Protéines difficiles à exprimer: un goulot d'étranglement pour la plupart des biologistes. J'ai choisi d'utiliser comme modèle d’étude Acanthamoeba polyphaga Mimivirus. Ce virus géant à ADN possède des protéines subissant des modifications post-traductionnelles, des structures multi-protéiques ou encore des voies enzymatiques jamais identifiées auparavant dans un virus, ce qui en font un modèle idéal pour l’étude de protéines récalcitrantes. Le but ultime de cette thèse, était de produire les protéines de capsides de Mimivirus. Le rôle de la protéine de capside dans l’assemblage de la particule virale, son infectivité et ses caractéristiques moléculaires sont d’une grande importance. Pour aller du gène à la protéine, J’ai participé à la compréhension de ce qui gouverne la terminaison de la transcription de Mimivirus et également participé à l'analyse globale du transcriptome au cours du cycle d'infection des amibes par Mimivirus. Nous avons montré que les transcrits de Mimivirus sont systématiquement polyadénylés dans des régions formant une structure secondaire en tige-boucle, même s’il n’existe pas de signal de polyadénylation canonique en amont. Nous en avons conclu que la polyadénylation de Mimivirus suit exclusivement une règle «épingle à cheveux». De plus, l’étude du transcriptome a révélé 3 phases temporelles distinctes dans le cycle infectieux: précoce, intermédiaire et tardive. Les transcrits de capsides sont tous exprimés durant la phase tardive mais leur profil d’expression ne sont pas superposables dans le temps. Les données de transcriptomique ont révélées la présence de plusieurs glycosyltransférases chez Mimivirus, dans la phase tardive du cycle, concomitant avec la production de la protéine de capside. Les informations recueillies sur l'expression des gènes à différents temps post-infection ont contribué à la conception de protocoles pour la production des protéines de capsides (la protéine majeure de capside (MCP) et ses paralogues) dans de systèmes eucaryote. / Difficult to express proteins: a bottleneck for most biologists. I have chosen to use Acanthamoeba polyphaga Mimivirus as my study model. This giant dsDNA virus possesses post-translationally modified proteins, multi-protein structures and enzyme pathways never before seen in a virus, which makes it ideal for refractory studies. The ultimate goal of my thesis was to produce the capsid proteins of Mimivirus. The role of the capsid protein in the assembly of the viral particle, its infectivity, and molecular features are of great importance. To go from gene to protein, I participated in the comprehension of what governs the post-transcriptional termination in Mimivirus and equally participated in the global analysis of the transcriptome during the infectious cycle of Acanthamoeba by Mimivirus. We have shown that the Mimivirus transcripts are systematically polyadenylated in the regions forming a stem-loop secondary structure; even when a canonical poyadenylation signal is absent We concluded that Mimivirus polyadenylation obeys a strict “Hairpin rule”. Moreover, the transcriptomic study revealed three distinct temporal phases: early, intermediate and late. The capsid transcripts are all expressed during the late phase but their expression profiles are not superimposable. The transcriptomic data also revealed the presence of several Mimivirus glycosyltransferases in the late temporal phase, concomitant with the capsid proteins. The expression data gathered throughout my thesis has contributed to the rational design of a protein production experiment to produce the major capsid protein and its three paralogs in eukaryotic systems.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010. / Härtill 2 uppsatser.

