Les ascidies présentent des propriétés remarquables au sein des métazoaires qui en font un modèle particulièrement intéressant pour étudier le fonctionnement et l’évolution des éléments cis-régulateurs dans un contexte développemental. Ciona intestinalis et Phallusia mammillata, deux espèces d’ascidies qui ont divergé il y a environ 300 millions d’années, combinent une grande conservation de leurs processus développementaux avec une grande divergence de leur séquence génomique. Pour comprendre comment « fabriquer » des embryons similaires avec des génomes divergents, nous avons identifié les éléments cis-régulateurs actifs au cours du développement de Ciona intestinalis et Phallusia mammillata en développant et en appliquant la méthode de ChIP-Seq sur des modifications d’histones sur des jeunes gastrulae. La définition puis la validation fonctionnelle de différentes catégories d'éléments cis-régulateurs nous a permis de révéler quelques propriétés de la cis-régulation au sein de génomes compacts et intensément remaniés. En sus, les données que nous avons produites constituent une resource fonctionnelle unique pour la caractérisation des éléments cis-régulateurs chez les ascidies et l'étude de leur évolution au sein des Chordés. / Ascidians display remarkable features within metazoans making them particularly suited for the study of function and evolution of cis-regulatory elements in the context of embryonic development. Ciona intestinalis and Phallusia mammillata, two ascidian species that diverged about 300M years ago, combine high conservation of their developmental processes with high divergence of their genome sequence. To understand how to “make” similar embryos with divergent genomes, we identified active cis-regulatory elements during Ciona intestinalis and Phallusia mammillata development by developing and applying the ChIP-Seq method on histone modifications in early-gastrula embryos. Definition then functional validation of different categories of cis-regulatory elements led us to reveal some features of cis-regulation within compact and highly dynamic genomes. Together, our data constitute a unique functional resource for characterizing cis-regulatory elements in ascidians and questioning their evolution within the Chordates.

No description available.

No / Human aging involves a gradual decrease in cellular integrity that contributes to multiple complex disorders such as neurodegenerative disorders, cancer, diabetes, and cardiovascular diseases. Genome-wide association studies (GWAS) play a key role in discovering genetic variations that may contribute towards disease vulnerability. However, mostly disease-associated SNPs lie within non-coding part of the genome; majority of the variants are also present in linkage disequilibrium (LD) with the genome-wide significant SNPs (GWAS lead SNPs). Overall 600 SNPs were analyzed, out of which 291 returned RegulomeDB scores of 1-6. It was observed that just 4 out of those 291 SNPs show strong evidence of regulatory effects (RegulomeDB score < 3), while none of them includes any GWAS lead SNP. Nevertheless, this study demonstrates that by combining ENCODE project data along with GWAS reported information will provide important insights on the impact of a genetic variant-moving from GWAS towards understanding disease pathways. It is noteworthy that both genome-wide significant SNPs as well as the SNPs in LD must be considered for future studies; this may prove to be crucial in deciphering the potential regulatory elements involved in complex disorders and aging in particular.

