Eukaryotes have developed stringent regulatory mechanisms that control cell division and ensure proper chromosome segregation. Maintaining genome integrity is especially important during meiosis, the specialised cell division programme in the germline that generates haploid gametes. As these cells transmit genetic information to the next generation, the consequences of meiotic errors are not restricted to an organismal level, but can directly impact the fitness of the offspring. Mammals display a high degree of sexual dimorphism in meiosis with regard to the stringency of regulatory mechanisms. This manifests in a relatively high degree of maternally-derived aneuploidies due to weaker checkpoint control in females, whereas more rigorous checkpoints in males frequently perturb fertility. Mouse models of aneuploidy often exhibit complete male sterility and early germ cell arrest, preventing the study of aneuploidy during late and post-meiotic stages in males. In this thesis, we have used the trans-chromosomic mouse model, Tc1, which carries a single copy of human chromosome 21 (HsChr21) and show that, unlike other aneuploid mouse strains, the Tc1 mouse can successfully passage the exogenous human chromosome through male meiosis and generate aneuploid offspring. Our investigations have shown that the presence of the aneuploid human chromosome causes spermatogenic defects due to an arrest at the first meiotic division. Despite this impairment, we found an unexpectedly high number of aneuploid gametes in Tc1 males and the majority of males were able to produce aneuploid offspring, albeit at a lower frequency. Transmission of HsChr21 through the male germline was less efficient compared to female germline transmission, but allowed us to study the impact of male germline-associated chromatin remodelling on the transcriptional deployment of HsChr21 in the offspring. This revealed that, despite fundamentally different developmental dynamics, male- versus female-germline passage result in indistinguishable transcriptional and regulatory phenotypes. An important pathway in the male germline involves the expression of piRNAs, a class of small non-coding RNAs that are commonly found in the germline of animals where they defend cells against transposable elements. Profiling the expression of small RNAs in the Tc1 mouse showed that conserved human piRNA clusters can be successfully transcribed by the mouse piRNA machinery. In addition, we detected Tc1-specific piRNA sequences that were neither present in human nor mouse, mapping to a human-specific repeat element. In line with the previously observed activation of human-specific repeat elements in the Tc1 mouse, this suggests that novel transcripts arising from human repeats can trigger an adaptive piRNA response, thereby demonstrating the plasticity of this pathway to newly invading repeat elements. Transcriptional profiling of spermatogenic cell populations on a single-cell level allowed us to generate an atlas of gene expression over the course of spermatogenesis and dissect meiotic silencing dynamics in the presence of aneuploidy. Transcriptional silencing during meiosis occurs in response to unpaired chromosomes and, in male germ cells, affects the sex chromosomes due to their largely unpaired nature. We found that the presence of HsChr21 has no impact on the silencing of chromosome X, however, the two chromosomes display drastically different silencing patterns with HsChr21 showing a much weaker repression. Taken together, this study revealed a higher than expected tolerance for aneuploidy in the mouse male germline thus allowing the characterisation of meiotic checkpoint mechanisms, the meiotic silencing response to unpaired chromosomes as well as piRNA expression in the presence of an exogenous human chromosome.

