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La méthylation de l’ADN est un phénomène épigénétique qui joue un rôle important dans le développement des mammifères et qui est associé à une répression transcriptionnelle. La méthylation de loci CpG de l’ADN est médiée par les méthyltransférases de l’ADN – les Dnmts. La méthylation joue également un rôle clef dès les stades précoces de la cancérogenèse dans une grande partie des tumeurs où on observe une méthylation, notamment la répression des gènes suppresseurs de tumeurs et une déméthylation, notamment l’expression de séquences d’ADN parasites. Dans une première partie de la thèse, nous nous sommes intéressés à la méthyltransférase de l’ADN Dnmt3L et plus particulièrement à identifier les mécanismes par quels cette méthyltransférase peut réprimer l’expression génique. Dnmt3L, identifiée et clonée en 2000, est caractérisée comme une méthyltransférase dépourvue de son domaine catalytique jouant probablement un rôle dans la régulation de la méthylation de l’ADN plutôt que dans l’ajout de groupes méthyls à l’ADN. Son rôle est fondamental dans l’établissement de l’empreinte génétique maternelle. Des travaux récents, réalisés, notamment par notre équipe, ont permis de montrer que plusieurs Dnmt répriment la transcription non seulement en méthylant l’ADN mais également en interagissant avec les déacétylases d’histones, HDAC. Au regard de ces résultats, nous nous sommes intéressés à évaluer si Dnmt3L est également capable de réprimer la transcription en recrutant une activité HDAC. Des recherches menées de concert avec Rachel Deplus, étudiante en thèse au laboratoire, ont permis de montrer que Dnmt3L interagit avec les déacétylases d’histones, ce qui conduit à la répression de la transcription. Dans l’ensemble, nos résultats ont permis de montrer que Dnmt3L, bien que dépourvue d’activité méthyltransférase, peut, tout comme les autres Dnmt, également réprimer la transcription par le biais de son interaction avec les enzymes HDAC. Dans une deuxième partie majeure de la thèse, nous nous sommes intéressés à l’étude des mécanismes par lesquels la méthylation de l’ADN peut être ciblée au sein du génome. Cette thématique bien que fondamentale, est encore peu connue à l’heure actuelle. Des études très récentes, réalisées dans notre laboratoire, suggèrent que les Dnmt peuvent être recrutées au sein de loci spécifiques suite à leur association avec des facteurs de transcription. Dans le cadre de ma thèse, nous avons pu montrer que la méthyltransférase de l’ADN Dnmt3a interagit in vitro et in vivo avec l’oncoprotéine Myc. Nous avons également pu mettre en évidence que la protéine Myc endogène recrute une activité méthyltransférase de l’ADN. Bien que l’oncoprotéine Myc soit surtout connue pour être un activateur de la transcription, Myc est également décrit pour réprimer la transcription de certains gènes spécifiques. Il était donc raisonnable de proposer que Dnmt3a pourrait être recruté pour réprimer les gènes régulés négativement par Myc. En effet en testant l’activité promotrice du gène p21, un gène connu pour être réprimé par Myc, nous avons démontré que Dnmt3a agit comme un co-répresseur transcriptionnel de Myc. Par des essais d’immunoprécipitation de la chromatine (ChIP), nous avons démontré que Myc et Dnmt3a forment un complexe stable sur le promoteur du gène p21. D’autre part nous avons montré que la 5 azacytidine, un agent déméthylant, lève la répression médiée par Myc au sein du promoteur du gène p21. En collaboration avec d’autres laboratoires, nous avons également effectué du séquençage au bisulfite du promoteur proximal de ce gène à partir d’ADN provenant de cellules sauvages pour Myc (myc+/+) ou invalidées pour Myc (myc-/-), montrant que la méthylation de ce promoteur est dépendante de la présence de Myc. Ainsi il semble que l’activité méthyltransférase de l’ADN de Dnmt3a soit requise pour sa fonction de co-répresseur des gènes régulés négativement par Myc. Cette étude confirme et valide le nouveau concept du recrutement des Dnmt par des interactions protéine-protéine. Notre travail a également des implications sur la compréhension du rôle de la méthylation de l’ADN dans la tumorigenèse. De plus, cette étude a permis de montrer pour la première fois que l’oncoprotéine Myc n’est pas seulement impliquée dans une répression génique passive (séquestration de co-activateurs) mais également une répression active (recrutement du co-represseur Dnmt3a).

