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A common mRNA modification is 5-methylcytosine (m5C), whose role in gene-transcript processing and cancer remains unclear. Here, we identify serine/arginine-rich splicing factor 2 (SRSF2) as a reader of m5C and impaired SRSF2 m5C binding as a potential contributor to leukemogenesis. Structurally, we identify residues involved in m5C recognition and the impact of the prevalent leukemia-associated mutation SRSF2P95H. We show that SRSF2 binding and m5C colocalize within transcripts. Furthermore, knocking down the m5C writer NSUN2 decreases mRNA m5C, reduces SRSF2 binding, and alters RNA splicing. We also show that the SRSF2P95H mutation impairs the ability of the protein to read m5C-marked mRNA, notably reducing its binding to key leukemia-related transcripts in leukemic cells. In leukemia patients, low NSUN2 expression leads to mRNA m5C hypomethylation and, combined with SRSF2P95H, predicts poor outcomes. Altogether, we highlight an unrecognized mechanistic link between epitranscriptomics and a key oncogenesis driver., SCOPUS: ar.j, info:eu-repo/semantics/published

The prevalence of type 2 diabetes is increasing worldwide, and nearly 540 million people are already affected. This confers a significant burden on human health. The onset of type 2 diabetes is associated with genetic predisposition and environmental/lifestyle risk factors. Among the latter, a sedentary lifestyle and hypercaloric/ quality diets are crucial drivers of type 2 diabetes development. Type 2 diabetes is usually characterized by insulin resistance, in which the beta cells are exposed to an increased demand to secrete insulin. This increased demand causes stress on the beta cells and leads to compensation mechanisms in an attempt to maintain normal glucose levels. However, if these mechanisms fail, beta cell dysfunction will ultimately result in the onset of type 2 diabetes. This progressive beta cell failure leaves molecular footprints that account for the pathology of type 2 diabetes. Thus, I investigated the gene signatures of human islets from type 2 diabetes donors, from non-diabetic human islets upon exposure to metabolic stress (i.e. lipo- and/or glucolipotoxicity), and from human islets from type 2 diabetes patients manifesting functional recovery after culture in a non-diabetic milieu.Gene signatures related to augmented inflammatory responses and depressed beta cell function were identified in type 2 diabetes islets, in comparison with non-diabetic controls. These results were confirmed by comparing the global transcriptomes of type 2 diabetes with other inflammatory diseases, such as type 1 diabetes, Alzheimer’s disease and multiple sclerosis. Additionally, gene signatures of ER stress and inflammatory signaling were also observed in human islets exposed to lipo- and/or gluocolipotoxic conditions. Of particular interest, the ER function (i.e. protein folding and sorting) and mitochondrial function (i.e. TCA cycle, oxidative phosphorylation)-related genes are severely perturbed with opposite direction between type 2 diabetes islets and in vitro ex, La prévalence du diabète de type 2 augmente dans le monde entier et près de 540 millions de personnes en sont déjà atteintes. Cela représente un fardeau important pour la santé humaine. L'apparition du diabète de type 2 est associée à une prédisposition génétique et à des facteurs de risque liés à l'environnement et au mode de vie. Parmi ces derniers, un mode de vie sédentaire et une alimentation hypercalorique/de qualité sont des facteurs cruciaux du développement du diabète de type 2. Le diabète de type 2 est généralement caractérisé par une résistance à l'insuline, dans laquelle les cellules bêta sont exposées à une demande accrue de sécrétion d'insuline. Cette demande accrue provoque un stress sur les cellules bêta et conduit à des mécanismes de compensation pour tenter de maintenir des niveaux de glucose normaux. Toutefois, si ces mécanismes échouent, le dysfonctionnement des cellules bêta entraînera finalement l'apparition d'un diabète de type 2. Cette défaillance progressive des cellules bêta laisse des empreintes moléculaires qui expliquent la pathologie du diabète de type 2. J'ai donc étudié les signatures génétiques d'îlots humains provenant de donneurs atteints de diabète de type 2, d'îlots humains non diabétiques exposés à un stress métabolique (c'est-à-dire à une lipo- et/ou glucolipotoxicité) et d'îlots humains provenant de patients atteints de diabète de type 2 et manifestant une récupération fonctionnelle après culture dans un milieu non diabétique.Des signatures génétiques liées à des réponses inflammatoires accrues et à une fonction déprimée des cellules bêta ont été identifiées dans les îlots de patients atteints de diabète de type 2, en comparaison avec des témoins non diabétiques. Ces résultats ont été confirmés par la comparaison des transcriptomes globaux du diabète de type 2 avec ceux d'autres maladies inflammatoires, telles que le diabète de type 1, la maladie d'Alzheimer et la sclérose en plaques. En outre, des signatures génétiques du stre, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