Epigenetic alterations including DNA methylation and microRNAs may provide important insights into gene-environment interaction in complex immune diseases such as inflammatory bowel disease (IBD). An integrative genome-wide approach was used to analyse whole blood genetic, DNA methylation and gene expression data in 240 newly diagnosed IBD patients and 190 controls. Using the Illumina 450k array, differences in whole blood DNA methylation were observed in IBD cases versus controls including 439 differentially methylated positions (DMPs) and 5 differentially methylated regions (DMRs). The top DMP (RPS6KA2, discovery Holm adjusted p=1.22×10-16, replication p=1×10-9) and DMRs (VMP1, ITGB2, TXK) were replicated in an independent cohort using pyrosequencing. Paired genetic and epigenetic data allowed the identification of methylation quantitative trait loci (meQTL); two of the five DMRs (VMP1, ITGB2) demonstrated significant association with genetic polymorphisms. Methylation in the VMP1/microRNA-21 region was significantly associated with two single nucleotide polymorphisms (cg18942579 -rs10853015 [meQTL FDR adjusted p=9.4 × 10-5], cg16936953 - rs8078424 [meQTL FDR adjusted p=8.8 × 10-5]), both of which are in linkage disequilibrium with a known IBD susceptibility variant (rs1292053). Separated leukocyte methylation data highlight the cell type of origin of epigenetic signals seen in whole blood. IBD-associated hypermethylation within the TXK gene transcription start-site negatively correlated with gene expression in whole blood and CD8+ T-cells, but not other cell types, highlighting that cell-specificity and gene location-specificity of DNA methylation change is critical when associating methylation and gene expression. These data offer significant translational potential as diagnostic biomarkers. Least absolute shrinkage and selection operator (lasso) modelling identified 30 methylation probes can be used to accurately discriminate IBD cases from controls (Area under receiver operating characteristic curve = 0.898, sensitivity = 90.6%, specificity = 84.7%). MicroRNAs (miRNA) are small non-coding nucleic acids that have the capacity to modulate gene expression. MiRNAs have been increasingly implicated in many of the important IBD pathogenic pathways including autophagy, intestinal epithelial barrier integrity and the Th17 pathway. In common with all epigenetic mechanisms, miRNA expression is dynamic and cell-specific. Small RNA sequencing (RNA-seq) was performed on RNA extracted from CD14+, CD4+ and CD8+ cells isolated from 8 newly diagnosed cases of ileal or ileocolonic CD and 8 age and sex matched controls. There was a median of 2.4 million reads per sample (range 132,800-12.8 million reads per sample). One microRNA was differentially expressed in CD compared with controls (hsa-miR-503-5p log fold change = 0.7, FDR adjusted p = 9.1 × 10-5) in CD4+ lymphocytes, however this finding did not remain significant when alternative normalisation methods were used. The small number of cases used in microRNA analyses raises the possibility of both type I and II error, and limits the ability to draw firm conclusion from this series of experiments. Site-specific differences in DNA methylation in IBD relate to underlying genotype and associate with cell-specific alteration in gene expression. This is the most detailed characterisation of the epigenome carried out in IBD to date. The findings strongly validate this approach in complex disease, are replicable, and provide clear translational opportunities.

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

Les modifications post-traductionnelles (MPTs) d’histones sont apparues comme un acteur majeur de la régulation de l’expression des gènes. Cependant peu de choses sont connues sur le réel impact des MPTs sur la chromatine. Il a été suggéré que les MPTs d’histones (H2A, H2B, H3 et H4) ont le potentiel de moduler la fonction chromatinienne selon un « codehistone » en recrutant des protéines spécifiques de liaison. L’objectif de mon projet est d’approfondir la fonction de l’acétylation du domaine globulaire de l’histone H3 et de comparer cette modification avec celles des queues N-terminale in vivo sur une lignée ES cellulaire. Pour étudier l’impact de ces MPTs in vivo, toutes les copies endogènes du gène H3 sauvage (WT) doivent être remplacées par des copies mutées. Ainsi la première étape de mon projet est d’établir une lignée cellulaire exprimant seulement H3 mutée (e.g reproduisant une acétylation permanente) afin d’étudier les effets des modifications sur le domaine globulaire de H3 sur (a) l’expression génique, (b) l’architecture chromatinienne mais également pour étudier (c) les effets réciproques et synergiques entre les différentes modifications du domaine globulaire et (d) comparer ces effets avec les modifications sur la queue N-terminale dans un système in vivo. / Post-translational modifications (PTMs) of histones have emerged as key players in the regulation of gene expression. However, little is known to what extent PTMs can directly impact chromatin. It has been suggested that PTMs of core histones (H2A, H2B, H3 and H4) have the potential to govern chromatin function according to the so called ‘‘histone code’’ hypothesis by recruiting specific binding proteins. The goal of my project is to gain insight in the function acetylation within the globular domain of H3 and to compare these modifications with histone tail modifications, in vivo by using the CRISPR in mouse embryonic stem cells (ES). To study the impact of PTMs in vivo, all endogenous wild type (WT) H3 gene copies have to be replaced with mutant copies. Hence, the primary focus of my project is to establish cell lines that exclusively express mutated H3 (e.g. mimicking acetylation) in order to study effects of H3 globular domain modifications on (a) gene expression (b) chromatin architecture as well as to study (c) cross talks and synergisms between globular domain modifications and (d) compare the effects with tail modifications in an vivo system.