Les méthyltransférases de l'ADN (DNMT) catalysent la méthylation de l'ADN, l'une des marques épigénétiques les plus étudiées. Dans les cancers, on observe une hyperméthylation spécifique de promoteurs de gènes suppresseurs de tumeurs (GST) conduisant à leur extinction génique, ce qui participe au maintien et à la progression de tumeurs. A ce jour, les mécanismes responsables de cette hyperméthylation spécifique des promoteurs de GST dans les cancers sont indéterminés. Ces travaux de thèse sont consacrés à l'inhibition des DNMT dans les cancers afin de restaurer l'expression des GST mais également à l'utilisation d'une approche innovante de chemobiologie pour l'identification de partenaires des DNMT potentiellement responsables de leur adressage vers les régions promotrices des GST. Les partenaires ainsi identifiés peuvent constituer de nouvelles cibles épigénétiques pour le ciblage indirect de la méthylation de l'ADN dans les cancers. Deux séries d'inhibiteurs de DNMT ont été étudiées. La première est la famille des chloronitro-flavanones, précédemment identifiée par criblage, pour laquelle de nouveaux dérivés de type bromonitro-flavanones ont été synthétisés afin d'améliorer la stabilité en conditions physiologiques. J'ai réalisé l'étude des effets pharmacologiques de cette famille de molécules. J'ai également entrepris la synthèse et la caractérisation pharmacologique de nouveaux inhibiteurs de type bi-substrats, analogues de l'adénosine et de la désoxycytidine, conçus par une approche rationnelle. Ces deux études ont permis respectivement d'identifier un dérivé flavanone plus stable et plus actif que le composé de référence et deux dérivés quinazoline-quinoléine très prometteurs, actifs sur les DNMT et dans les lignées cellulaires, à la fois pour la réexpression d'un gène rapporteur mais surtout dans l'induction de la déméthylation du GST CDKN2A et de sa réexpression. Pour identifier les partenaires de DNMT, nous avons employé une approche de chemobiologie (" Activity-Based Protein Profiling - ABPP ") basée sur la conception de sondes chimiques comportant un inhibiteur de DNMT. Ces sondes, utilisées sur des cellules vivantes, permettent, grâce à une étape de fonctionnalisation par chimie bioorthogonale, de purifier les protéines partenaires des DNMT. Vingt sondes ont été synthétisées et leurs activités ont été évaluées sur des modèles enzymatiques et cellulaires. Les sondes sélectionnées ont été utilisées dans des lignées cellulaires cancéreuses pour purifier les protéines partenaires qui ont ensuite été identifiées par analyse protéomique. Suite à leur validation, ces protéines pourront constituer de nouvelles cibles de la méthylation aberrante de l'ADN dans les cancers. / DNA methyltransferases (DNMTs) catalyse DNA methylation, one of the most studied epigenetic marks. In cancers, a specific hypermethylation of the promoters of the tumour suppressor genes (TSGs) is observed, which leads to their silencing. This abnormal DNA methylation pattern participates to the maintenance and the progression of the tumour. Today, the mechanisms that direct this specific hypermethylation of TSG promoters and their transcriptional repression in cancers are still unknown. The aim of my PhD is to identify DNMT inhibitors that are able to reactivated TSGs in cancer cells but also to identify the DNMT partners that address specifically these enzymes to TSG promoter regions. Such partners can constitute new anticancer "epitargets" to indirectly target DNA methylation specifically in cancer cells. Two families of DNMT inhibitors were studied. The first one starts from the chloronitro-flavanones previously identified by screening. New derivatives including bromonitro-flavanones were synthesised aiming at improving compound stability. I pharmacologically characterised these compounds and show for one of them an increased stability and activities compared to reference compound. In parallel, I synthesised and pharmacologically characterised new bi-substrate analogue inhibitors, mimicking the adenosine and the deoxycytidine. Two very promising quinazoline-quinoline derivatives were identified. They are active against DNMT and in cell lines, both for reexpression of a reporter gene but mostly in CDKN2A TSG demethylation inducing its reexpression. To identify DNMT partners we adopted a chemical biology approach (Activity-Based Protein Profiling (ABPP)) based on the use of chemical probes including in-house non- nucleoside DNMT inhibitors as bait to trap the DNMT partners. We designed and synthesised twenty chemical probes and evaluate them using enzymatic and cellular-based assays. Selected probes were used to carry out ABPP directly in living cells. After functionalization by bioorthogonal chemistry, DNMT protein partners were purified and identified by proteomic analysis. Target validation would enable to determine new targets for the aberran

A exposição ao estresse induz um aumento dos níveis de óxido nítrico (NO) e glutamato em estruturas do cérebro de ratos, as quais estão relacionadas com o transtorno de depressão maior (DM) em humanos. Ademais, o estresse está diretamente relacionado com o aumento da metilação do DNA, uma alteração epigenética repressiva, no hipocampo de animais. Estudos anteriores demonstraram o efeito tipo antidepressivo dos inibidores da enzima óxido nítrico sintase (NOS) em animais submetidos ao estresse. Porém não se sabe se há uma relação entre o aumento do NO e glutamato induzido pelo estresse e alteração na metilação do DNA em genes relacionado com a patofisiologia da DM. Assim, o objetivo deste estudo foi investigar os efeitos dos inibidores da NOS nas alterações comportamentais e nos mecanismos intracelulares relacionado com a metilação do DNA no cérebro de ratos submetidos ao teste do desamparo