Background & Aims: Severe forms of alcohol-related liver diseaseare associated with increased susceptibility to infectionswhich are associated with poor prognosis. The cellular and molecularmechanisms responsible for this altered host defense areincompletely understood.Methods: We performed whole blood phenotypic analysis andex vivo stimulation with various pathogen-associated molecularpatterns (PAMPs). We included 34 patients with alcohol-relatedcirrhosis (18 of whom had biopsy-proven severe alcoholic hepatitis[sAH]), 12 healthy controls and 11 patients with chronic alcoholconsumption without significant liver disease. We also evaluatedthe transcriptomic (RNA-seq) and chromatin accessibility (ATACseq)profiles of CD14+ monocytes from a subset of patients.Results: Circulating monocytes and conventional dendritic cells(DCs) from patients with sAH displayed complex alterationscharacterized by increased expression of both activating andinhibitory surface markers and an impaired pro-inflammatoryresponse upon stimulation with PAMPs representative of gramnegativebacteria (lipopolysaccharide, Pam3CSK4) or fungalpathogens (Zymosan). Their decreased ability to produce morethan 1 cytokine (polyfunctionality) upon PAMP stimulationcorrelated with the risk of developing infection at 28 days ormortality at 90 days. The presence of acute-on-chronic liverfailure in patients with sAH did not significantly modify theimmune profile of monocytes and DCs. Moreover, CD14+ monocytesof patients with sAH displayed altered transcriptional andepigenomic profiles characterized by downregulation of keyinnate immune and metabolic pathways and upregulation ofimportant immunomodulatory factors.Conclusions: In patients with sAH, the altered transcriptional programand functional properties of monocytes that contribute to patients'susceptibility to infection have strong epigenetic determinants., Doctorat en Sciences médicales (Médecine), info:eu-repo/semantics/nonPublished