L'expression du gène Igf2, qui est soumis à l'empreinte génomique parentale chez les mammifères, est hautement régulée au cours du développement embryonnaire et de la période périnatale grâce à divers mécanismes transcriptionnels et post-transcriptionnels. Ces mécanismes mettent à contribution de longs ARN non codants produits au sein même du locus, dont le plus connu est l'ARN H19. En utilisant une approche de complémentation génétique par un transgène H19 dans myoblastes H19 KO de souris, nous démontrons l'existence de plusieurs nouveaux promoteurs d'Igf2. L'un de ces promoteurs, qui est conservé chez l'homme, peut être activé par un ARN ectopique antisens d'H19 (lncARN 91H) en dépit d'une méthylation complète de la région de contrôle empreinte située en cis sur le même allèle. Nous montrons également que les lncARN 91H présentent une certaine spécificité tissulaire et que leur transcription peut être initiée à partir des séquences conservées CS4, CS5 et CS9 situées en aval du gène H19. Quant à l'ARN H19, qui est l'ARN non codant majeur du locus, il semble pouvoir réguler ses transcrits antisens dans les myoblastes H19 KO complémentés par le transgène H19, mais surtout il participe activement à la régulation post-transcriptionnelle du gène Igf2 chez la souris. Nous observons en effet qu'il favorise la coupure endoribonucléolytique de l'ARN Igf2 par un mécanisme qui reste à découvrir. Enfin, nous mettons en évidence l'existence d'un l'arrêt de l'élongation de la transcription du gène d'Igf2, pour lequel nous proposons un modèle de régulation faisant intervenir un autre long ARN non codant du locus: le lncARN PIHit. Au-delà des mécanismes qui restent à explorer, nos résultats renforcent l'idée que la structure tridimensionnelle de la chromatine participe à la régulation de l'expression des gènes chez les mammifères. / In mammals, the expression of the Igf2 gene, which is subject to parental genomic imprinting, is tightly regulated during embryonic development and the perinatal period through several transcriptional and post-transcriptional mechanisms. These mechanisms are involving long non-coding RNAs (lncRNAs) produced within the locus; among them the best known is probably the H19 RNA. Using a genetic complementation assay consisting in transfections of an H19 transgene into H19 KO myoblasts, we discovered several novel Igf2 promoters in the mouse. One of these promoters, that is conserved in the human, can be activated by ectopic H19 antisens RNAs (91H lncRNAs) despite a complete methylation of the Imprinting-Control Region located in cis on the same allele. We also show that the 91H lncRNAs possess some tissue-specific features and that their transcription can be initiated from the CS4, CS5 and CS9 conserved sequences located downstream of the H19 gene. On the other hand, the H19 RNA, that is the major lncRNA of the locus, appears to regulate its antisense transcripts in H19 KO myoblasts complemented with the H19 transgene, but its major function seems to be in regulating post-transcriptionally the Igf2 gene expression. Indeed, we have observed that it favours the endoribonucleolytic cleavage of the Igf2 messenger RNAs through a mechanism that remains to be elucidated. Finally, we reveal the existence of a premature transcriptional elongation stop of the Igf2 gene, for which we propose a regulation model involving another lncRNA of the locus: the PIHit lncRNA. Beyond the mechanisms that remain to be explored, our results strengthen the idea that, in mammals, the three-dimensional organization of the chromatin is involved in regulating gene expression.