aprendido (learned helplessness - LH) e em cultura celular do hipocampo desafiadas com NMDA e dexametasona. Métodos: Estudo 1: Cultura primária de células do hipocampo ou cultura imortalizada HiB5 foram desafiadas/estressadas com NMDA (30µM,1h), L-arginina (500µM,1h) e/ou dexametasona (1µM, 1h ou 24h) e pré-tratadas com inibidor seletivo da nNOS (NPA, 100nM, 30min antes do desafio) ou com inibidor da DNMT (5-Aza, 10 µM, 30 min antes do desafio). A expressão dos genes para as enzimas DNMTs, BDNF, NT4, TrkB e nNOS foram avaliadas por RT-qPCR, a expressão proteica das enzimas DNMT3b e nNOS foram avaliadas por western blotting. Estudo 2: Ratos foram submetidos à choques inescapáveis (0,4 mA; 40 choques) na sessão de pré-teste do LH, após sete dias os animais foram submetidos a sessão de teste (choques escapáveis de 0,4 mA). Os animais foram tratados com inibidores da NOS 7-nitroindazole (7-NI;60mg/kg,i.p), aminoguanidina (AMG; 30mg/kg,i.p) ou veículo por 7 dias e submetidos a sessão de teste 1h, após a última injeção. A metilação global foi analisada por imunoensaio (ELISA) e a expressão dos genes DNMT3b, BDNF, nNOS e iNOS foram avaliadas por RT-qPCR, nas estruturas: cortex, hipocampo ventral e hipocampo dorsal. Resultados: Estudo 1: O pré- tratamento com NPA, atenuou o aumento da expressão do mRNA para a enzima DNMT3b, em cultura primária do hipocampo desafiada com NMDA, dexametasona e Larginina, e também em cultura HiB5 desafiada com dexametasona. Porém, o NPA não inibiu a diminuição da expressão do BDNF (exon 1, exon 4 e exon 9), em cultura primária de células do hipocampo desafiadas com NMDA. O pré tratamento com 5-Aza, não inibiu as alterações induzidas pelo NMDA em cultura primária de hipocampo. Estudo 2: Ratos submetidos ao estresse dos choques inescapáveis na sessão de pré-teste apresentaram aumento no número de falhas em escapar dos choques na sessão de teste (desamparo aprendido), um efeito que foi atenuado pelo tratamento com AMG ou 7-NI. Interessantemente, o efeito comportamental do estresse foi acompanhado por aumento nos níveis da metilação global do DNA e DNMT3b no hipocampo ventral (vHPC), que foi atenuado pelos pré-tratamentos com AMG e 7-NI, porém não houve diferença estatisticamente significante no córtex e no hipocampo dorsal dos ratos. Conclusão: Os dados apresentados demonstraram que tanto o estresse (in vivo) quanto o desafio com glicocorticóides, NMDA e L-arginina (in vitro) são capazes de modular a expressão daenzima DNMT3b e a metilação de DNA no hipocampo. O tratamento com inibidores da NOS reduzem os efeitos do estresse in vivo (comportamental e molecular) e in vitro. Em conjunto, os dados sugerem que a liberação de glutamato e NO durante o estresse pode modular a expressão da enzima DNMT3b, levando ao aumento da metilação do DNA em genes relacionados com a resposta de adaptação ao estresse. Essa é a primeira evidência de que o NO pode modular metilação do DNA induzida por estresse. / Stress exposure increases glutamate and nitric oxide (NO) levels, as well as DNA methylation in the hippocampus. However, it is not yet known if there is a causal relationship between these events. Moreover, both nitric oxide synthase (NOS) inhibitors and DNA methylation inhibitors counteract the behavioral effects of stress. Therefore, our aim was to investigate the effects of NOS inhibitors on stress-induced changes on behaviour, DNA methylation and genes expression in the hippocampus of rats submitted to learned helplessness - LH. Moreover, the effects of direct administration of dexamethasone (glucocorticoid), NMDA and L-arginine was investigated in hippocampal cell cultures. Methods: Study 1: Primary hippocampal cell culture was challenged with NMDA (30µM,1h), L-arginine (500µM,1h) or dexamethasone (1µM,24h) and pretreated with nNOS inhibitor (NPA, 100nM, 30min before the challenge) or with DNMT inhibitor (5-Aza, 10 µM, 30 min before the challenge). DNMTs, BDNF, NT4, TrkB and nNOS gene expression was assessed by RT-qPCR. DNMT3b and nNOS levels were assessed by western blotting. Study 2: Rats were submitted to inescapable footshocks and treated with the NOS inhibitors 7-nitroindazole (7-NI; 60 mg/kg, i.p) or aminoguanidine (AMG; 30 mg/kg, i.p], or vehicle for 7 days and tested 1h after the last injection with escapable footshocks. The number of escape failures during the test, global DNA methylation (ELISA) and DNMT3b, BDNF, nNOS and iNOS mRNA expression (RT-qPCR) was evaluated. Results: NPA pretreatment attenuated DNMT3b mRNA expression in hippocampus primary cell culture challenged with NMDA, dexamethasone or L-arginine. Similarly effects were observed in HiB5 cell challenged with dexamethasone. However, NPA pretreatment did not inhibit the decrease of BDNF (exon 1, exon 4 and exon 9) induced by NMDA. Moreover, pretreatment with 5-Aza did not inhibit the decreased of BDNF induced by NMDA in primary cell culture. Study 2: Stress exposure increased the number of escape failures in the test, which was attenuated by treatment with AMG or 7-NI, an antidepressant-like effect. Interestingly, the increased DNA methylation DNMT3b mRNA expression in the ventral hippocampus (vHPC) of stressed rats were also attenuated by treatment with both AMG and 7-NI. Conclusions: NOS inhibitors attenuated stress-induced depressive-like behavior, DNA methylation and DNMT3b mRNA expression in the vHPC. In vitro, selective nNOS inhibition also blocks corticosterone-, NMDA- and L-arginine-induced DNMT3b mRNA expression in hippocampal cell culture. Altogether, our results suggest that glutamate release, leading to NO production during stress may mediate intracellular mechanisms that regulate DNMT3b expression and DNA methylation. This is the first evidence indicating that NO modulates DNA methylation induced by stress.