The heart is a vital organ of our body whose correct function is essential to life. During embryonic development, it arises from various pools of cardiovascular progenitors (CPs) that are specified in a precise spatiotemporal pattern during gastrulation. Correct specification of the various populations of CPs is dependent upon coordinated action of transcription factors (TFs) that together control gene expression in space and time, thereby defining the cellular identity of CPs. These molecular events are to date poorly described in nascent CPs. Mesp1 is a master TF that promotes CP specification and differentiation. Using mouse pluripotent stem cells (PSCs) as a model for gene regulation during early development, we performed RNA-seq, Mesp1, H3K27Ac and H3K4me1 ChIP-seq as well as ATAC-seq during differentiation of PSCs in which the expression of Mesp1 can be induced in a doxycycline-dependent manner. We uncovered the various patterns of gene regulation that are arise within 24 hours of Mesp1 induction, including rapid activation of some genes and delayed activation of others. Using Mesp1 ChIP-seq, we characterized the direct and indirect Mesp1 target genes in a genome-wide manner. We then defined and validated the activity of distal regulatory elements that enable the activation of Mesp1 target genes. Motif enrichment analysis at Mesp1 binding sites uncovered several potential cofactors of Mesp1. Using a variety of in vitro and in vivo validation methods, we identified Zic2 and Zic3 as essential cofactors of Mesp1, which together regulate its ability to open the chromatin as well as regulate its target gene expression during CP specification. Then, we explored the mechanisms of Mesp1-mediated enhancer activation in CPs through enhancer reporter assays, demonstrating the potential and specificity of Mesp1-bound enhancers to activate gene expression in a cell-type specific manner, opening the way to dissecting the motif grammar of CP-specific enhancers in future expe, Le cœur est un organe dont le bon fonctionnement est essentiel à la vie. Pendant le développement embryonnaire, il se forme à partir de différentes populations de progéniteurs cardiovasculaires (PCs) qui se spécifient selon un schéma spatiotemporel précis durant la gastrulation. L’agencement et la genèse de ces derniers dépend de l’action coordonnée de facteurs de transcription (FT) qui régulent l’expression des gènes dans le temps et dans l’espace, définissant ainsi l’identité de ces progéniteurs. Ces évènements moléculaires restent peu décrits dans les PCs précoces. Mesp1 est un FT qui promeut la spécification et la différentiation des PCs. En utilisant les cellules souches pluripotentes (CSP) dans lesquelles l’expression de Mesp1 peut être induite par la doxycycline comme modèle pour la régulation des gènes pendant l’embryogenèse précoce, nous avons réalisé du RNA-seq, Mesp1, H3K27Ac et H3K4me1 ChIP-seq ainsi que de l’ATAC-seq pendant la différentiation de ces CSP avec et sans induction de Mesp1. Nous avons découvert que Mesp1 active différents gènes avec des cinétiques d’expression distinctes, certains gènes répondant plus rapidement à l’induction de Mesp1 que d’autres. En analysant le Mesp1 ChIP-seq, nous avons caractérisé les gènes qui sont directement versus indirectement régulés par Mesp1 dans le génome entier. Ensuite, nous avons défini et validé la fonction de régions régulatrices liées par Mesp1 pour l’activation de certains de ces gènes cibles. L’analyse de ces sites par enrichissement de motif prédit les cofacteurs potentiels de Mesp1. En utilisant une large gamme de méthodes in vivo et in vitro, nous avons validé que Zic2 et Zic3 agissent comme des cofacteurs essentiels à Mesp1, en régulant sa capacité à ouvrir la chromatine et l’expression de ces gènes cibles. Ensuite, nous avons exploré les mécanismes d’activation des régions régulatrices liées par Mesp1 via des constructions reportrices, démontrant ainsi le potentiel et la spécificité de ces séquenc, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

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Le travail réalisé dans le cadre de cette thèse de doctorat s’inscrit dans un objectif decaractérisation moléculaire des tumeurs thyroïdiennes papillaires. Celui-ci est composé de deux projetscomplémentaires qui ont comme objectif commun de mieux comprendre les mécanismes mis en placelors de la tumorigenèse et la progression tumorale de ces cancers, en se focalisant sur le rôle desmiARNs. Parmi les cancers thyroïdiens, le cancer thyroïdien papillaire (PTC) représente la forme la plusfréquente, et bien que son pronostic soit bon, les patients peuvent présenter des récidives et desmétastases qui diminuent leur survie. Les miARNs sont des petits ARNs simple brin qui régulentnégativement l’expression génique. Ils ont été montrés comme étant différentiellement exprimés dansles PTC par rapport aux tissus sains, et comme étant impliqués dans le processus de tumorigenèse.Dans un premier temps, nous avons étudié le rôle du miR-7-5p dans la tumorigenèse du cancerthyroïdien papillaire, le miR-7-5p étant un miARN récemment caractérisé comme étant sous-exprimédans ces tumeurs. La surexpression du miR-7-5p dans deux lignées cellulaires thyroïdiennes, TPC1 etHtori-3, entraîne une diminution de la prolifération cellulaire. L’analyse du transcriptome parmicroarray a par ailleurs mis en évidence une modulation des voies de signalisation MAPK et PI3K quisont connues pour être nécessaires à la prolifération des cellules thyroïdiennes et pour être déréguléesdans le processus de tumorigenèse des PTC. Nous avons plus précisément étudié la corrélation entrel’expression du miR-7-5p et celles de deux effecteurs en amont de ces voies de signalisation, EGFR etIRS2. Nous avons confirmé l’augmentation de l’expression de EGFR et de IRS2 dans les PTC, liée àl’agressivité des tumeurs, et mis en évidence une corrélation négative de leur expression par rapportà celle du miR-7-5p. Nos résultats supportent donc un effet anti-tumoral du miR-7-5p dans la thyroïde.La diminution de son expression dans la t, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

