-domains within RD1, I characterized a new positive regulatory domain at its N-terminal region, which seems to be required for C/EBPβ activity in a promoter-specific manner. In conclusion, this study not only supports previously hypothesized mechanisms by which C/EBPβ is regulated, but it also redefines the contribution of LAP*, LAP and LIP in regulating transcription. Most importantly, the results emphasize the countless possibilities by which C/EBPβ transactivation potential could be modulated during cellular differentiation.

The CCAAT/Enhancer Binding Protein Beta (C/EBPβ) is part of the leucine zipper family of transcription factors and is involved in a myriad of processes including cellular proliferation and differentiation. C/EBPβ is expressed as three isoforms (LAP*, LAP, LIP), translated from a single mRNA by a leaky ribosomal scanning mechanism. While LAP* and LAP have activating functions, LIP is recognized as being a repressor of transcription due to its lack of activation domains. Numerous studies have shown that C/EBPβ acetylation state modulates its activity in a promoter-specific manner. For instance, the acetyltransferases GCN5/PCAF and the deacetylase complex mSin3A/HDAC1 regulate C/EBPβ activity on the C/EBPa promoter. GCN5/PCAF-mediated acetylation of C/EBPβ was shown to positively affect its transcriptional activity in a steroid-dependent mechanism via the glucocorticoid receptor (GR). GR relieves HDAC1 association from C/EBPβ by targeting the deacetylase for proteasomal degradation, hence favouring GCN5-mediated acetylation of C/EBPβ and allowing maximum activation capacity to be reached. In order to further elucidate C/EBPβ activation, I sought to characterize the interplay between GCN5 and HDAC1 in regulating C/EBPβ LAP/LIP activity during murine adipogenesis by identifying their binding domain in C/EBPβ. I identified a minimal domain located within regulatory domain 1 (RD1) of C/EBPβ that is required for both GCN5 and HDAC1 binding. Furthermore, the loss of the identified domain in C/EBPβ appears to partially mimic the GR effect, thus giving C/EBPβ a higher basal transcriptional activity that accelerates NIH 3T3 and 3T3 L1 adipogenesis. Moreover, I also showed that the LIP isoform inhibitory mode of action is partially mediated through the mSin3A/HDAC1 repressor complex, which gives LIP an active repressor function. In addition to LIP inhibitory function, I also showed that a cysteine residue located in LAP* negatively regulates its transactivating function during murine adipogenesis. Although RD1 of C/EBPβ has been suggested to act as a negative regulatory domain, I showed that only five residues are responsible for most of its inhibitory effect. Hence, in an attempt to further define sub-domains within RD1, I characterized a new positive regulatory domain at its N-terminal region, which seems to be required for C/EBPβ activity in a promoter-specific manner. In conclusion, this study not only supports previously hypothesized mechanisms by which C/EBPβ is regulated, but it also redefines the contribution of LAP*, LAP and LIP in regulating transcription. Most importantly, the results emphasize the countless possibilities by which C/EBPβ transactivation potential could be modulated during cellular differentiation.