A exposição ao estresse induz um aumento dos níveis de óxido nítrico (NO) e glutamato em estruturas do cérebro de ratos, as quais estão relacionadas com o transtorno de depressão maior (DM) em humanos. Ademais, o estresse está diretamente relacionado com o aumento da metilação do DNA, uma alteração epigenética repressiva, no hipocampo de animais. Estudos anteriores demonstraram o efeito tipo antidepressivo dos inibidores da enzima óxido nítrico sintase (NOS) em animais submetidos ao estresse. Porém não se sabe se há uma relação entre o aumento do NO e glutamato induzido pelo estresse e alteração na metilação do DNA em genes relacionado com a patofisiologia da DM. Assim, o objetivo deste estudo foi investigar os efeitos dos inibidores da NOS nas alterações comportamentais e nos mecanismos intracelulares relacionado com a metilação do DNA no cérebro de ratos submetidos ao teste do desamparo aprendido (learned helplessness - LH) e em cultura celular do hipocampo desafiadas com NMDA e dexametasona. Métodos: Estudo 1: Cultura primária de células do hipocampo ou cultura imortalizada HiB5 foram desafiadas/estressadas com NMDA (30µM,1h), L-arginina (500µM,1h) e/ou dexametasona (1µM, 1h ou 24h) e pré-tratadas com inibidor seletivo da nNOS (NPA, 100nM, 30min antes do desafio) ou com inibidor da DNMT (5-Aza, 10 µM, 30 min antes do desafio). A expressão dos genes para as enzimas DNMTs, BDNF, NT4, TrkB e nNOS foram avaliadas por RT-qPCR, a expressão proteica das enzimas DNMT3b e nNOS foram avaliadas por western blotting. Estudo 2: Ratos foram submetidos à choques inescapáveis (0,4 mA; 40 choques) na sessão de pré-teste do LH, após sete dias os animais foram submetidos a sessão de teste (choques escapáveis de 0,4 mA). Os animais foram tratados com inibidores da NOS 7-nitroindazole (7-NI;60mg/kg,i.p), aminoguanidina (AMG; 30mg/kg,i.p) ou veículo por 7 dias e submetidos a sessão de teste 1h, após a última injeção. A metilação global foi analisada por imunoensaio (ELISA) e a expressão dos genes DNMT3b, BDNF, nNOS e iNOS foram avaliadas por RT-qPCR, nas estruturas: cortex, hipocampo ventral e hipocampo dorsal. Resultados: Estudo 1: O pré- tratamento com NPA, atenuou o aumento da expressão do mRNA para a enzima DNMT3b, em cultura primária do hipocampo desafiada com NMDA, dexametasona e Larginina, e também em cultura HiB5 desafiada com dexametasona. Porém, o NPA não inibiu a diminuição da expressão do BDNF (exon 1, exon 4 e exon 9), em cultura primária de células do hipocampo desafiadas com NMDA. O pré tratamento com 5-Aza, não inibiu as alterações induzidas pelo NMDA em cultura primária de hipocampo. Estudo 2: Ratos submetidos ao estresse dos choques inescapáveis na sessão de pré-teste apresentaram aumento no número de falhas em escapar dos choques na sessão de teste (desamparo aprendido), um efeito que foi atenuado pelo tratamento com AMG ou 7-NI. Interessantemente, o efeito comportamental do estresse foi acompanhado por aumento nos níveis da metilação global do DNA e DNMT3b no hipocampo ventral (vHPC), que foi atenuado pelos pré-tratamentos com AMG e 7-NI, porém não houve diferença estatisticamente significante no córtex e no hipocampo dorsal dos ratos. Conclusão: Os dados apresentados demonstraram que tanto o estresse (in vivo) quanto o desafio com glicocorticóides, NMDA e L-arginina (in vitro) são capazes de modular a expressão daenzima DNMT3b e a metilação de DNA no hipocampo. O tratamento com inibidores da NOS reduzem os efeitos do estresse in vivo (comportamental e molecular) e in vitro. Em conjunto, os dados sugerem que a liberação de glutamato e NO durante o estresse pode modular a expressão da enzima DNMT3b, levando ao aumento da metilação do DNA em genes relacionados com a resposta de adaptação ao estresse. Essa é a primeira evidência de que o NO pode modular metilação do DNA induzida por estresse. / Stress exposure increases glutamate and nitric oxide (NO) levels, as well as DNA methylation in the hippocampus. However, it is not yet known if there is a causal relationship between these events. Moreover, both nitric oxide synthase (NOS) inhibitors and DNA methylation inhibitors counteract the behavioral effects of stress. Therefore, our aim was to investigate the effects of NOS inhibitors on stress-induced changes on behaviour, DNA methylation and genes expression in the hippocampus of rats submitted to learned helplessness - LH. Moreover, the effects of direct administration of dexamethasone (glucocorticoid), NMDA and L-arginine was investigated in hippocampal cell cultures. Methods: Study 1: Primary hippocampal cell culture was challenged with NMDA (30µM,1h), L-arginine (500µM,1h) or dexamethasone (1µM,24h) and pretreated with nNOS inhibitor (NPA, 100nM, 30min before the challenge) or with DNMT inhibitor (5-Aza, 10 µM, 30 min before the challenge). DNMTs, BDNF, NT4, TrkB and nNOS gene expression was assessed by RT-qPCR. DNMT3b and nNOS levels were assessed by western blotting. Study 2: Rats were submitted to inescapable footshocks and treated with the NOS inhibitors 7-nitroindazole (7-NI; 60 mg/kg, i.p) or aminoguanidine (AMG; 30 mg/kg, i.p], or vehicle for 7 days and tested 1h after the last injection with escapable footshocks. The number of escape failures during the test, global DNA methylation (ELISA) and DNMT3b, BDNF, nNOS and iNOS mRNA expression (RT-qPCR) was evaluated. Results: NPA pretreatment attenuated DNMT3b mRNA expression in hippocampus primary cell culture challenged with NMDA, dexamethasone or L-arginine. Similarly effects were observed in HiB5 cell challenged with dexamethasone. However, NPA pretreatment did not inhibit the decrease of BDNF (exon 1, exon 4 and exon 9) induced by NMDA. Moreover, pretreatment with 5-Aza did not inhibit the decreased of BDNF induced by NMDA in primary cell culture. Study 2: Stress exposure increased the number of escape failures in the test, which was attenuated by treatment with AMG or 7-NI, an antidepressant-like effect. Interestingly, the increased DNA methylation DNMT3b mRNA expression in the ventral hippocampus (vHPC) of stressed rats were also attenuated by treatment with both AMG and 7-NI. Conclusions: NOS inhibitors attenuated stress-induced depressive-like behavior, DNA methylation and DNMT3b mRNA expression in the vHPC. In vitro, selective nNOS inhibition also blocks corticosterone-, NMDA- and L-arginine-induced DNMT3b mRNA expression in hippocampal cell culture. Altogether, our results suggest that glutamate release, leading to NO production during stress may mediate intracellular mechanisms that regulate DNMT3b expression and DNA methylation. This is the first evidence indicating that NO modulates DNA methylation induced by stress.