Type 1 diabetes (T1D) is an autoimmune disease characterized by the selective destruction of pancreatic β-cells, mediated by autoreactive T cells.The resulting inflammatory response takes place in the context of a dialogue between invading immune cells and the targeted β-cells, and it is modulated by genetic susceptibility, acting on both immune and β-cells, and by inflammatory cytokines and chemokines. Stress pathways triggered within β-cells may potentiate autoimmunity, and T1D susceptibility genes shape β-cell responses to “danger signals”, innate immunity, and activation of apoptosis. However, the molecular mechanisms linking genetic variation, environmental triggers, and the signaling events promoting β-cell dysfunction and loss remain poorly clarified. Pre-mRNA splicing is a crucial mechanism for gene expression regulation, and more than 95% of the human multi-exonic primary transcripts undergo alternative splicing. Splicing dysregulation have been increasingly recognized to play a pivotal role in multiple pathologies, including autoimmune diseases. More than 15% of the mutations described in the Human Gene Mutation Database are predicted to affect splicing. Our group has shown that exposure to pro-inflammatory cytokines induces major changes on the β-cell transcriptome, affecting the splicing of genes that are key for β-cell function and survival. Importantly, our group identified that GLIS3, a susceptibility gene for both T1D and type 2 diabetes (T2D), modulates β-cell apoptosis via regulation of the splicing factor SRSF6, linking T1D genetic susceptibility and alternative splicing. The downregulation of GLIS3, either by germline mutations associated with monogenic forms of diabetes or risk single nucleotide polymorphisms, contribute to SRSF6 splicing factor downregulation. Splicing factors are the primary regulators of splicing and orchestrate functionally related transcripts into regulatory networks, therefore, oscillations of splicing factors’ expression, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

Bone metastasis is the major deleterious event in prostate cancer (PCa). TMPRSS2-ERG fusion is one of the most common chromosomic rearrangements in PCa. However, its implication in bone metastasis development is still unclear. Since bone metastasis starts with the tropism of cancer cells to bone through specific migratory and invasive processes involving osteomimetic capabilities, it is crucial to better our understanding of the influence of TMPRSS2-ERG expression in the mechanisms underlying the bone tropism properties of PCa cells. We developed bioluminescent cell lines expressing the TMPRSS2-ERG fusion in order to assess its role in tumor growth and bone metastasis appearance in a mouse model. First, we showed that the TMPRSS2-ERG fusion increases cell migration and subcutaneous tumor size. Second, using intracardiac injection experiments in mice, we showed that the expression of TMPRSS2-ERG fusion increases the number of metastases in bone. Moreover, TMPRSS2-ERG affects the pattern of metastatic spread by increasing the incidence of tumors in hind limbs and spine, which are two of the most frequent sites of human PCa metastases. Finally, transcriptome analysis highlighted a series of genes regulated by the fusion and involved in the metastatic process. Altogether, our work indicates that TMPRSS2-ERG increases bone tropism of PCa cells and metastasis development., info:eu-repo/semantics/published