Endotoxin-tolerante Zellen zeigen über mehrere Tage eine verminderte Produktion pro-inflammatorischer Zytokine, sodass epigenetische Veränderungen ein Grund für die Endotoxintoleranz sein könnte. Im 1. Teil wurden epigenetische Veränderungen an gezielten LPS-tolerisierbaren Genen mithilfe eines in-vitro-Modells mit humanen Monozyten untersucht. Die Gene kodierend für TNF und CXCL10 zeigten eine Reduktion der transkriptionsaktivierenden Histonmarker H3K27ac und H4ac, die durch eine stark reduzierte Genexpression in toleranten Monozyten begleitet wurde. Demgegenüber wiesen Gene wie IL6 und IL1B eine Zunahme an H4ac und H3K27ac auf, während ihre Genexpression in widersprüchlicher Weise reduziert war. Repressive epigenetische Marker (H3K9me2, H3K27me3, H4K20me3, DNA-Methylierung) konnten in den untersuchten Genen nicht nachgewiesen werden. Zudem war die IL6-Genexpression verstärkt abhängig von der Signaltransduktion toleranter Monozyten, was auf unterschiedliche Repressionsmechanismen schließen lässt. Im 2. Teil konnte gezeigt werden, dass die genomweite transkriptionelle Reprogrammierung durch eine globale Verschiebung von aktiven H3K27ac und H4ac in naiven Monozyten zu repressiven H3K9me2, H3K27me3 und H4K20me3 in toleranten, restimulierten Zellen einherging. Mehr als 10000 Genombereiche wiesen Veränderungen an Histonmarkern auf, obwohl nur 3638 Gene unterschiedlich exprimiert waren. Circa 27% der differentiell exprimierten Gene zeigten ein Expressionsmuster, welches mit Veränderungen an aktiven und/oder repressiven Markern innerhalb der Promoterregion korrelierte. Zudem zeigten intergenische Regionen einen verstärkten Anstieg an repressiven Histonmarkern, was auf eine mögliche regulatorische Funktion dieser Bereiche in der Endotoxintoleranz schließen lässt. Die Studie zeigt, dass die Epigenetik der Monozyten stark von der Endotoxintoleranzinduktion betroffen ist, wenn auch nicht alle Veränderungen dem beobachteten Genexpressionsmuster zugeordnet werden konnten. / Endotoxin-tolerant cells show a reduced ability to produce pro-inflammatory cytokines for several days, which assumes an impact of epigenetic changes in endotoxin tolerance induction. Using an in vitro model with human monocytes, the first part focused on the analysis of epigenetic changes in specific LPS-tolerizable genes. The genes encoding for TNF and CXCL10 showed a reduction in the transcription-activating histone marks H3K27ac and H4ac in tolerant monocytes, which was accompanied by a strongly reduced gene expression. In contrast, the IL6 and IL1B genes showed an increase in activating histone modifications, while their gene expressions were moderately reduced. Repressive epigenetic marks (H3K9me2, H3K27me3, H4K20me3, DNA methylation) were not specifically enhanced in the genes studied. Particularly the IL6 gene expression was more susceptible to the signaling strength in tolerant monocytes implying distinct mechanisms in the repression of the genes analyzed. Within the second part, genome-wide reprogramming of tolerant monocytes was accompanied by a global shift from activating H3K27ac and H4ac in naive monocytes to repressive H3K9me2, H3K27me3 and H4K20me3 in tolerant cells treated with LPS. More than 10000 genomic regions were distinctly regulated by histone marks, though only 3638 genes were differentially expressed. Correlation analyses identified 27 % of the differentially expressed genes that showed a transcriptional level consistent with changes in activating and/or repressive histone marks within their promoter regions. Intergenic regions were highly enriched for repressive histone marks in LPS-tolerant monocytes implying a regulatory function in endotoxin tolerance. The data indicate that the epigenetic environment of monocytes is highly affected by endotoxin tolerance induction, though not all changes are directly linked to the gene expression pattern observed.

