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.

Un poids corporel élevé est la résultante de l'interaction entre des variants génétiques à risque et un environnement obésogène, et des processus épigénétiques sont probablement impliqués. Dans cette thèse, nous avons exploré des associations entre le méthylome sanguin humain et des polymorphismes nucléotidiques (SNPs) liés à l'obésité, ainsi que des mesures qualitatives et quantitatives de l'apport journalier en acides gras. Nous avons trouvé des associations entre des SNPs liés à l'obésité et la méthylation de promoteurs et enhancers proximaux. Nous avons également constaté que la qualité de l'apport journalier en acides gras est associée à une méthylation différentielle de promoteurs de gènes impliqués dans des voies métaboliques. Puis, nous avons examiné l'impact d'une nuit blanche sur la méthylation et la transcription de gènes du rythme circadien dans le muscle squelettique et le tissu adipeux. Nous avons constaté qu'une seule nuit blanche peut modifier le profil épigénétique et transcriptionnel de gènes clés du rythme circadien, et ce de manière tissu-spécifique. Enfin, nous avons examiné les effets de l'obésité maternelle chronique et de la perte de poids sur la transcription de gènes de la machinerie épigénétique dans le foetus et le placenta de souris. Nous avons constaté que la transcription de ces gènes est sensible aux trajectoires pondérales maternelles, et en particulier ceux de la voie d'acétylation des histones. Dans l'ensemble, cette thèse a démontré que la génétique, des stimuli environnementaux obésogènes et l'environnement maternel agissent sur les marques épigénétiques à des endroits génomiques pertinents dans la pathogenèse de l'obésité. / An elevated body weight is the result of the interplay between susceptibility gene variants and an obesogenic environment, and recent evidence shows that epigenetic processes are likely involved. In this doctoral thesis, we explored associations between the human blood methylome and obesity-associated gene variants as well as dietary fat quality and quantity. We found associations between obesity-associated SNPs and methylation levels at proximal promoters and enhancers, and some of these associations were replicated in multiple tissues. We also found that contrary to dietary fat quantity, dietary fat quality associates with methylation levels in the promoter of genes involved in metabolic pathways. Then, using a gene-targeted approach, we looked at the impact of an acute environmental stress (sleep loss) on the methylation and transcription levels of circadian clock genes in skeletal muscle and adipose tissue of healthy men. We found that a single night of wakefulness can alter the epigenetic and transcriptional profile of core circadian clock genes in a tissue-specific manner. Finally, we looked at the effects of chronic maternal obesity and subsequent weight loss on the transcription of epigenetic machinery genes in the fetus and placenta of mice. We found that the transcription of epigenetic machinery genes is highly sensitive to maternal weight trajectories, and particularly those of the histone acetylation pathway. Overall, this thesis demonstrated that genetics, obesogenic environment stimuli and maternal programming impact epigenetic marks at genomic locations relevant in the pathogenesis of obesity.

En 3ème position de fréquence parmi les myopathies, la dystrophie facio-scapulo humérale (FSHD) reste encore très énigmatique malgré l’association génétique avec la contraction d’une région du locus 4q35 contenant le macrosatellite D4Z4. Au cours de ce projet, l’étude de la régulation de la méthylation de l’ADN par séquençage après traitement au bisulfite de sodium ou par MeDIP chez les patients FSHD a montré des profils de méthylation similaires entre porteurs asymptomatiques et contrôles d’une part et patients FSHD1 et FSHD2 d’autre part. Ces deux groupes de malades présentent une hypométhylation principalement au niveau de la région proximale de D4Z4. Des mutations d’un nouveau gène candidat, SMCHD1, ont été décrites dans la plupart des cas de FSHD2, pour lesquels nous retrouvons fréquemment mais non systématiquement une baisse de la méthylation. D4Z4 joue également un rôle clé dans la régulation des extrémités chromosomiques et leur dynamique d’organisation tri-dimensionnelle dans l’espace nucléaire. Afin d’étudier la dynamique du locus 4q35 au cours de la différenciation cellulaire, nous avons mesuré les interactions spécifiques de différentes régions géniques au sein du locus et suivi l’évolution de la conformation de la chromatine dans différents contextes biologiques grâce à une méthode de FISH en trois dimensions couplée à une analyse in sillico des images (3D-FISH). Le retour à un état de pluripotence par la production d'hIPSCs montre un comportement nucléaire de ces régions proche de celui observé chez les contrôles. Enfin nous avons suivi la conformation du locus jusqu’à la formation de fibres musculaires matures multinuclées. / In the third position in term of frequency among the myopathies, facio-scapulo humeral dystrophy (FSHD) remains enigmatic despite the genetic correlation with the contraction of the 4q35 locus containing the macro satellite, D4Z4. In this project, the involvement of DNA methylation has been investigated in FSHD patients using bisulfite sequencing or MeDIP. These analyses revealed similar DNA methylation patterns between asymptomatic carriers and controls and between FSHD1 and FSHD2 patients. These two groups of patients show a marked hypomethylation mostly in the proximal region of D4Z4. Moreover, the recent discovery of mutations of a new candidate gene, SMCHD1 have been observed in most FSHD2 cases. In these patients, we observed frequent but not systematical association between decreased methylation and SMCHD1 mutation. This gene might be associated with D4Z4 DNA methylation maintenance; however its function remains unknown. D4Z4 plays a crucial role in chromosomal ends regulation and three-dimensional dynamics of the locus within the nuclear space. In order to study the dynamics and topology of the 4q35 locus during the skeletal muscle differentiation, we measured specific interactions between different regions within the locus and followed this chromatin conformation in different biological situations thanks to FISH in three-dimensions followed by in sillico analysis of the images (3D-FISH). Upon pluripotency in induced pluripotent stem cells derived from human fibroblasts (hIPSCs) display the same nuclear organization as in controls. Finally, we have followed the configuration of the locus during skeletal muscle commitment.