Made available in DSpace on 2014-06-12T15:50:15Z (GMT). No. of bitstreams: 2 arquivo1959_1.pdf: 2352925 bytes, checksum: 90760b286db4ed0dcc12ae48554413a9 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Estudos recentes têm demonstrado que as redes biológicas apresentam características nãoaleatórias, dentre as quais destacamos a arquitetura modular. Neste trabalho, estamos interessados na organização modular das redes de regulação transcricional (RRT), que modelizam as interações entre genes e proteínas que controlam a sua expressão no nível transcricional. Compreender os mecanismos de regulação transcricional é crucial para se explicar a diversidade morfológica e funcional das células. Nós nos propomos a abordar o problema da identificação de módulos regulatórios transcricionais, i.e. grupos de genes co-regulados e seus reguladores, com ênfase no aspecto computacional. Uma distinção importante deste trabalho é que estamos também interessados em estudar o aspecto evolutivo dos módulos transcricionais. Do ponto de vista biológico, a abordagem proposta está fundamentada em três premissas principais: (i) genes co-regulados são controlados por proteínas regulatórias comuns (fatores de transcrição FTs) e, portanto, eles devem apresentar padrões de sequência (motifs) comuns nas suas regiões regulatórias, que correspondem aos sítios de ligação desses FTs, (ii) genes co-regulados respondem coordenadamente a certas condições ambientais e de desenvolvimento e, logo, devem ser co-expressos sob essas condições, e (iii) uma vez que módulos transcricionais são presumivelmente responsáveis por funções biológicas importantes, eles estão sujeitos a uma maior pressão seletiva e, consequentemente, devem ser evolutivamente conservados. Nós definimos, portanto, o conceito de metamódulo regulatório transcricional (MMRT) como grupos de genes compartilhando motifs e exibindo um comportamento de expressão coerente em contextos específicos consistentemente em várias espécies e propomos modelos probabilísticos para descrever o comportamento modular em termos do compartilhamento de elementos regulatórios (motifs), da co-expressão e da conservação evolutiva das associações funcionais entre os genes com base em dados diversos tais como dados de sequência, de expressão e dados filogenéticos

Transcriptional regulatory complexes control gene expression patterns and permit cellular responses to stimuli. Deregulation of complex components upsets target gene expression and can lead to disease. This dissertation examines proteins involved in two distinct regulatory complexes: C-terminal binding protein (CtBP) 1 and 2, and Interferon Regulatory Factors (IRF) 3 and 5. Although critical in developmental processes and injury response, CtBP transcriptional repression of cell adhesion proteins, pro-apoptotic factors, and tumor suppressors has been linked to the pathogenesis of multiple forms of cancer. IRFs function in the immune system and have been implicated in autoimmune disorders. Understanding IRF activation is critical to treating pathogens that target IRF function or for future autoimmune disease therapies. We attempted to determine crystal structures that would provide the details of IRF activation, allowing insight into mechanisms of pathogen immune evasion and autoimmune disorders. Although no new structures were solved, we have optimized expression of C-terminal IRF-3 / co-activator complexes, as well as full-length IRF3 and IRF5 constructs. Modifying the constructs coupled with new crystal screening will soon result in structures which detail IRF activation, advancing understanding of the roles of IRF family members in disease. Through structural and biochemical characterization we sought to identify and develop inhibitors of CtBP transcriptional regulatory functions. High concentrations of CtBP substrate, 4-Methylthio 2-oxobutyric acid (MTOB), have been shown in different cancer models to interfere with CtBP transcriptional regulation. We began the process of structure based drug design by solving crystal structures of both CtBP family members bound to MTOB. The resulting models identified critical ligand contacts and unique active site features, which were utilized in inhibitor design. Potential CtBP inhibitors were identified and co-crystallized with CtBP1. One such compound binds to CtBP more than 1000 times more tightly than does MTOB, as a result of our structure-based inclusion of a phenyl ring and a novel pattern of hydrogen bonding. This molecule provides a starting point for the development of compounds that will both bind more tightly and interfere with transcriptional signaling as we progress towards pharmacologically targeting CtBP as a therapy for specific cancers.