La méthylation de l’ADN et les modifications post-traductionnelles des histones sont desmodifications épigénétiques qui interviennent dans la régulation des éléments transposables(ET) chez de nombreuses espèces. La proportion des ET dans les génomes varie selon lesespèces considérées et pose la question des mécanismes de régulation de ces ET. Au sein del’espèce Drosophila simulans, les populations naturelles présentent un polymorphisme uniquedans le nombre de copies des ET, ce qui en fait un excellent modèle pour étudier cettequestion. L’étude de la méthylation d’ADN et des modifications post-traductionnelles deshistones associées au rétrotransposon à LTR tirant dans la lignée germinale des populationsnaturelles a permis de montrer l’influence d’une copie d’ET sur la structure de la chromatineau site d’insertion. Dans un second volet, nous avons cherché à caractériser la méthylation del’ADN chez la drosophile, chez laquelle la fonction est encore mal connue. Nous avons, pardes approches spécifiques et globales, mesuré l’abondance de cette marque épigénétique chezla drosophile. Nous concluons que les taux de méthylation de l’ADN sont très faibles maisvariables entre espèces. Notre travail n’a pas permis de mettre en évidence un rôle de laméthylation de l’ADN dans le contrôle des ET, toutefois, nous ne pouvons pas exclure cesystème de régulation. / Epigenetic modifications such as DNA methylation and post-translational histonemodifications are involved in transposable elements (TE) silencing in many species. Theirrelative abundance in genomes ask the question of differences in regulation mecanismbetween species. Within the Drosophila simulans species, natural populations exibits a uniquepolymorphism in TE copy number, providing a powerfull tool for the analysis of TEregulation in population from the same specie. We analyzed DNA methylation and posttranslationalhistone modifications associated with the LTR retrotransposon tirant in thegermline of natural populations and report the influence of this element on chromatinestructure. DNA methylation is a wide-conserved epigenetic modification involved in generegulation and TE silencing but its function in drosophila remains missunderstood. Usingdifferent methods, we determined the abundance of methylated cytosines in drosophila, andshowed that methylation level are low and variable between species. Our results show lowevidence for a TE regulation system involving DNA methylation but this cannot be so farexcluded.

Le suppresseur de tumeur DOK1 (downstream of tyrosine kinases1) est une protéine régulatrice de voies de signalisation impliquées dans des processus cellulaires tel que la prolifération, la migration et l'apoptose. Le rôle suppresseur de tumeur de DOK1 a été démontré dans des modèles animaux. Les souris knock-out pour DOK1 présentent une forte susceptibilité de développer des leucémies, des tumeurs malignes hématologiques, des adénocarcinomes pulmonaires, ainsi que des sarcomes histiocytaires agressifs. En outre, nous avons rapporté précédemment que le gène DOK1 peut être muté et son expression réprimée dans différentes tumeurs malignes humaines, telles que les lignées cellulaires de lymphome de Burkitt (BL) et la leucémie lymphoïde chronique (LLC). Cependant, les mécanismes de dérégulation de DOK1 restent inconnus, notamment dans les processus de carcinogenèse induite ou non par des oncovirus. Dans ce projet de thèse, nous avons d'abord caractérisé le promoteur de DOK1 et le rôle du facteur de transcription E2F1 comme le principal régulateur de l'expression de DOK1. Nous avons démontré pour la première fois la contribution de DOK1 dans la réponse cellulaire au stress par son rôle suppresseur de prolifération cellulaire et promoteur d'apoptose. Nous avons trouvé que l'expression du gène DOK1 est réprimée dans une variété de cancers humains, y compris le cancer de la tête et du cou, les lymphomes de Burkitt et les cancers du poumon. Cette répression est due à l'hyperméthylation aberrante de DOK1. Nous avons donc étudié les événements épigénétiques, qui sont souvent altérés dans les cancers, et leurs implications dans la répression de DOK1 dans les lignes cellulaire cancéreuses de la tête et du cou. Nous nous sommes par la suite intéressés aux mécanismes de dérégulation de DOK1 par le virus d'Epstein Barr dans le cadre de sa propriété oncogénique dans les lymphocytes B humains ainsi que dans les lignes cancéreuses du lymphome de Burkitt. Nos résultats apportent de nouvelles informations sur les mécanismes de régulation de l'expression de DOK1 dans la carcinogenèse induite ou non par des oncovirus, ce qui pourrait le définir comme un biomarqueur potentiel de cancer et comme une cible intéressante pour des thérapies épigénétiques / The newly identified tumor suppressor DOK1 (downstream of tyrosine kinases1) inhibits cell proliferation, negatively regulates MAP kinase activity, opposes leukemogenesis, and promotes cell spreading, motility, and apoptosis. DOK1 also plays a role in the regulation of immune cell activation, including B cells. The tumor suppressor role of DOK1 was demonstrated in animal models. DOK1 knockout mice show a high susceptibility to develop leukemia, hematological malignancies as well as lung adenocarcinomas and aggressive histiocytic sarcoma. In addition, we previously reported that the DOK1 gene can be mutated and its expression is down-regulated in human malignancies such as Burkitt’s lymphoma cell lines (BL) and chronic lymphocytic leukemia (CLL). However, very little is known about the mechanisms underlying DOK1 gene regulation and silencing in viral- and non viral-related tumorigenesis. In the present project, we first characterized the DOK1 promoter. We have shown the role of E2F1 transcription factor as the major regulator of DOK1 expression and how DOK1 plays a role in DNA stress response though opposing cell proliferation and promoting apoptosis. We demonstrated that DOK1 gene expression is repressed in a variety of human cancers, including head and neck, Burkitt’s lymphoma and lung cancers, as a result of aberrant hypermethylation. We investigated the link between the epigenetic events and DOK1 silencing in non viral head and neck cancer cell lines, and by Epstein Barr virus in relation to its oncogenic activity in human B cells and neoplasia such as Burkitt’s lymphoma. These data provide novel insights into the regulation of DOK1 in viral and non viral-related carcinogenesis, and could define it as a potential cancer biomarker and an attractive target for epigeneticbased therapy