O glioblastoma (GBM) é considerado um dos tumores mais agressivos do sistema nervoso central (SNC). Mesmo com o uso de protocolos modernos de tratamento o prognóstico se mantém bastante reservado, sendo que crianças com GBM apresentam uma sobrevida média de 12 a 15 meses. Mecanismos epigenéticos podem interferir no processo de carcinogênese, sendo que a acetilação do DNA pode modular a expressão de genes que atuam no controle do ciclo celular, contribuindo assim para o desenvolvimento e progressão de neoplasias. Estudos clínicos demonstram que inibidores de histonas deacetilases (HDACs), em monoterapia ou combinados a outros agentes antineoplásicos, são clinicamente ativos e bem tolerados no tratamento de uma ampla variedade de tumores. Estes inibidores podem sensibilizar a resposta celular à irradiação ionizante, possibilitando uma redução nas doses-padrão utilizadas, minimizando os efeitos colaterais a curto e longo prazo. A radiação ionizante induz dano no DNA e é geralmente aceito que quebras da dupla-fita (DSBs) é o tipo de lesão mais severa relacionada à sobrevivência celular e preservação da integridade genômica. No presente estudo, avaliamos o potencial efeito radiosensibilizante do PCI-24781, um novo e potente pan-inibidor de HDAC nas linhagens celulares de GBM pediátrico SF188 e KNS42. Foram comparadas as taxas de proliferação celular, clonogenicidade e apoptose das linhagens SF188 e KNS42 com ou sem tratamento com PCI-24781. Também foram comparadas as taxas de clonogenicidade das linhagens SF188 e KNS42 que foram irradiadas com ou sem tratamento prévio com PCI-24781. Adicionalmente, foram avaliados os efeitos do PCI-24781 na expressão de algumas das principais proteínas responsáveis pelo reparo de quebras da dupla-fita ocasionadas pela irradiação. Para os ensaios de proliferação celular foram utilizados os tempo de 24, 48, 72 e 96h, para apoptose, 48h e para capacidade clonogênica sem irradiação o tempo de 48h, em diferentes doses de PCI-24781 (0,25 - 16 M). O inibidor bloqueou significativamente a proliferação celular (p<0,05), induziu morte por apoptose (p<0,05) e reduziu a capacidade na formação de colônias (p<0,001) em ambas as linhagens. No ensaio para avaliação da radiosensibilidade, foram utilizadas as doses do IC30 11 de cada linhagem do ensaio clonogênico seguida de diferentes doses de irradiação. Ambas as linhagens apresentaram uma significativa (p<0,001) diminuição na formação de colônias em todas as doses de irradiação. A linhagem mais resistente à droga, SF188 foi escolhida para estudo do reparo de quebras da dupla-fita ocasionadas pela irradiação. As expressões da proteína Rad51, importante na via de reparo por recombinação homóloga (HR), e das proteínas DNA-PKcs, Ku70 e Ku86, importantes na via de reparo por união terminal não-homóloga (NHEJ) apresentaram uma maior diminuição quando a linhagem irradiada foi previamente tratada com PCI-24781 em comparação à radioterapia exclusiva. Estes achados demonstram que o inibidor de histona PCI-24781 apresenta um importante papel como agente radiosensibilizante, comprometendo o reparo das quebras de dupla-fita em células de GBM pediátrico tratadas com radioterapia. / Glioblastoma (GBM) is considered one of the most aggressive tumors to affect the central nervous system (CNS). Even employing modern treatment protocols the prognosis remains very poor, with children affected by GBM presenting a median survival rate of 12 to 15 months. Epigenetic mechanisms may interfere with the process of tumorigenesis, and DNA acetylation can modulate the expression of genes that contribute in cell cycle control and participate to the development