Le myélome multiple (MM) est une néoplasie B caractérisée par l’accumulation d’un clone plasmocytaire dans la moelle osseuse. Des études ont démontré que les modifications épigénétiques comme la méthylation de l’ADN jouent un rôle dans la régulation d’expression de différents gènes associés au cancer. Dans une étude récente, nous avons pu décrire un score génique de méthylation de l’ADN permettant de prédire la sensibilité des cellules de MM aux inhibiteurs de DNMT (DNA methyltranfexrase) (Moreaux, et al 2012). Parmi les gènes dont l’expression est inhibée par les inhibiteurs de DNMT et associés avec un pronostic péjoratif chez les patients atteints de MM, nous avons identifié RECQ1. RECQ1 est une hélicase de la famille RECQ qui s’associe aux origines de réplication durant la phase S du cycle cellulaire et joue un rôle important dans l’élongation des fourches de réplication. RECQ1 est fortement exprimé dans différents types de tumeurs solides et l’inhibition de RECQ1 conduit à la catastrophe mitotique et inhibe la croissance de tumeurs solides. Le but de notre projet a été de caractériser la fonction de RECQ1 dans la physiopathologie du MM et les mécanismes de résistance aux traitements. Afin d’étudier le rôle biologique de RECQ1 dans les plasmocytes tumoraux, nous avons utilisé des vecteurs lentiviraux pour induire de façon inductible la surexpression ou l'inhibition de RECQ1. La déplétion de RECQ1 dans les cellules de MM entraîne une inhibition de la croissance, une induction significative d’apoptose et la formation de foyers 53BP1 indiquant la présence de cassures d’ADN double brin. Une forte expression de RECQ1 étant associée à un mauvais pronostic et la déplétion de RECQ1 conduisant à une induction de cassures d’ADN double brin, nous nous sommes demandé si l’inhibition de l’expression de RECQ1 pourrait sensibiliser les cellules de MM aux agents génotoxiques utilisés dans le traitement du MM. La déplétion de RECQ1 sensibilise, de façon significative, les cellules de MM au melphalan suggérant que l’association d’un inhibiteur de DNMT pour cibler RECQ1 et du melphalan pourrait avoir un effet synergique chez les patients RECQ1++. La surexpression de RECQ1 protège les lignées cellulaires de myélome contra l'apoptose induite par melphalan et bortézomib. De plus, l'épuisement RECQ1 sensibilise les cellules de myélome de traitement est démontré que RECQ1 interagit avec des protéines impliquées dans différentes voies de réparation des dommages de l’ADN : PARP1 (NHEJ/BER), RAD51 (HR), MSH2 et MSH6 (Mismatch repair). RECQ1 interagit avec PARP1 dans la fraction chromatinienne des cellules de MM mais pas avec RAD51 ni MSH2. Cette interaction est significativement induite en présence de melphalan. Des inhibiteurs de PARP sont actuellement en développement préclinique ou en essai clinique. De façon intéressante, la déplétion de RECQ1 sensibilise significativement les cellules de MM à un inhibiteur de PARP in vitro suggérant que l’association d’un inhibiteur de DNMT pour cibler RECQ1 et d’un inhibiteur de PARP pourrait avoir un intérêt thérapeutique dans le MM. Nous avons également confirmé que des doses sous-létales d’inhibiteur de DNMT sensibilisent les cellules de MM au melphalan in vitro. / Multiple myeloma (MM) is a plasma cell cancer with poor survival, characterized by the clonal expansion of multiple myeloma cells (MMCs), primarily in the bone marrow. Using a microarray-based genome-wide screen for genes responding to DNA methyltransferases (DNMT) inhibition in MM cells, we identified RECQ1 among the genes downregulated by DNMT inhibitor. RECQ helicase are DNA unwinding enzymes involved in the maintenance of chromosome stability. RECQ1 silencing in cancer cells results in mitotic catastrophe and prevents tumor growth in murine models. RECQ1 is significantly overexpressed in primary myeloma cells compared to normal plasma cells and in myeloma cell lines compared to primary myeloma cells of patients. High RECQ1 expression is associated with a poor prognosis in two independent cohorts of patients. RECQ1 knock down inhibits growth of myeloma cells and induces apoptosis. Given the known role of RECQ1 in replication and DNA repair activation, the effect of RECQ1 depletion in DNA damage response was investigated. RECQ1 depletion induced spontaneous accumulation of DNA double strand breaks (DSBs) evidenced by the phosphorylation of ATM and H2AX histone and detection of 53BP1 foci. Using an alkaline comet assay, a significant increase in DNA strand breaks was confirmed in RECQ1 depleted cell lines compared to control. RECQ1 depletion was associated with CHK1 and CHK2 phosphorylation in MM cells. Since RECQ1 depletion is associated with DNA damage response activation and DNA strand breaks formation, a link between RECQ1 expression and drug sensitivity was hypothesized. RECQ1 overexpression significantly protects myeloma cell lines from melphalan and bortezomib-induced apoptosis. Furthermore, RECQ1 depletion sensitizes myeloma cells to treatment. Using immunoprecipitation, RECQ1 was shown to interact with PARP1 but not RAD51 or MSH2. An increased association of the two proteins was found upon DNA damages induced by melphalan. In agreement, RECQ1 depletion sensitizes myeloma cell lines to PARP inhibitor. We identified RECQ1 as a miR-203 target. Interestingly, aberrant methylation of miR-203 was reported in MM cells and treatment with 5-aza-2’-deoxycitidine led to promoter demethylation and miR-203 re-expression. Furthermore, anti-miR-203 treatment induced a significant increase of RECQ1 mRNA level in MM cells.In conclusion, RECQ1 represent a biomarker of drug resistance in MM, which is targeted by DNMT inhibitors. This suggests association of alkylating agents and/or PARP inhibitors with DNMT inhibitor may represent a therapeutic approach in RECQ1high patients associated with a poor prognosis.

