Stress contributes to the development of major depressive disorder (MDD) and post-traumatic stress disorder (PTSD), and an intermediary factor between stress and psychiatric disorders may be epigenetics. Studies have shown altered DNA methylation (DNAm) in animal models of and humans with stress exposure and in individuals with PTSD and MDD. The availability of genome-wide experimental platforms has given us new tools to investigate DNAm, and in this dissertation these techniques have been used to further our current understanding of the epigenetics of stress. We performed a genome-wide investigation in mice exposed to chronic stress that exhibit depressive- and anxious-like behaviors, examining DNAm changes within the dentate gyrus, a sub-region of the hippocampus that contributes to the stress response. Using the Methyl-Seq method, an intergenic region of chromosome X was shown to be differentially methylated with chronic stress, and this finding replicated in two additional cohorts of mice. In postmortem brain tissue of humans with MDD, an increase in DNAm within this intergenic region was also found. Animal models do not fully capture the complexity of stress and psychiatric disorders in humans, but comparable studies in humans are limited by the difficulty of obtaining brain tissues. Instead, these studies have used peripheral tissues to examine DNAm changes related to stress and psychiatric disorders. To address the usefulness of these peripheral tissues, we employed the Illumina 450K and EPIC arrays to establish a resource that compares DNAm of the brain to that of blood, buccal, and saliva tissues. Glucocorticoids (GCs) play an essential role in the stress response, and their dysregulation is seen in individuals with MDD and PTSD. To determine the role of GCs in stress-mediated epigenetic changes, buccal samples were obtained before and after individuals were given GCs in the context of oral surgery, and DNAm was analyzed using the Illumina EPIC array. Five CpGs were altered following this exposure, to a genome-wide significant degree. Further analysis revealed FDR-significant CpG changes to be in genes involved in steroid hormone biosynthesis and in genes differentially expressed with GC exposure. Collectively, these results exemplify the complexity of DNAm changes associated with the stress response and provide potential avenues for elucidating their impact on psychiatric disorders.

La vigne est une plante cultivée dans le monde entier dont l’importance économique est principalement liée à la production de vin. La baie de raisin est également l’un des principaux modèles d’étude pour les fruits non-climatériques notamment pour l’étude des mécanismes contrôlant le mûrissement des baies. Le développement de la baie de raisin est caractérisé par deux phases de croissance séparées par une phase de latence se produisant au moment de la véraison. La baie de raisin est composée de trois tissus principaux: la peau, la pulpe et les graines. La peau et la pulpe présentent une structure et une composition en métabolites distinctes et contribuent de manière différente à la qualité du vin, la pulpe fournissant essentiellement le sucre, les acides aminés et organiques alors que la peau est riche en anthocyanes. A l'heure actuelle, les mécanismes moléculaires impliqués dans le contrôle de la maturation des baies de raisin sont encore mal compris. Si l'ABA, le sucre et différents facteurs de transcription jouent un rôle important dans le contrôle de cette phase de développement, les mécanismes épigénétiques, en particulier la méthylation de l’ADN, apparaissent aussi comme des régulateurs importants du développement et du mûrissement des fruits charnus. Dans ce contexte, Le projet de thèse présenté vise à analyser le rôle de la méthylation de l’ADN (1) dans la maturation des baies de raisin et (2) dans la synthèse des anthocyanes en utilisant comme système modèle des cellules de baie de raisin cultivées in vitro.La culture in vitro de baies de raisin en présence d’inhibiteurs de la méthylation de l'ADN, aboutit à une inhibition de la maturation, suggérant que la méthylation de l’ADN joue un rôle crucial pour cette étape du développement chez la vigne. La pellicule et la chair de baies de raisin récoltées à divers stades de développement ont ensuite été analysées séparément pour déterminer les variations des transcriptomes, de l’abondance de différents métabolites, et de la méthylation de l'ADN. Les principaux résultats indiquent des variations des métabolites et du transcriptome, avec des spécificités liés au tissu analysé. En outre, l'analyse des variations de méthylation de l'ADN à deux stades de développement dans chacun de ces deux tissus révèle l’existence de variations de méthylation spécifiques à chaque tissu, tandis que les variations communes aux deux tissus restent limitées. Ces résultats suggèrent un contrôle de la méthylation de l’ADN spécifique à chaque tissu lors de la maturation de la baie. Cependant les régions différentiellement méthylées identifiées dans chaque tissu, ne sont pas associées à des gènes exprimés différentiellement au cours de la maturation des baies, ce qui pose la question du rôle de la méthylation de l’ADN dans le contrôle de l’expression génique dans les baies.Pour analyser le rôle de la méthylation de l’ADN dans le contrôle de la synthèse des anthocyanes, nous avons utilisé des suspensions de cellules de raisin du génotype Gamay Teinturier (GT), connues pour accumuler des anthocyanes lorsqu’elles sont cultivées à la lumière. L’utilisation de la zébularine, un inhibiteur de la méthylation d’ADN, permet de stimuler l’accumulation d'anthocyanes dans les cellules GT en présence de lumière, et de l’induire à l’obscurité. Les traitements à la zébularine provoquent en outre une limitation de la croissance cellulaire, une modification de l’accumulation des sucres solubles et acides organiques ainsi