and progression of cancer. Clinical studies demonstrate that histone deacetylase inhibitors (HDACs), alone or in combination with other antineoplastic agents, are clinically active and well tolerated in the treatment of a wide variety of tumors. These inhibitors may sensitize the cellular response to ionizing radiation, enabling the reduction in standard doses of radiation, ultimately minimizing both short and long-term side effects. Ionizing radiation induces DNA damage and it is generally accepted that the double-stranded breaks (DSBs) is the most severe type of injury related to cell survival and preservation of genomic integrity. In the present study, we evaluated the potential radiosensitizer effect of PCI-24781, a novel potent pan-HDAC inhibitor in the pediatric GBM cell lines SF188 and KNS42. We compared the cell proliferation rates, apoptosis of clonogenicity of KNS42 and SF188, with or without treatment with PCI-24781. Moreover, clonogenicity rates were compared between cell lines that were irradiated with or without prior treatment with PCI-24781 Additionally, we evaluated the effects of PCI-24781 in the expression of some of the major proteins responsible for the repair of double-stranded breaks caused by the irradiation. For the cell proliferation assays, the times of 24, 48, 72 and 96 hours were used, for apoptosis, the time of 48h and clonogenic capacity without irradiation, the time of 48h, and different doses of PCI-24781 (0,25 - 16 M). The inhibitor significantly blocked cell proliferation (p<0,05), inducing cell death by apoptosis (p<0,05) and reducing the colony forming ability (p<0,001) of both lineages. In the assays to evaluate the radiosensitivity , the IC30 doses of the clonogenic assays were used for each cell-line after different doses of irradiation. Both lineages showed a significant decrease (p<0,001) in colony formation at all doses of irradiation. The most resistant cell-line to the drug, SF188, was 13 chosen to study the double-strand breaks repair caused by irradiation. The Rad51 protein levels, critical for homologous recombination (HR), and the DNA-PKcs proteins Ku70 and Ku86, important for DNA repair through non-homologous end joining (NHEJ) showed significant decrease in expression when cell-line was treated with PCI-24781 prior to radiotherapy. These data demonstrates that the histone deacetylase inhibitor PCI-24781 plays an important role as a radiosensitizer agent, compromising the repair of double-strand breaks in pediatric GBM cells following irradiation.

Na inativação do cromossomo X (ICX) um dos dois cromossomos X presentes nas fêmeas de mamíferos placentários é silenciado transcricionalmente. Esse é um mecanismo de compensação de dose que assegura que a quantidade dos produtos gênicos oriundos do cromossomo X esteja em equilíbrio entre machos e fêmeas. A ICX pode ocorrer de modo aleatório, onde cada célula escolhe ao acaso qual será o cromossomo X inativado: cromossomo X paterno ou cromossomo X materno; ou de forma \"imprintada\" (termo adaptado do inglês imprinted), ou seja, dependente da origem parental do cromossomo X. Enquanto nas fêmeas marsupiais a inativação ocorre de forma \"imprintada\", sendo o X paterno inativado em todos os tecidos, nos mamíferos eutérios a ICX nos tecidos somáticos ocorre de modo aleatório. Porém alguns eutérios mantiveram o mecanismo \"imprintado\" de ICX exclusivamente nos tecidos extraembrionários, como ratos e camundongos. Em humanos, o estado controverso da ICX em tecidos extraembrionários foi reavaliado por nosso grupo utilizando uma análise mais ampla e identificou-se um padrão aleatório (Moreira de Mello