Les modifications épigénétiques participent au contrôle de l'expression génique. Il a été montré que la méthylation des désoxycytidines (dC) de l'ADN joue un rôle clé dans la régulation épigénétique chez les mammifères. Cette modification correspond à la marque épigénétique la plus stable. Elle a lieu sur des résidus CpG regroupés en ilôts, essentiellement situés au niveau des séquences promotrices, des séquences répétées et des séquences encadrants les ilôtsCpG. L'hyperméthylation des promoteurs induit une inhibition de l'expression des gènes, tandis qu'une hypométhylation est associée à une expression. Les enzymes responsables de la méthylation de l'ADN sont les méthyltransférases d'ADN (DNMTs). Deux familles de DNMTscatalytiquement actives ont été identifiées: on distingue la DNMT1, principalement responsable de la maintenance de la méthylation de l'ADN lors de la réplication, et les DNMT3A et 3B, qui sont responsables d'une méthylation de l'ADN dite de novo. L'altération des profils de méthylation de l'ADN conduit à diverses maladies telles que le cancer. Les cellules cancéreuses présentent souvent un profil de méthylation de l'ADN différent des cellules saines, on observe en particulier une hyperméthylation spécifique des gènes dits suppresseurs de tumeur. Une restauration de leur expression par l'inhibition de la méthylation de l'ADN représente ainsi une stratégie thérapeutique attrayante. Plusieurs inhibiteurs de DNMTs ont été décrits et deux analogues de nucléosides sont approuvés par la FDA pour traiter certaines leucémies: la 5-azacytidine (VidazaTM) et la 5-azadeoxycytidine (Dacogene(r)). Notre laboratoire développe depuis plusieurs années de nouveaux inhibiteurs non nucléosidiques de DNMTs qui ciblent leur site catalytique. J'ai étudié ici les effets pharmacologiques de ces inhibiteurs catalytiques des DNMTs, en utilisant plusieurs lignées cellulaires cancéreuses (issues d'une leucémie, d'un lymphome et d'un cancer du côlon). J'ai utilisé pour cela différentes technologies permettant d'analyser la méthylation de l'ADN, l'accessibilité de la chromatine, les modifications des histones et l'expression des gènes. Ces nouvelles thérapies épigénétiques visent à la reprogrammation des cellules cancéreuses, c'est pourquoi j'ai exploré les modifications à long terme induites par ces nouveaux composés. Nous avons montré que ces composés sont des inhibiteurs puissants de DNMT3A et qu'ils sont capables d'induire l'expression d'un gène raporteur (la luciférase) sous le contrôle du promoteur CMV, par une déméthylation de ce promoteur et une ouverture de la chromatine. Enfin, ces nouveaux inhibiteurs de DNMTs déméthylent la région promotrice de gènes suppresseurs de tumeurs et induisent leur ré-expression. / Epigenetic modifications participate to the control of gene expression. Methylation of deoxycytidines (dC) in the DNA was shown to play a key role in epigenetic regulation in mammals. It is the most stable epigenetic mark and occurs at CpG sites, which are grouped in islands and essentially located in promoters, repeated sequences and CpG island shores. Hypermethylation of promoters induces gene silencing while hypomethylation is associated to gene expression. Enzymes responsible for DNA methylation are the DNA methyltransferases (DNMTs). Two families of catalyticallyactive DNMTs have been identified: DNMT1, mainly responsible for DNA methylation maintenance during replication; and DNMT3A and 3B that perform de novo DNA methylation and support maintenance. Alteration of DNA methylation patterns lead to various diseases such as cancer. Cancerous cells often present aberrant DNA methylation, in particular a specific hypermethylation of tumor suppressor genes is observed. Restoring their expression by inhibition of DNA methylation represents an attractive therapeutic strategy. Several DNMTs inhibitors have been described. Two nucleoside analogs are FDA approved to treat leukemia: 5-azacytidine (VidazaTM) and 5-azadeoxycytidine (Dacogene(r)). Our laboratory develops since several years new inhibitors of DNMT, non-nucleoside analogs, targeting the catalytic site. Here, I studied the pharmacological effects of these DNMTs catalytic inhibitors using several cancer cell lines (leukemia, lymphoma and colon cancer) and different technologies to follow DNA methylation, chromatin accessibility, histone modifications and gene expression. Since epigenetic therapies aim at the reprogramming of cancer cells, I explored the long-term modifications induced by the compounds. We show that these novel compounds are potent inhibitors of DNMT3A and able to induce the expression of a reporter gene (luciferase) under the control of a methylated CMV promoter by demethylation of the promoter and opening of the chromatin. Finally, these new DNMTs inhibitors demethylate the promoter region of tumor suppressor genes and induce their re-expression.