En mamíferos, así como en plantas, mutaciones en AND helicasas/ATPasas del la família SNF2, no solo afectan a la estructura de la cromatina, sino que también afectan al patrón global de la metilación del ADN. Sugiriendo una relación funcional entre la estructura de la cromatina y la epigenética. El complejo NuRD, el cual posee una ATPasa de la familía SNF2, está relacionado con la represión de la transcripción y en el remodelamiento de la cromatina. Nuestro laboratorio demostró que la proteína leucémica PML-RAR&#945; reprime la transcripción de sus genes diana por el reclutamiento de DNMTs y el complejo PRC2. En esta tesis, demostramos una relación directa del complejo NuRD en la represión génica y en los cambios epigenéticos en la leucemia promielocítica aguda (APL). Mostramos que PML-RAR&#945; se une y recluta NuRD a sus genes diana, incluyendo el gen supresor de tumores RAR&#61538;2, facilitando que el complejo de Polycomb se reclute y metile la lisina 27 de la histona H3. Tratamiento con Acido Retinóico (RA), el qual se utiliza en pacientes, reduce la ocupación de NuRD en células leucémicas. Eliminando NuRD no solo provoca que las histonas no se deacetilen y que la cromatina no se compacte, sino que también provoca que tanto la metilación del ADN y de las histonas no se produzca, así como la represión génica del gen RAR&#61538;2, favoreciendo la diferenciación celular. Nuestros resultados caracterizan un nuevo papel del complejo NuRD en el establecimiento de los patrones epigenéticos en APL, demostrando una relación esencial entre la estructura de la cromatina y epigenética durante el desarrollo de la leucemia, pudiéndose aplicar a la terapia de esta enfermedad. / In mammals, as in plants, mutations in SNF2-like DNA helicases/ATPases were shown to affect not only chromatin structure but also global methylation patterns, suggesting a potential functional link between chromatin structure and epigentic marks. The SNF2-like containing NuRD complex is involved in gene transcriptional repression and chromatin remodeling. We have previously shown that the leukemogenic protein PMLRAR&#945; represses target genes through recruitment of DNMTs and Polycomb complex. In this thesis, we demonstrate a direct role of the NuRD complex in aberrant gene repression and transmission of epigenetic repressive marks in acute promyelocytic leucemia (APL). We show that PML-RAR&#945; &#61472;binds and recruits NuRD to target genes, including to the tumor-suppressor gene RAR&#61538;2. In turn, the NuRD complex facilitates Polycomb binding and histone methylation at lysine 27. Retinoic acid treatment reduced the promoter occupancy of the NuRD complex. Knock-down of the NuRD complex in leukemic cells not only prevented histone deacetylation and chromatin compaction, but also impaired DNA and histone methylation as well as stable silencing, thus favoring cellular differentiation. These results unveil an important role for NuRD in the establishment of altered epigenetic marks in APL, demonstrating an essential link between chromatin structure and epigenetics in leukemogenesis that could be exploited for therapeutic intervention.