The development of several organ systems through modeling and shaping of the tissue structure occurs from signaling through axon guidance molecules. The Slit family of ligands has been shown to regulate branching morphogenesis in mammary gland duct development and loss of Slit gene expression during this time leads to the formation of hyperplastic, disorganized lesions suggesting a potential role for Slits in cancer formation. Characterization of human pancreatic ductal adenocarcinoma cell lines showed a loss of SLIT2 expression in cells that contain activated Kras. Loss of SLIT2 expression was associated with DNA methylation of CpG sites within the SLIT2 core promoter and chromatin enrichment of repressive histone modifications at the SLIT2 transcriptional start site. Additionally, treatment of pancreatic ductal adenocarcinoma cell lines with demethylating agent 5-aza-2'-deoxycytidine led to SLIT2 re-expression while treatment with histone deacetylase inhibitor Trichostatin A did not. Mir-218-1 is an intronic microRNA encoded within intron 15 of the SLIT2 gene. Expression of mir-218-1 does not correlate with SLIT2 mRNA expression suggesting that it is transcribed from a promoter independent of the SLIT2 gene promoter. Pancreatic ductal adenocarcinoma cell lines showed a peak of H3K4me3 chromatin enrichment localized to a 1kb region within intron 4 of the SLIT2 gene denoting a candidate alternative promoter for mir-218-1. A concordant peak of H4ac chromatin enrichment overlapped the peak of H3K4me3 enrichment and transcriptional activity was measured from the 1kb region in all pancreatic ductal adenocarcinoma cell lines. A NF-κB binding site was also predicted to exist within the 1kb region. Transfection with two independent siRNAs to NF-κB led to an increase in both pre-mir-218-1 and mature mir-218-1 while treatment with an inhibitor to IκB kinase led to an increase in pre-mir-218-1 expression. Additionally, the p65 subunit of NF-κB was found to bind to the candidate mir-218-1 alternative promoter in pancreatic ductal adenocarcinoma cell lines that do not contain DNA CpG methylation at the predicted NF-κB binding site. It was discovered that miR-218 is a modulator of ARF6 expression suggesting a role in the inhibition of pancreatic ductal adenocarcinoma cell invasion through modulation of the actin cytoskeleton. Overexpression with a miR-218 precursor showed that miR-218 is an inhibitor of pancreatic ductal adenocarcinoma cell invasion in two dimensions. Additionally, it was found that while miR-218 does not have an affect on the ability of pancreatic ductal adenocarcinoma cells to form functional invadopodia, miR-218 is an inhibitor of the extracellular matrix degradation properties of mature invadopodia. Interestingly, the effect of miR-218 on pancreatic ductal adenocarcinoma cell invasion or extracellular matrix degradation is not reliant on the cell's dependency on Kras signaling for growth and survival. Collectively, these observations indicate that understanding the transcriptional regulation of SLIT2 and mir-218-1 expression as well as their signaling properties may provide a step toward the development of diagnostic tests and therapeutic treatments for patients with invasive or metastatic pancreatic ductal adenocarcinoma.

<p>Next generation sequencing technologies have provided us with a wealth of data profiling a diverse range of biological processes. In an effort to better understand the process of gene regulation, two predictive machine learning models specifically tailored for analyzing gene transcription and polyadenylation are presented.</p><p>Transcriptional enhancers are specific DNA sequences that act as ``information integration hubs" to confer regulatory requirements on a given cell. These non-coding DNA sequences can regulate genes from long distances, or across chromosomes, and their relationships with their target genes are not limited to one-to-one. With thousands of putative enhancers and less than 14,000 protein-coding genes, detecting enhancer-gene pairs becomes a very complex machine learning and data analysis challenge. </p><p>In order to predict these specific-sequences and link them to genes they regulate, we developed McEnhancer. Using DNAseI sensitivity data and annotated in-situ hybridization gene expression clusters, McEnhancer builds interpolated Markov models to learn enriched sequence content of known enhancer-gene pairs and predicts unknown interactions in a semi-supervised learning algorithm. Classification of predicted relationships were 73-98% accurate for gene sets with varying levels of initial known examples. Predicted interactions showed a great overlap when compared to Hi-C identified interactions. Enrichment of known functionally related TF binding motifs, enhancer-associated histone modification marks, along with corresponding developmental time point was highly evident.