et al., 2010), demonstrando a importância de se realizar uma análise global para se determinar o perfil de atividade do cromossomo X. Em bovinos o padrão de ICX em placenta não está claro. Ele foi verificado analisando-se a expressão de um único gene, e os autores concluíram que o padrão era \"imprintado\" (Xue et al., 2002). Porém a análise de um único gene pode não representar o estado epigenético de um cromossomo inteiro. Assim o padrão de ICX em tecidos extraembrionários bovinos se mostra uma questão importantíssima para ser esclarecida. No presente trabalho o cromossomo X bovino foi analisado em busca de SNPs (polimorfismos de base única) localizados em regiões codificadoras em genes expressos no tecido extraembrionário, permitindo assim através da análise da expressão alelo-específica determinar o padrão de expressão do cromossomo X. Os resultados apresentados neste trabalho mostram um padrão de expressão bialélica, indicando que em populações diferentes de células, diferentes cromossomos X estavam ativos. Portanto a ICX em tecidos extraembrionários bovinos ocorre de modo aleatório, padrão semelhantes àquele encontrado em humanos, e diferente daquele encontrado em ratos e camundongos. Este trabalho mostra a importância de uma análise global da expressão gênica no cromossomo X, permitindo assim traçar um perfil de atividade mais próximo possível da realidade. / In X chromosome inactivation (XCI), one of the two X chromosomes present in female mammals is transcriptionally silenced, resulting in a dosage compensation mechanism. The XCI can occur randomly, so that each cell chooses randomly which one will be the inactivated X chromosome: paternal (pX) or maternal (mX); or dependent on parental origin of X chromosome, ie, imprinted. While in female marsupials the inactivation occurs in an imprinted fashion, with the Xp inactivated in all tissues, both somatic and extra-embryonic, in the mammalian eutherians XCI in the somatic tissues occurs randomly. However some eutherians still retain the imprinted XCI mechanism exclusively in extra-embryonic tissues, such as rats and mice. In humans, the controversy of the XCI in placenta was re-evaluated by our group. Using a broader analysis, a random pattern was identified, in contrast to the previously published works. It demonstrated the importance of conducting a comprehensive analysis to determine the profile of X chromosome (Moreira de Mello et al., 2010). In cattle the pattern of XCI in bovine placenta is unclear. It was verified by analyzing the expression of a single gene, and the authors concluded that the pattern was imprinted (Xue et al., 2002). Because the analysis of a single gene may not represent the epigenetic state of an entire chromosome, the pattern of XCI in cattle extra-embryonic tissues is an important issue to be clarified. In the present study the cattle X chromosome was analyzed searching for SNPs (single nucleotide polymorphisms) located in coding regions of genes expressed in extra-embryonic tissue. So that, by analyzing the allele-specific expression it is possible to determine the X chromosome expression patter. The preset results show a bi-allelic expression pattern. This indicates that in different cells populations, different X chromosomes are active. Thus, the XCI in extra-embryonic tissues of bovines occurs randomly, similar to the human pattern but different to that verified in rats and mice. This work shows the importance of a global analysis of the gene expression in X chromosome, through which it can trace the closest activity profile as possible to reality.

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.