Brain tumours account for the most frequent type of solid tumours among children. Despite advances in surgery and chemotherapy, brain tumours are still the main cause of cancer deaths in children. Furthermore, little is known about DNA methylation changes in paediatric astrocytoma. Recent investigations suggest that many tumours are initiated not only by genetic abnormalities, but also caused by epigenetic changes. DNA methylation is a key epigenetic mechanism that controls the regulation of gene expression. Interestingly, unlike DNA mutations, epigenetic abnormalities are reversible. The reversibility of epigenetic abnormalities upon pharmacological unmasking has prompted interest in developing epigenetic therapy with the crucial goal of restoring the expression of aberrantly silenced genes. The focus of this study was to utilise a combination of different microarray strategies to develop an integrative candidate gene approach to identify several novel frequently methylated genes in a cohort of paediatric HGA (High grade glioma) samples. In addition, to investigate the potential of therapeutic efficacy of a DNA methyltransferase inhibitor, 5-Aza-dC in paediatric HGA. There were 147 genes commonly identified to be potentially methylated in IN699 cells using the two different array strategies integration; re-expression array and Illumina Infinium Human Methylation 450k array. Furthermore, using two complementary microarray strategies including methylation 450k array and expression array, this work identified 55 genes that were both methylated and under-expressed in these HGA cultures. Following validation with CoBRA and RT-PCR coupled with the response of hypermethylated promoters to the demethylating agent 5-Aza-dC, six novel genes (CXCL14, PRR5L, ELTD1, ITGA2, KRT8 and NTM) that are frequently silenced in paediatric astrocytoma were identified. This study suggests that re-expression of ii CXCL14 inhibited the colony formation and cell growth and reduces the migration rate significantly in IN699 short term culture and likely have functional significance in the development of paediatric HGA and an excellent candidate gene for further analysis. In parallel, the efficacy of 5-Aza-dC treatment was examined in paediatric HGA aiming to introduce this epigenetic therapy as a potential mechanism in management of this tumours. This study demonstrated that, relatively low dose of 5-Aza-dC sharply reduced the colony formation and inhibited proliferation and not through the apoptotic effect. It is likely that this reduction in proliferation without cell death is due to using relatively low doses that do not acutely kill cells, thus, allow the sustained alterations in both gene expression patterns and appearance of a new phenotype to emerge. Taken together, this work contributes to a more detailed understanding of the effect of epigenetic silencing on paediatric HGA. This investigation also demonstrated the use of epigenetic drug, 5-aza-dC to reverse the gene silencing for the potential treatment of paediatric HGA.

No description available.