Alternativ splicing av pre-mRNA ger upphov till proteindiversitet. Histonmodifieringar kopplas till den alternativa splicingens reglering genom adaptorsystem som overfor den epigenetiska informationen direkt till splicingfaktorerna. De cis- agerande RNA- elementen pa exoner och introner med tillhorande trans- reglerande splicingfaktorer paverkas darfor direkt av specifika histonmodifieringar. En sammankopplande integrerad modell over en rad DNA- baserade processer foreslas. Denna komplexa modell ger en bild av interaktioner och paverkan mellan dessa delar. Kromatin remodellering kravs for bildandet av eukromatin. Nukleosomers placering vid exonrika regioner med specifika modifieringsmonster pekar ut exonerna samt mojliggor inbindning av RNA polymeras II som med sin CTD doman rekryterar bade splicing- och modifieringsfaktorer. Transkriptionshastigheten paverkas av nukleosomplaceringen vilket i sin tur paverkar rekrytering av spliceosomens komponenter, andra trans- agerande regulatorer och aven pre-mRNA sekvensens sekundarstruktur. Kromatin- adaptorkomplex laser av specifika histonmodifieringar och overfor informationen till splicingapparaten. Detta skapar mojlighet till den viktiga cell- och vavnadsspecifika alternativa splicingens reglering. I den integrerade modellen blir komplexiteten tydligare dar alla dessa processer interagerar med varandra och de cis- regulatoriska sekvenserna pa premRNA transkriptet. / Alternative splicing of pre-mRNA generates protein diversity. Histone modifications are connected to the regulation of alternative splicing through adaptor systems that transfers the epigenetic information directly to the splicing factors. The cis- acting RNA elements on the exons and introns together with the trans- regulating splicing factors are therefore directly affected of specific histone modifications. An integrated model over several DNA process mechanisms is suggested. This complex model explains the interactions of the different parts and how they affect each other. Chromatin remodelers are required to obtain euchromatin. Nucleosome positioning at exon rich regions with a specific modification pattern point out where the exons are, and this enable the RNA polymerase II to find and bind to the DNA. It’s CTD domain recruits both splicing- and modifications factors. The transcription rate is also affected of the nucleosome positioning and that in turn affects the recruitment of the components of the spliceosomen, other trans- acting regulators and even the formation of the secondary structure of the pre-mRNA transcript. Chromatin- adaptor complex reads specific histone modifications and transfers this information to the splicing apparatus. All this creates the possibility to regulate important cell- and tissue specific alternative splicing patterns. The integrated model makes the complex processes more clearer when all these integrates with each other and the cis- acting regulating elements on the pre-mRNA transcript.

No description available.

Bacterial pathogens dramatically affect host cell transcription programs for their own profit, however the underlying mechanism in most cases remain elusive. While investigating the effects of listeria monocytogenes on histone modifications, we discovered a new transcription regulatory machanism by which the expression of genes is repressed, during infection. Upon infection by L. monocytogenes, the secret virulence factor, InlB, binds the c-Met receptor and activates signaling through PI3K/Akt. This signaling platform is necessary for causing the relocalization of the histone deacetylase, SIRT2, to the nucleus and associating to chromatin.In characterizing the mechanism governing SIRT2 nuclear relocazing during infection, our results have demonstrated that SIRT2 undergoes a post-translational modification. SIRT2 undergoes dephosphorylation at a novel N-terminal phospho-site. SIRT2 is recruiter to the transcription star sites of genes repressed during inection leading to H3K18 deacetylation and transcriptional repression.finnaly, my results demonstrate that SIRT2 is hijacked by L monocytogenes and promotes an increase in intracellular bacteria. Together, these data uncover a key role for SIRT2 mediated H3K18 deacetylation during infection and characterize a novel mechanisme imposed by a pathogenic bacteriomto reprogram the host cell.