</p><p>On the other hand, pre-mRNA cleavage and polyadenylation is an essential step for 3'-end maturation and subsequent stability and degradation of mRNAs. This process is highly controlled by cis-regulatory elements surrounding the cleavage site (polyA site), which are frequently constrained by sequence content and position. More than 50\% of human transcripts have multiple functional polyA sites, and the specific use of alternative polyA sites (APA) results in isoforms with variable 3'-UTRs, thus potentially affecting gene regulation. Elucidating the regulatory mechanisms underlying differential polyA preferences in multiple cell types has been hindered by the lack of appropriate tests for determining APAs with significant differences across multiple libraries. </p><p>We specified a linear effects regression model to identify tissue-specific biases indicating regulated APA; the significance of differences between tissue types was assessed by an appropriately designed permutation test. This combination allowed us to identify highly specific subsets of APA events in the individual tissue types. Predictive kernel-based SVM models successfully classified constitutive polyA sites from a biologically relevant background (auROC = 99.6%), as well as tissue-specific regulated sets from each other. The main cis-regulatory elements described for polyadenylation were found to be a strong, and highly informative, hallmark for constitutive sites only. Tissue-specific regulated sites were found to contain other regulatory motifs, with the canonical PAS signal being nearly absent at brain-specific sites. We applied this model on SRp20 data, an RNA binding protein that might be involved in oncogene activation and obtained interesting insights. </p><p>Together, these two models contribute to the understanding of enhancers and the key role they play in regulating tissue-specific expression patterns during development, as well as provide a better understanding of the diversity of post-transcriptional gene regulation in multiple tissue types.</p> / Dissertation

BACKGROUND:Colony stimulating factor-1 (CSF-1) plays an important role in ovarian cancer biology and as a prognostic factor in ovarian cancer. Elevated levels of CSF-1 promote progression of ovarian cancer, by binding to CSF-1R (the tyrosine kinase receptor encoded by c-fms proto-oncogene).Post-transcriptional regulation of CSF-1 mRNA by its 3' untranslated region (3'UTR) has been studied previously. Several cis-acting elements in 3'UTR are involved in post-transcriptional regulation of CSF-1 mRNA. These include conserved protein-binding motifs as well as miRNA targets. miRNAs are 21-23nt single strand RNA which bind the complementary sequences in mRNAs, suppressing translation and enhancing mRNA degradation.RESULTS:In this report, we investigate the effect of miRNAs on post-transcriptional regulation of CSF-1 mRNA in human ovarian cancer. Bioinformatics analysis predicts at least 14 miRNAs targeting CSF-1 mRNA 3'UTR. By mutations in putative miRNA targets in CSF-1 mRNA 3'UTR, we identified a common target for both miR-128 and miR-152. We have also found that both miR-128 and miR-152 down-regulate CSF-1 mRNA and protein expression in ovarian cancer cells leading to decreased cell motility and adhesion in vitro, two major aspects of the metastatic potential of cancer cells.CONCLUSION:The major CSF-1 mRNA 3'UTR contains a common miRNA target which is involved in post-transcriptional regulation of CSF-1. Our results provide the evidence for a mechanism by which miR-128 and miR-152 down-regulate CSF-1, an important regulator of ovarian cancer.

<p>Salicylic acid (SA) is a central plant defense signal. It is not only required for closing the stomata upon infection to prevent pathogens from entering into the plant apoplast, but also mediates defense responses activated by pathogen-originated microbe-associated molecular patterns (MAMPs) and effectors in the infected tissues. In addition, SA is a necessary and sufficient signal for systemic acquired resistance (SAR). In <italic>Arabidopsis</italic> <italic>thaliana</italic>, SA level increases in response to pathogen attack, which is essential for activating defense responses. This SA accumulation involves transcriptional activation of several genes including <italic>ICS1</italic> (<italic>ISOCHORISMATE</italic> <italic>SYNTHASE</italic> <italic>1</italic>), <italic>EDS5</italic> (<italic>ENHANCED</italic> <italic>DISEASE</italic> <italic>SUSCEPTIBILITY</italic> <italic>5</italic>), <italic>EDS1</italic> (<italic>ENHANCED</italic> <italic>DISEASE</italic> <italic>SUSCEPTIBILITY</italic> <italic>1</italic>), <italic>PAD4</italic> (<italic>PHYTOALEXIN-DEFICIENT</italic> <italic>4</italic>) and <italic>PBS3</italic> (<italic>avrPphB</italic> <italic>SUSCEPTIBLE</italic> <italic>3</italic>). However, it is not well understood how pathogenic signals induce these SA accumulation genes. Interestingly, our time-course transcriptome analysis showed that these five genes share a similar pathogen-induced expression pattern, suggesting the existence of common transcription factors (TFs). Through yeast-one-hybrid screening, a TF NTL9 was identified for its interactions with the promoters of the SA accumulation genes. Preferentially expressed in guard cells, NTL9 activates the expression of SA accumulation genes in guard cells. The <italic>ntl9</italic> mutant is defective in pathogen-induced stomatal closure mediated by a well-characterized MAMP, flg22. Consistent with the stomatal closure defect, the <italic>ntl9</italic> mutant exhibits elevated susceptibility to surface-inoculated pathogens. The stomatal closure defect of the <italic>ntl9</italic> mutant can be rescued by exogenous application of SA, demonstrating that NTL9 acts upstream of SA in stomatal closure response. These results suggest that NTL9-mediated activation of SA accumulation genes is essential for MAMP-triggered stomatal closure.</p><p>While plants induce SA to activate defense responses, pathogens can also produce virulence factors to counteract the effects of SA. Coronatine is one such virulence factor produced by <italic>Pseudomonas</italic> <italic>syringae</italic>. Coronatine is known to promote opening of stomata for bacterial entry, bacterial growth in the apoplast, systemic susceptibility and development of disease symptoms such as chlorosis. In the process of examining the mechanisms underlying coronatine-mediated virulence, three homologous TFs, ANAC019, ANAC055 and ANAC072, were found to be activated by coronatine directly through the TF, MYC2. Genetic characterization of these three TF mutants revealed that these TFs mediate multiple virulence effects of coronatine by inhibiting SA accumulation. To exert this inhibitory effect, these TFs repress <italic>ICS1</italic> and activate <italic>BSMT1</italic>, genes involved in SA biosynthesis and inactivation modification, respectively. Thus, a signaling cascade downstream of coronatine was illustrated to dampen SA-mediated defense responses through differential transcriptional regulation of genes related to SA level.</p><p>Taken together, my dissertation studies revealed novel transcriptional regulation of SA production and demonstrated that this transcriptional regulation is a vital point not only for plant defense activation but also for pathogen manipulation to counteract defense responses. Further studies on the interplay of this transcriptional regulation by different TFs would broaden our understanding about the dynamics of plant-pathogen interaction.</p> / Dissertation

Transcription factors regulate distinct macrophage functions by regulating gene expression in response to micro-environmental cues. This functional plasticity is critical for regulating innate and adaptive immune responses during infection and during chronic disease processes including inflammatory diseases and cancer. Microarray analysis of macrophages polarized to a pro-inflammatory (M1) phenotype with LPS and IFNγ revealed that basic leucine zipper transcription factor ATF-like 2 (Batf2), a member of the AP1 transcription factors, is selectively upregulated in M1 macrophages compared to anti-inflammatory IL-4-polarized (M2) macrophages. The initial hypothesis was that Batf2 is a master regulator of gene expression that orchestrates M1 polarization. To investigate a potential role of Batf2 during macrophage polarization, its expression in M1 polarized macrophages was examined. Batf2 mRNA appears within 60 minutes following LPS/ IFNγ treatment and is sustained for at least 48 hours. To address the hypothesis that Batf2 acts as a master transcriptional factor driving a functional M1 phenotype, we have established macrophage cell lines that constitutively express Batf2. Batf2 overexpression did not enhance key M1-associated genes, including iNOS and H2-Aa, but did enhance LPS/IFNγ-driven Cxcl10. Batf2 overexpression also failed to suppress key M2-associated genes including Fizz1 and Mrc1. Batf2 overexpression also failed to alter multiple non-immunity-related genes established or predicted to be downstream of Batf2 in macrophages or other cells. Overall, contrary to our initial hypothesis, constitutive Batf2 expression by itself does not appear to broadly induce M1 gene expression; rather, it appears to enhance only select genes. Since other Batf family members interact with members of the IRF family, I discuss the possibility that Batf2 works in conjunction with a limiting cofactor, possibly Irf family members and/or other regulatory proteins.