De nombreux gènes suppresseurs de tumeurs sont inactivés par hyperméthylation dans les cancers. Cette inactivation serait en partie initiée par la protéine, MBD2 (Methyl-CpG-Binding Domain protein 2). Cette protéine recrute au niveau de séquences méthylées des complexes enzymatiques capables de modifier la structure chromatinienne et crée ainsi des régions fonctionnellement inactives. Dès lors, ce répresseur apparaît être une cible potentielle pour combattre le cancer. Dans cette perspective, rechercher les cibles de MBD2 et comprendre sa capacité à contrôler l’expression génique semblent cruciales. Au cours de deux études gènes candidats, nous avons pu démontrer (i) une réelle spécificité de cible du répresseur méthylationdépendant MBD2 pour les loci hTERT et pS2/TFF1 ; et (ii) un nouveau rôle de la protéine MBD2 en tant que modulateur de l’expression génique. De plus, les actions antagonistes entre le répresseur MBD2 et le trans-activateur naturel du gène pS2, le récepteur aux oestrogènes α, ont été explorées. Puis, l’analyse globale des profils de distribution de MBD2, de la méthylation de l’ADN, ainsi que de l’ARN polymérase II, sur puce promoteur a montré que MBD2 possède toutes les caractéristiques d’un répresseur trancriptionnel méthylation-dépendant. En effet, 74% des promoteurs fixés par MBD2 sont méthylés et cette liaison est associée dans 65% des cas à une répression transcriptionnelle. / In the past few years, several clinical trials have shown that targeting DNA methylation machinery might be of interest in cancer therapy to restore tumor suppressor genes expression and inhibit tumor growth. The Methyl-CpG-Binding Domain protein 2 (MBD2) is an important constituent of the DNA methylation machinery since this protein is directly involved in the mediation of the epigenetic signal. Moreover, MBD2 seems to show some gene specificity, its inhibition reactivate a limited number of genes. Taken together these data suggest that MBD2 represents potential new target in cancer therapy and, therefore, new insights on MBD2 specificities are, in this context, of importance. To this end, we have developed two different approaches: a candidate genes analysis and a genome-wide analysis, using ChIP-on-chip method, in order to map MBD2 binding sites. The candidate gene approaches are strongly in favour of the “one gene – one MBD” hypothesis, at least for the genes analyzed. Indeed, our results indicate that MBD2 is specifically and directly involved in the transcriptional repression of hTERT and pS2/TFF1 genes. Furthermore, a new role of MBD2 in the fine-scale modulation of these genes was demonstrated, and the antagonist actions between MBD2 and the natural trans-activator of pS2 gene, the estrogen α, were explored. Genome wide distribution of MBD2 binding sites, DNA Methylation profiles, and silencing potential, showed that the MBD2 is a real methylation-dependant transcriptional repressor: 74% of the MBD2 binding promoters are methylated and 65% silenced.

DNA methylation and demethylation are involved in regulation of gene expression. CpG clusters have been identified as hotspots of oxidative damages and mutagenesis. DNA base excision repair can remove oxidative DNA damage on CpG clusters and mediate an active DNA demethylation pathway. In this study, we examined the molecular mechanisms underlying interactions among DNA methylation, demethylation and BER. Our results demonstrated that a single 5-methylcytosine did not exhibit a significant effect on BER. Surprisingly we found that the abasic site completely inhibited the activity of thymine DNA glycosylase (TDG) leading to the sustainment of the mismatch efficiently extended by pol β. Interestingly, APE1 3’-5’ exonuclease could removed the mismatch. Our results demonstrate a molecular mechanisms underlying DNA base lesion and BER in maintenance of a normal DNA methylation pattern and a critical role of APE1 to combat pol β extension of the mismatch thereby reducing the introduction of mutagenesis.