Epigenetic modifications are alterations in the cell nucleus that affect gene expression and can occur in chromatin at the level of DNA methylation or histone modifications. Such ‘epigenetic marks’ can be heritable through cell division but leave the DNA sequence unchanged. Post-­translational modifications can be found on the histone proteins associated with DNA; the majority of histone modifications are found on the lysine-­rich N-‐terminal amino acid “tails”. Histone acetylation and methylation influence the chromatin structure by loosening or tightening the packaging of DNA, respectively, in association with other chromatin modifiers. Condensed chromatin is linked to transcriptional silencing and genetic imprinting and also occurs at chromosomal centromeres, where it is linked to kinetochore binding. Heart development is well studied, but the epigenetic processes involved are not yet completely understood. While active chromatin mechanisms such as histone acetylation and chromatin remodelling have been described in the heart, the role of gene repressive epigenetic mechanisms has been poorly investigated. Cardiomyocytes are post-­mitotic cells that do not divide to regenerate a damaged heart. The regeneration of cardiomyocytes after myocardial infarction is an important topic of interest in cardiovascular science. There are various approaches to heart repair after infarction, including activating cardiomyocytes so they become mitotic once again, or growing cardiomyocytes in vitro to attach to a lesion site. An important factor in these approaches is understanding the epigenetic mechanisms controlling cell division. In this thesis, we aim to advance the current knowledge of the epigenetic repressive mechanisms involved in cardiomyocyte formation and heart development to explain their lack of regenerative capacities. We studied the epigenetic changes that occur during cardiac development leading to a non-­‐regenerative state to pinpoint the moment at which these changes arise. We found that the epigenetic process is independent of whether cardiac lineage differentiation occurs during embryogenesis or during differentiation in vitro. We discovered that cardiac heterochromatin displays a singular epigenetic signature during development as compared to brain, another post-­mitotic tissue, or liver, an actively regenerative tissue. We observed an epigenetic change in the repressive histone modification histone H3 lysine 9 trimethylation that was specific to heart development. This change involved a nuclear reorganisation of heterochromatin and a reduction of the levels of this mark in E13.5 and E14.5 embryos, as compared to E10.5 embryos. This was consistent with our observations of the histone lysine methyltransferase SUV39H1, the levels of which were lower after stage E10.5 of development. However, contradictorily, in differentiated cardiomyocytes in vitro, SUV39H1 was increased but showed low levels of H3K9me3, compared to ES cells, which had low levels of SUV39H1 and high levels of H3K9me3. We detected extremely low levels of the H3K9me3 in adult heart tissue. We observed that in adult hearts, the myocardium had maintained these major changes in H3K9me3, while this effect was not observed in the epicardium. Genomic studies were carried out to determine changes at a genomic level between the two key epigenetic stages in heart development we identified at E10.5 and E13.5. Methylated DNA immunoprecipitation sequencing and chromatin immunoprecipitation sequencing for H3K9me3 analyses were carried out to find overall changes in methylation patterns. No global changes in DNA methylation were detected between these developmental stages. These results imply that the differences observed in H3K9me3 are due to remodelling of the heterochromatin during heart development and cardiomyocyte formation, rather than quantitative changes.

La méthylation des histones constitue un niveau important de contrôle épigénétique chez les eucaryotes. Mes études portent sur la caractérisation des facteurs potentiellement intervenant dans la mise en place et la lecture de la méthylation pour mieux apprécier son rôle et des mécanismes sous-jacents dans la régulation de la transcription et du développement des plantes chez l’Arabidopsis thaliana. Ainsi, la première partie de mes travaux de thèse a contribué à l’étude d’une protéine à domaine SET (SET DOMAIN GROUP7, SDG7) et à montrer que SDG7 est nécessaire au bon déroulement de l'induction de VIN3 et du processus de vernalisation pour la floraison. Nos résultats suggèrent que SDG7 pourrait méthyler une protéine non-histone encore inconnue dans la régulation de la transcription et le contrôle de la durée de vernalisation. La deuxième partie de ma thèse porte sur l’étude de SDG8 et les H2B-UBIQUITIN-ligases HUB1/HUB2 pour examiner un cross-talk éventuel entre la triméthylation de H3K36 (H3K36me3) et la monoubiquitination d’H2B (H2Bub1). Nous avons montré que H3K36me3 et H2Bub1 sont déposés largement indépendamment, qui diffère d’une dépendance hiérarchique de déposition préalablement observée chez la