La toxoplasmose, déterminée par le parasite Toxoplasma gondii, est l'une des infections les plus répandues au monde, en raison de la persistance à vie sous forme latente de ce parasite au sein de ces hôtes. Ce parasite fait partie des Apicomplexa et détourne les voies de signalisation de l'hôte par des mécanismes épigénétiques qui convergent vers des protéines nucléaires clés. Nous rapportons ici une nouvelle stratégie de persistance parasitaire impliquant la protéine de rhoptries ROP16 de T. gondii, sécrétée précocement lors de l'invasion, qui cible le facteur de transcription UHRF1 (Ubiquitin-like containing PHD and RING fingers domain 1) et induit un arrêt du cycle de la cellule-hôte. Ceci est induit par l'activité de la DNMT et le remodelage de la chromatine au niveau du promoteur du gène de la cycline B1 par le recrutement d’UHRF1 phosphorylé associé à un complexe protéique multienzymatique répressif. Cela conduit à la désacétylation et à la méthylation de l'histone H3 entourant le promoteur de la cycline B1 pour réduire de manière épigénétique son activité transcriptionnelle. De plus, l'infection par T. gondii provoque une hyper-méthylation de l'ADN dans la cellule hôte par la régulation positive des DNMTs. ROP16 est déjà connue pour activer et phosphoryler des facteurs de transcription de l'immunité protectrice tels que STAT 3/6/5 et le suppresseur tumoral p53 impliqué dans la progression du cycle cellulaire. De plus, ROP16 module ces voies de signalisation de l'hôte de manière souche-dépendante. Comme dans le cas de STAT3, les effets de ROP16 sur UHRF1 dépendent du polymorphisme d'un seul acide aminé du domaine kinase de ROP16. Ce travail montre que Toxoplasma module un nouvel initiateur épigénétique, UHRF1, via un événement précoce initié par la kinase parasitaire ROP16. / Toxoplasmosis, caused by the apicomplexan parasite Toxoplasma gondii, is one of the most common infections in the world due to the lifelong persistence of this parasite in a latent stage in its hosts. T. gondii hijacks host signaling pathways through epigenetic mechanisms which converge on key nuclear proteins. Here we report a new parasite persistence strategy involving Toxoplasma rhoptry protein ROP16 secreted early during invasion, which targets the transcription factor UHRF1 (Ubiquitin-like containing PHD and RING fingers domain 1), and leads to host cell cycle arrest. This is mediated by DNMT activity and chromatin remodeling at the cyclin B1 gene promoter through recruitment of phosphorylated UHRF1 associated with a repressive multienzymatic protein complex. This leads to deacetylation and methylation of histone H3 surrounding the cyclin B1 gene promoter to epigenetically silence its transcriptional activity. Moreover, T. gondii infection causes DNA hypermethylation in its host cell, by upregulation of DNMTs. ROP16 is already known to activate and phosphorylate protective immunity transcription factors such as STAT 3/6/5 and the tumor suppressor p53 involved in cell cycle progression. Moreover, ROP16 modulates host signaling pathways in a strain-dependent manner. Like in the case of STAT3, the strain-dependent effects of ROP16 on UHRF1 can be attributed to a single amino-acid polymorphism in ROP16. This study demonstrates that Toxoplasma hijacks a new epigenetic initiator, UHRF1, through an early event initiated by the ROP16 parasite kinase.

Introdução: Mecanismos epigenéticos, como a metilação de DNA, desempenham um papel importante na neurobiologia da depressão. Enquanto o estresse aumenta a metilação de DNA e reduz a expressão de genes envolvidos na plasticidade neuronial, inibidores de DNA metiltransferases (DNMTi), enzimas que catalisam a metilação de DNA, aumentam rapidamente a expressão gênica e induzem efeitos tipo-antidepressivos em modelos animais. Considerando, ainda, que antidepressivos convencionais podem interferir com mecanismos epigenéticos, este trabalho testou a hipótese de que drogas DNMTi induzem efeito tipo-antidepressivo agudo e sustentado em modelos animais. Além disso, avaliamos se o efeito de antidepressivos convencionais e de DNMTis sobre os níveis de mRNA e de metilação de DNA em diferentes genes associados a depressão e regulados por mecanismos epigenéticos (BDNF, TrkB, 5-HT1A, NMDA e AMPA) em estruturas encefálicas (hipocampo dorsal, ventral e córtex pré-frontal) de animais submetidos a modelo animal de depressão. Métodos: Para tanto, ratos Wistar foram submetidos ao modelo do desamparo aprenddo [learned helplessness, LH, pré-teste (PT), 40 choques inescapáveis nas patas]. Os animais receberam injeções sistêmicas de DNMTi (5-AzaD ou RG108), antidepressivos (imipramina ou fluoxetina), ou veículo, por 1 ou 7 dias, e foram submetidos a sessão teste do desamparo aprendido (T, 30 choques escapáveis) no último dia. Adicionalmente, um grupo independente foi submetido ao mesmo protocolo experimental e sacrificados 1 h após a última injeção. As estruturas encefálicas foram dissecadas para posterior análise molecular [imunoprecipitação de DNA metilado (meDIP) e quantificação de RNAm por qRT-PCR). Resultados: O estresse dos choques nas patas aumentou o número de falhas no teste. O tratamento com DNMTi agudamente, assim como com antidepressivos (tratamento repetido), foi capaz de atenuar essas alterações comportamentais, efeito considerado tipo-antidepressivo nesse modelo. Ainda, o estresse aumentou a metilação de DNA e reduziu os níveis de RNAm para BDNF e TrkB, enquanto que o tratamento com RG108 atenuou essas alterações moleculares no córtex pré-frontal de ratos. Conclusão: Os presentes resultados indicam que DNMTi, diferente de antidepressivos convencionais, são capazes de induzir rápido e sustentado efeito tipo-antidepressivo. Além disso, BDNF e TrkB parecem ser importantes para a resposta comportamental induzida pela inibição de DNMTs no córtex pré-frontal de ratos submetidos ao LH. / Introduction: Epigenetic mechanisms, such as DNA methylation, are thought to play an important role in the neurobiology of depression. While stress increases DNA methylation and decreases the expression of genes involved in neuronal plasticity, DNA methyltransferases inhibitors (DNMTi) increases gene expression and induces antidepressant-like effects in animal models. Considering that conventional antidepressants could interfere with epigenetic mechanisms, this work tested the hypothesis that acute treatment with DNMTi would induce acute and long-lasting antidepressant-like effects. Furthermore, we evaluated whether the stress could induce changes in the mRNA and DNA methylation levels in different genes involved with depression and regulated by epigenetic mechanisms (BDNF, TrkB, 5-HT1A, NMDA and AMPA) in different brain structures [dorsal hippocampus, ventral hippocampus and prefrontal cortex (PFC)] and whether such changes would be attenuated by systemic treatment with DNMTi (acutely) and antidepressants (chronically). Methods: Male Wistar rats were submitted to the learned helplessness model (LH; pretest session, 40 inescapable foot shocks). The animals received systemic injection the DNMTi (5-AzaD or RG108), antidepressants (imipramine or fluoxetine) or vehicle for one or seven days and were submitted to the LH test (30 escapable foot shocks) in the last day. Additionally, one independent group were submitted to the same experimental protocol and sacrificed one hour after last injection for collection of brain samples to further molecular analyses (methylated DNA immunopreciptation and mRNA levels by qRT-PCR). Results: Exposure to inescapable footshocks increased the number of escape failures in the test. Treatment with DNMTi (acute), as well as with antidepressants (repeated treatment), attenuated stress-induced behavioral responses, an antidepressant-like effect in this model. Moroever, stress increased DNA methylation and decreased RNAm levels of BDNF and TrkB, while treatment with RG108 attenuated molecular changes induced by stress in rat PFC. Conclusion: The present results indicate that DNMTi, different from conventional antidepressants, are able to induce rapid and sustained antidepressant-like effects. In addition, BDNF and TrkB appear to be important for behavioral response induced by inhibition of DNMTs in the rat PFC submitted to the LH.