DNA methylation and histone modifications are key epigenetic regulators of gene expression, and tight connections are known between the two. DNA methyltransferases are upregulated in several tumors and aberrant DNA methylation profiles are a cancer hallmark. On the other hand, histone demethylases are upregulated in cancer cells. Previous work on ES cells has shown that the lysine demethylase KDM1A binds to DNMT1, thereby affecting DNA methylation. In cancer cells, the occurrence of this interaction has not been explored. Here we demonstrate in several tumor cell lines an interaction between KDM1A and both DNMT1 and DNMT3B. Intriguingly and in contrast to what is observed in ES cells, KDM1A depletion in cancer cells was found not to trigger any reduction in the DNMT1 or DNMT3B protein level or any change in DNA methylation. In the S-phase, furthermore, KDM1A and DNMT1 were found, to colocalize within the heterochromatin. Using P-LISA, we revealed substantially increased binding of KDM1A to DNMT1 during the S-phase. Together, our findings propose a mechanistic link between KDM1A and DNA methyltransferases in cancer cells and suggest that the KDM1A/DNMT1 interaction may play a role during replication. Our work also strengthens the idea that DNMTs can exert functions unrelated to act on DNA methylation., SCOPUS: ar.j, info:eu-repo/semantics/published

The TET enzymes convert methylcytosine to the newly discovered base hydroxymethylcytosine. While recent reports suggest that TETs may play a role in response to oxidative stress, this role remains uncertain, and results lack in vivo models. Here we show a global decrease of hydroxymethylcytosine in cells treated with buthionine sulfoximine, and in mice depleted for the major antioxidant enzymes GPx1 and 2. Furthermore, genome-wide profiling revealed differentially hydroxymethylated regions in coding genes, and intriguingly in microRNA genes, both involved in response to oxidative stress. These results thus suggest a profound effect of in vivo oxidative stress on the global hydroxymethylome., info:eu-repo/semantics/published

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. Alteration of miRNA levels is common in tumors and contributes to the pathogenesis of human malignancies. In the present study we examined the role played by miR-137 in breast tumorigenesis. We found miR-137 levels to be lower in breast cancer cells than in their non-tumorigenic counterparts and observed reduced proliferation and migration of breast cancer cells overexpressing miR-137. We further identified KDM5B, a histone demethylase known to be involved in breast cancer tumorigenesis, as a target of miR-137. As the involvement of histone demethylases in cancer is still poorly understood and as the role of miRNAs in controlling epigenetic mechanisms in cancer is emerging, we broadened our study to the whole KDM5 histone demethylase family to see if the genes coding for these epigenetic enzymes might be regulated by miRNAs in cancer cells. We discovered that KDM5C is overexpressed in breast cancer cells, providing evidence that miR-138 regulates its expression. We found miR-138 overexpression to affect breast cancer cell proliferation. Altogether, our findings suggest that miRNAs may regulate KDM5 histone demethylase levels in breast cancer and thereby control breast cancer cell proliferation and migration., SCOPUS: ar.j, info:eu-repo/semantics/published

DNA methylation is a central epigenetic modification that is established by de novo DNA methyltransferases. The mechanisms underlying the generation of genomic methylation patterns are still poorly understood. Using mass spectrometry and a phosphospecific Dnmt3a antibody, we demonstrate that CK2 phosphorylates endogenous Dnmt3a at two key residues located near its PWWP domain, thereby downregulating the ability of Dnmt3a to methylate DNA. Genome-wide DNA methylation analysis shows that CK2 primarily modulates CpG methylation of several repeats, most notably of Alu SINEs. This modulation can be directly attributed to CK2-mediated phosphorylation of Dnmt3a. We also find that CK2-mediated phosphorylation is required for localization of Dnmt3a to heterochromatin. By revealing phosphorylation as a mode of regulation of de novo DNA methyltransferase function and by uncovering a mechanism for the regulation of methylation at repetitive elements, our results shed light on the origin of DNA methylation patterns., SCOPUS: ar.j, info:eu-repo/semantics/published