levure. La dernière partie de ma thèse a permis l’identification des protéines HUA2/HULK2 à domaine PWWP comme lecteurs éventuels de H3K36me3 dans la régulation de la floraison et du développement des plantes. / Histone methylation is one of the keys epigenetic marks evolutionarily conserved in eukaryotes. My study focuses on the characterization of factors potentially involved in the deposition and reading of lysine (K) methylation to appreciate its role and underlying mechanisms in the regulation of transcription and plant development, using Arabidopsis thaliana as a model organism. In the first part of my thesis, I report on our study of SET DOMAIN GROUP7 (SDG7), a protein containing the evolutionarily conserved SET domain, which is generally recognized as a signature of K-methyltransferases. We found that SDG7 plays an important role in the regulation of VIN3 induction associated with cold duration measure during vernalization treatment. Intriguingly, levels of several different histone methylations were found unchanged in the sdg7 mutant plants and the recombinant SDG7 protein failed to show a histone-methyltransferase activity in vitro. We thus conclude that SDG7 might methylate a yet unknown non-histone protein to regulate transcription and proper measurement of the duration of cold exposure in the vernalization process. In the second part, I studied interaction between SDG8 and HISTONE MONOUBIQUITINATION1 (HUB1) and HUB2. My results unravel that H3K36me3 and H2Bub1 are deposited largely independently in Arabidopsis, which is in contrast to the dependent crosstalk of these two different epigenetic marks previously reported in yeast. In the last part of my thesis, I report on the identification of the PWWP-domain proteins HUA2/HULK2 as readers of H3K36me3 and demonstrate that sdg8 and hua2 genetically interacts in the regulation of flowering time.

Mouse embryonic stem (ES) cells have played a crucial role in studying developmental processes and gene function in vivo. They are extremely useful in the generation of transgenic animals as they can be genetically manipulated and subsequently microinjected into blastocyst stage embryos, where they combine with the inner cell mass and contribute to the developing embryo. Some of the resulting pups are chimaeric, consisting of a mixture of cells derived from the host blastocyst and the injected ES cells. We have identified several ES cell clones arising from gene targeting experiments with an impaired capacity to generate viable chimaeras. When injected into blastocysts, these clones cause embryonic death during mid to late gestation, suggesting that the cells are able to contribute to the embryo but interfere with normal embryonic development. The aim of this work was to identify the underlying changes in the transcriptome, epigenome or cell surface markers that have occurred in these compromised ES cells and to further define the developmental phenotype of the chimaeric embryos. Different stages during development were analysed and whereas there was little difference in embryonic death at gestational day e13.5, there was a significant decrease in embryos surviving to gestational day e17.5. Additionally, severe haemorrhaging was observed in all the dead embryos and small foci of haemorrhaging could also be seen in a number of embryos that were still alive. This was also observed at e13.5, albeit to a less severe extent. Using RNA sequencing to discover differences in the transcriptome between control ES cells and the compromised ES cells, five genes were identified that were downregulated in the compromised cells. Four of these, Gtl2, Rtl1as, Rian and Mirg are all located in the imprinted Dlk1-Dio3 region on chromosome 12 and are normally expressed from the maternal genome. This pattern was also validated in tissues from e17.5 chimaeric embryos. The expression of this locus is to a large extent regulated by a differentially methylated region located approximately 13kb upstream of the Gtl2 promoter, the IG-DMR. Whereas this is usually only methylated on the paternal copy, in the compromised ES cells both the paternal and the maternal copy were fully methylated, likely causing the silencing of Gtl2, Rtl1as, Rian and Mirg. Using the DNA methyltransferase inhibitor 5-azacytidine, expression of Gtl2 could be rescued. Injection of those 5-azacytidine treated cells into blastocysts did partially rescue the embryonic lethal phenotype. Additionally, cell surface markers were analysed in a phenotypic screen using phage display. NGS analysis of the phage outputs indicates that there may be additional differences in cell surface markers between the control and compromised ES cell clones, but their specific details remain to be identified. Overall, we have identified the maternally expressed genes of the Dlk1-Dio3 region as markers that can distinguish between ES cells with normal or compromised developmental potency and propose to include these genes in the pre-blastocyst injection screening routine for experiments involving the production of chimaeras or genetically modified mouse strains.