O aborto espontâneo recorrente (AER) caracteriza-se pela ocorrência de três ou mais abortos consecutivos espontâneos até a 20ª semana de gestação. É uma condição patológica multifatorial, em que alterações morfológicas uterinas, distúrbios endócrinos, alterações no cariótipo, polimorfismos genéticos relacionados aos genes envolvidos no metabolismo da homocisteína, hemostasia, infecções, autoanticorpos e o processo inflamatório podem contribuir para a ocorrência de AER. O estado fisiológico do endométrio é essencial para a implantação do embrião no útero durante a gestação. Na interface materno-fetal, há uma modulação de citocinas, necessária para o estabelecimento da angiogênese e desenvolvimento da placenta. Um desequilíbrio entre as citocinas pode diminuir a tolerância ao feto e ocasionar rejeição fetal. A concentração de citocinas pode ser modificada por conta de uma diminuição na expressão de alguns genes, e esta pode ser regulada pelo seu estado de metilação sítio-específica. A metilação do DNA é um mecanismo epigenético de regulação gênica, e que corresponde à incorporação de grupos metila em ilhas CpG localizadas próximas às regiões promotoras de genes humanos, e isso pode ser importante na avaliação do risco de complicações gestacionais. Além disso, o estado nutricional de vitaminas foi relacionado a alterações no padrão de metilação de alguns genes. Os objetivos deste trabalho foram avaliar as concentrações dos mediadores inflamatórios em mulheres com aborto e em grupo controle, verificar correlações entre as concentrações de vitaminas, homocisteína total e taxa de metilação do DNA, verificar correlações entre concentração de citocinas e taxa de metilação do DNA e determinar odds ratio (IC 95%) de ter aborto em modelos multivariados. Foram incluídas 253 mulheres com história de aborto recorrente e 264 mulheres saudáveis (controle). O DNA foi extraído de leucócitos de sangue periférico para o estudo de metilação. Foram separadas alíquotas de soro e plasma para dosagem de vitaminas, metabólitos e citocinas. Não foram encontradas diferenças nas taxas de metilação do DNA entre os grupos aborto e controle. A citocina TNFα está aumentada nos grupos de aborto em comparação ao controle. A taxa de metilação do DNA no gene IFNG foi correlacionada inversamente às concentrações de folato sérico e citocina IFNγ no grupo controle. E as concentrações de IL10 foram inversamente correlacionadas à taxa de metilação do DNA nos grupos de aborto secundário e controle. Neste trabalho, verificou-se que as vitaminas e as citocinas influenciam na taxa de metilação do DNA do gene IFNG e a citocina pró-inflamatória TNFα apresenta-se aumentada em mulheres com história de aborto. / Recurrent spontaneous abortion (RSA) is characterized by the occurrence of three or more consecutive spontaneous abortions until the 20th week of gestation. It is a multifactorial pathological condition in which morphological uterine, endocrine disorders, changes in the karyotype genetic polymorphisms related to genes involved in homocysteine metabolism, infection, autoimmunity and inflammatory processes may contribute to the occurrence of RSA. The physiological state of the endometrium is essential for embryo implantation in the uterus during pregnancy. In maternal-fetal interface, there is a modulation of cytokines necessary for the establishment and development of placental angiogenesis. An imbalance between cytokines can decrease tolerance to fetus and cause fetal rejection. Concentration of cytokines may be modified due to a decrease in the expression of genes related to some of these cytokines that can be regulated by DNA methylation, which is an epigenetic mechanism of gene regulation and which corresponds to the incorporation of groups Methyl CpG islands located near the promoter regions of human genes, and this may be important in assessing the risk of pregnancy complications. In addition, the nutritional status of vitamins was associated with changes in the methylation pattern of certain genes. The aims of this study were to determine the concentrations of inflammatory mediators in women with abortion and the control group, examine correlations between concentrations of vitamins, total homocysteine and DNA methylation rate, examine correlations between cytokine concentration and DNA methylation and determine odds ratio (95% CI) of having abortion in multivariate models. We included 253 women with a history of recurrent miscarriage and 264 healthy women (control). DNA was extracted from peripheral blood leukocytes for the study of methylation. Serum and plasma aliquots were used for determination of vitamins, metabolites and cytokines. There were no differences in rates of DNA methylation between control and abortion groups. The cytokine TNFα is increased in abortion groups compared to the control. DNA methylation rate in gene IFNG was inversely correlated with serum folate and serum cytokine IFNγ in the control group. Also IL10 concentrations were inversely correlated to DNA methylation rate in groups of miscarriage and secondary control. In this work, it was found that vitamins and cytokines influence DNA methylation rate in the promoter region and are different in the study and control groups.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13326号 / 論医博第2194号 / 新制||医||1043(附属図書館) / (主査)教授 小川 誠司, 教授 滝田 順子, 教授 河本 宏 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Brassica napus (AACC, 2n = 38) is an allotetraploid species derived from the parentel diploid species Brassica rapa (AA, 2n = 20) and Brassica oleracea (CC, 2n = 18). The aim of thesis was to carry out the genetic and epigenetic analysis of high-copy rRNA genes (or rDNA) in several varieties of hybrid species B. napus. The experiments involved determining the ratio of parental genes in hybrids, sequencing and methylation analysis of the promoter region of rDNA. Using Southern hybridization, it was revealed significant variability in the number of parental rDNA units between each variety. Data from sequence analysis were in good agreement with the results of Southern blot. Genetic recombination between parental rDNA units was revealed in one variety by DNA sequencing of promotor region. To study methylation, bisulfite sequencing was performed. It was found out that rDNA units of B. rapa origin have a higher value of methylation than units originated from B. oleracea.