Promoter methylation profiles are proposed as potential prognosis and/or diagnosis biomarkers in cervical cancer. Up to now, little is known about the promoter methylation profile and expression pattern of stem cell (SC) markers during tumor development. In this study, we were interested to identify SC genes methylation profiles during cervical carcinogenesis. A genome-wide promoter methylation screening revealed a strong hypermethylation of Undifferentiated cell Transcription Factor 1 (UTF1) promoter in cervical cancer in comparison with normal ectocervix. By direct bisulfite pyrosequencing of DNA isolated from liquid-based cytological samples, we showed that UTF1 promoter methylation increases with lesion severity, the highest level of methylation being found in carcinoma. This hypermethylation was associated with increased UTF1 mRNA and protein expression. By using quantitative RT-PCR and Western Blot, we showed that both UTF1 mRNA and protein are present in epithelial cancer cell lines, even in the absence of its two main described regulators Oct4A and Sox2. Moreover, by immunofluorescence, we confirmed the nuclear localisation of UTF1 in cell lines. Surprisingly, direct bisulfite pyrosequencing revealed that the inhibition of DNA methyltransferase by 5-aza-2′-deoxycytidine was associated with decreased UTF1 gene methylation and expression in two cervical cancer cell lines of the four tested. These findings strongly suggest that UTF1 promoter methylation profile might be a useful biomarker for cervical cancer diagnosis and raise the questions of its role during epithelial carcinogenesis and of the mechanisms regulating its expression. © 2012 Guenin et al., SCOPUS: ar.j, info:eu-repo/semantics/published

Late-Breaking Poster Presentations - Abstract LB-180, info:eu-repo/semantics/published

Histone methylation plays key roles in regulating chromatin structure and function. The recent identification of enzymes that antagonize or remove histone methylation offers new opportunities to appreciate histone methylation plasticity in the regulation of epigenetic pathways. Peptidylarginine deiminase 4 (PADI4; also known as PAD4) was the first enzyme shown to antagonize histone methylation. PADI4 functions as a histone deiminase converting a methylarginine residue to citrulline at specific sites on the tails of histones H3 and H4. This activity is linked to repression of the estrogen-regulated pS2 promoter. Very little is known as to how PADI4 silences gene expression. We show here that PADI4 associates with the histone deacetylase 1 (HDAC1). Kinetic chromatin immunoprecipitation assays revealed that PADI4 and HDAC1, and the corresponding activities, associate cyclically and coordinately with the pS2 promoter during repression phases. Knockdown of HDAC1 led to decreased H3 citrullination, concomitantly with increased histone arginine methylation. In cells with a reduced HDAC1 and a slightly decreased PADI4 level, these effects were more pronounced. Our data thus suggest that PADI4 and HDAC1 collaborate to generate a repressive chromatin environment on the pS2 promoter. These findings further substantiate the "transcriptional clock" concept, highlighting the dynamic connection between deimination and deacetylation of histones., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

The pluripotency-determining gene Oct3/4 (also called Pou5f1) undergoes postimplantation silencing in a process mediated by the histone methyltransferase G9a. Microarray analysis now shows that this enzyme may operate as a master regulator that inactivates numerous early-embryonic genes by bringing about heterochromatinization of methylated histone H3K9 and de novo DNA methylation. Genetic studies in differentiating embryonic stem cells demonstrate that a point mutation in the G9a SET domain prevents heterochromatinization but still allows de novo methylation, whereas biochemical and functional studies indicate that G9a itself is capable of bringing about de novo methylation through its ankyrin domain, by recruiting Dnmt3a and Dnmt3b independently of its histone methyltransferase activity. These modifications seem to be programmed for carrying out two separate biological functions: histone methylation blocks target-gene reactivation in the absence of transcriptional repressors, whereas DNA methylation prevents reprogramming to the undifferentiated state., Journal Article, Research Support, N.I.H. Extramural, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Histone methylation plays key roles in the regulation of chromatin structure and function. The recent identification of enzymes that antagonize or remove histone methylation offers new opportunities to appreciate histone methylation plasticity in the regulation of epigenetic pathways. Peptidylarginine deiminase 4 (PADI4) was the first enzyme shown to antagonize histone methylation. PADI4 functions as a histone deiminase converting a methylarginine residue to citrulline at specific sites on the tail of histones H3 and H4. This activity is linked to repression of the estrogen-regulated pS2 promoter. Very little is known as to how PADI4 silences gene expression. To investigate the mechanisms by which histone demethylation and in particular PADI4 functions, and as no PADI4 interactors have been described so far, in vitro interaction assays and coimmunoprecipitations were performed. We found that the histone deacetylase HDAC1 interacts with PADI4, both in vitro and in vivo, and associates with PADI4-mediated histone deiminase activity. Chromatin immunoprecipitations in MDA-ER66 cells using antibodies against PADI4, HDAC1, citrulline H3 and acetylated histones show that PADI4 and HDAC1 appear transiently and in a cyclic manner on the estrogen-responsive promoter pS2, in the presence of estradiol. Their presence correlates with the loss of arginine methylation, acquisition of citrulline, histone deacetylation, and disengagement of RNA polymerase II from the pS2 promoter. Furthermore, sequential ChIP further indicated that PADI4 and HDAC1 bind together to the pS2 promoter in the presence of estrogen. These results further substantiate the “transcriptional clock” concept, highlighting the dynamic interplay between deimination and deacetylation of histones., info:eu-repo/semantics/nonPublished