Introdução: Mecanismos epigenéticos, como a metilação de DNA, desempenham um papel importante na neurobiologia da depressão. Enquanto o estresse aumenta a metilação de DNA e reduz a expressão de genes envolvidos na plasticidade neuronial, inibidores de DNA metiltransferases (DNMTi), enzimas que catalisam a metilação de DNA, aumentam rapidamente a expressão gênica e induzem efeitos tipo-antidepressivos em modelos animais. Considerando, ainda, que antidepressivos convencionais podem interferir com mecanismos epigenéticos, este trabalho testou a hipótese de que drogas DNMTi induzem efeito tipo-antidepressivo agudo e sustentado em modelos animais. Além disso, avaliamos se o efeito de antidepressivos convencionais e de DNMTis sobre os níveis de mRNA e de metilação de DNA em diferentes genes associados a depressão e regulados por mecanismos epigenéticos (BDNF, TrkB, 5-HT1A, NMDA e AMPA) em estruturas encefálicas (hipocampo dorsal, ventral e córtex pré-frontal) de animais submetidos a modelo animal de depressão. Métodos: Para tanto, ratos Wistar foram submetidos ao modelo do desamparo aprenddo [learned helplessness, LH, pré-teste (PT), 40 choques inescapáveis nas patas]. Os animais receberam injeções sistêmicas de DNMTi (5-AzaD ou RG108), antidepressivos (imipramina ou fluoxetina), ou veículo, por 1 ou 7 dias, e foram submetidos a sessão teste do desamparo aprendido (T, 30 choques escapáveis) no último dia. Adicionalmente, um grupo independente foi submetido ao mesmo protocolo experimental e sacrificados 1 h após a última injeção. As estruturas encefálicas foram dissecadas para posterior análise molecular [imunoprecipitação de DNA metilado (meDIP) e quantificação de RNAm por qRT-PCR). Resultados: O estresse dos choques nas patas aumentou o número de falhas no teste. O tratamento com DNMTi agudamente, assim como com antidepressivos (tratamento repetido), foi capaz de atenuar essas alterações comportamentais, efeito considerado tipo-antidepressivo nesse modelo. Ainda, o estresse aumentou a metilação de DNA e reduziu os níveis de RNAm para BDNF e TrkB, enquanto que o tratamento com RG108 atenuou essas alterações moleculares no córtex pré-frontal de ratos. Conclusão: Os presentes resultados indicam que DNMTi, diferente de antidepressivos convencionais, são capazes de induzir rápido e sustentado efeito tipo-antidepressivo. Além disso, BDNF e TrkB parecem ser importantes para a resposta comportamental induzida pela inibição de DNMTs no córtex pré-frontal de ratos submetidos ao LH. / Introduction: Epigenetic mechanisms, such as DNA methylation, are thought to play an important role in the neurobiology of depression. While stress increases DNA methylation and decreases the expression of genes involved in neuronal plasticity, DNA methyltransferases inhibitors (DNMTi) increases gene expression and induces antidepressant-like effects in animal models. Considering that conventional antidepressants could interfere with epigenetic mechanisms, this work tested the hypothesis that acute treatment with DNMTi would induce acute and long-lasting antidepressant-like effects. Furthermore, we evaluated whether the stress could induce changes in the mRNA and DNA methylation levels in different genes involved with depression and regulated by epigenetic mechanisms (BDNF, TrkB, 5-HT1A, NMDA and AMPA) in different brain structures [dorsal hippocampus, ventral hippocampus and prefrontal cortex (PFC)] and whether such changes would be attenuated by systemic treatment with DNMTi (acutely) and antidepressants (chronically). Methods: Male Wistar rats were submitted to the learned helplessness model (LH; pretest session, 40 inescapable foot shocks). The animals received systemic injection the DNMTi (5-AzaD or RG108), antidepressants (imipramine or fluoxetine) or vehicle for one or seven days and were submitted to the LH test (30 escapable foot shocks) in the last day. Additionally, one independent group were submitted to the same experimental protocol and sacrificed one hour after last injection for collection of brain samples to further molecular analyses (methylated DNA immunopreciptation and mRNA levels by qRT-PCR). Results: Exposure to inescapable footshocks increased the number of escape failures in the test. Treatment with DNMTi (acute), as well as with antidepressants (repeated treatment), attenuated stress-induced behavioral responses, an antidepressant-like effect in this model. Moroever, stress increased DNA methylation and decreased RNAm levels of BDNF and TrkB, while treatment with RG108 attenuated molecular changes induced by stress in rat PFC. Conclusion: The present results indicate that DNMTi, different from conventional antidepressants, are able to induce rapid and sustained antidepressant-like effects. In addition, BDNF and TrkB appear to be important for behavioral response induced by inhibition of DNMTs in the rat PFC submitted to the LH.