The Myc transcription factor is an essential mediator of cell growth and proliferation through its ability to both positively and negatively regulate transcription. The mechanisms by which Myc silences gene expression are not well understood. The current model is that Myc represses transcription through functional interference with transcriptional activators. Here we show that Myc binds the corepressor Dnmt3a and associates with DNA methyltransferase activity in vivo. In cells with reduced Dnmt3a levels, we observe specific reactivation of the Myc-repressed p21Cip1 gene, whereas the expression of Myc-activated E-boxes genes is unchanged. In addition, we find that Myc can target Dnmt3a selectively to the promoter of p21Cip1. Myc is known to be recruited to the p21Cip1 promoter by the DNA-binding factor Miz-1. Consistent with this, we observe that Myc and Dnmt3a form a ternary complex with Miz-1 and that this complex can corepress the p21Cip1 promoter. Finally, we show that DNA methylation is required for Myc-mediated repression of p21Cip1. Our data identify a new mechanism by which Myc can silence gene expression not only by passive functional interference but also by active recruitment of corepressor proteins. Furthermore, these findings suggest that targeting of DNA methyltransferases by transcription factors is a wide and general mechanism for the generation of specific DNA methylation patterns within a cell.

La méthylation des cytosines de l’ADN est un niveau de contrôle essentiel de la transcription génique. Elle joue un rôle primordial dans plusieurs étapes du développement comme l’inactivation du chromosome X et l’empreinte génomique. De plus, il est de plus en plus évident que la méthylation de l’ADN participe à la cancérogenèse.Actuellement, le monde de la méthylation de l’ADN n’en est encore qu’à l'aube de son histoire. En effet, les mécanismes moléculaires la gouvernant sont encore peu connus. La méthylation de l’ADN est caractérisée par deux concept clés :le verrouillage de la transcription des gènes et le ciblage en des régions spécifiques du génome. Au cours de notre travail de thèse de doctorat, nous avons poursuivi les avancées réalisées dans ces deux domaines.Dans un premier temps, nous nous sommes attachés à l’étude de la répression transcriptionnelle entraînée par la méthylation de l’ADN. Grâce à plusieurs études récentes, il paraît de plus en plus clair que la méthylation agit de paire avec la structure de la chromatine. Nous avons donc concentré nos recherches sur l’interconnexion de celle-ci avec deux machineries impliquées dans la régulation de son degré de compaction :la désacétylation et la méthylation des histones. Par diverses expérimentations, nous avons démontré un lien étroit entre ces machineries répressives pour l’imposition d’un état silencieux de la transcription.Dans la deuxième partie de ce travail, nous avons dirigé notre attention sur le ciblage des Dnmt. Pour cela, nous avons mené deux stratégies de front. La première est une approche ciblée et consiste en l’étude de l’association des Dnmt avec l’oncoprotéine bien connue, Myc. La seconde approche est plus large. Grâce à l’utilisation de la technique du double hybride en levure, nous avons identifié de nouveaux partenaires des Dnmt, dont un qui pourrait s’avéré particulièrement intéressant :le protéine Cart1 (cartilage homeoproteine 1) impliquée dans le développement d, Doctorat en sciences biomédicales, info:eu-repo/semantics/nonPublished