qu’une reprogrammation importante du transcriptome. Ces résultats suggèrent un effet général de la zébularine sur les cellules GT plutôt qu’un effet spécifique sur l’accumulation d’anthocyanes.Dans l'ensemble, les résultats indiquent que la méthylation de l'ADN est importante pour le contrôle de la maturation des fruits de la vigne, bien que les mécanismes qui sous-tendent les variations de la méthylation et leurs rôles dans les différents tissus de la baie de raisin restent à préciser. / Grapevine is a worldwide cultivated fruit crop with high economic importance mainly because of its usage for vine production. Grape berry is also one of the main models for non-climacteric fruits to study the mechanisms controlling the ripening process. Grape berry development is characterized by two phases of rapid size increase separated by a lag phase at the time of ripening induction. Grape berries are composed of three main tissues, the peel, the pulp and the seeds. Peel and pulp present distinct structure and metabolite composition and contribute in a different way to wine quality, the pulp providing sugar, amino and organic acids whereas the peel is important for anthocyanins and other phenolic compound abundance. At the present time, the molecular mechanisms involved in the control of grape berry ripening are still poorly understood. Recent results indicate that both ABA and sugar may be important signals together with various transcription factors. In addition, epigenetic mechanisms are now emerging as important regulators of fleshy fruit development, DNA methylation being critically important for tomato, sweet range and strawberry ripening.The present project aims at analyzing the potential role of DNA methylation in the control grape berry ripening. It also investigates the potential role of DNA methylation in the synthesis of anthocyanins, a compound of primary importance in peel of red grape berries, using in vitro grown fruit cells. To address these questions, grape berries cultivated in vitro were treated with DNA methylation inhibitors. Treatments resulted in delayed and reduced grape berry ripening, therefore sustaining the idea that DNA methylation plays critical roles at this developmental step. Grape berries harvested at various developmental stages were then dissected and each tissue was separately analyzed for transcriptomic, metabolic and DNA methylation variations. Main results indicate significant and distinct metabolic and transcriptomic variations consistent with each tissue following specific modifications during ripening. In addition, analysis of DNA methylation variations at two developmental stages in each tissue indicates both common and tissue specific changes in DNA methylation patterns during fruit ripening. A very small proportion of DMRs is found similarly in the pup and the peel, but most are tissue specific, also consistent with tissue specific control at this developmental phase. Of note, among the different DMRs identified in each tissue, only a few were associated with differentially expressed genes (DEG) during ripening, whereas most were not, questioning the general role of DNA methylation in the control of gene expression at this developmental transition in grape.As Anthocyanins are the most abundant polyphenolic compounds in the skin of red grape berries, we used grape cell suspensions of the Gamay Teinturier genotype, that are known to accumulate anthocyanins when grown in light conditions, to analyze the potential role of DNA methylation in their synthesis. GT cells cultivated in light conditions were treated with the DNA methyltransferase inhibitor zebularine, they accumulate higher quantities of anthocyanins. Of note, GT cells grown in the absence of light do not accumulate anthocyanins. However, zebularine was sufficient to induce anthocyanin accumulation in the absence of light. Zebularine treatments had significant additional effects on grape cells including, cell growth limitation, and modification of soluble sugar, organic acid or stilbene accumulation, together with important transcriptomic reprogramming, consistent with a general effect on cells rather than a specific effect on anthocyanin accumulation.Taken together, results are consistent with DNA methylation being important in the control of grape fruit ripening, although the precise mechanisms underlying methylation variations and roles in grape berries remain to be deciphered.

The RCg2 promoter was identified in a search for root-specific genes to combat the rice water weevil (RWW) but expressed at low frequency (~10%). Spatial expression of RCg2 was investigated using two reporter constructs YXA (RCg2-gus-ocs) and YXB (RCg2-gus-RCg2) that included 1.6 kb of the RCg2 5' sequence fused to the ?-glucuronidase (gus) coding region. YXB plants were generated via Agrobacterium-mediated transformation but only 8 of 158 plants analyzed showed strong GUS activity despite the presence of an intact construct. Reactivation of RCg2 gene in rice was investigated by treatment of R0 and R1 of YXB transgenic plants with 5-azacytidine. Reactivation of RCg2-gus was observed in some transgenic plants indicating different mechanisms involved in the gene silencing of the YXB lines. DNA methylation analysis, northern blotting, RT-PCR and small RNA analysis supported the conclusion that PTGS and TGS are present in the silenced plants. Promoter analysis in silico and using promoter deletion assays predicted that the RCg2 promoter contains a complex region that includes miRNA homologs, MITEs and repetitive sequences. The high frequency of promoter-related silencing suggests functional interactions of these elements of the transgene and the homologous endogenous gene. To identify key elements contributing to the root-preferential expression of RCg2 and the high frequency of silencing observed in transgenic (YXB) lines, several RCg2 promoter deletion constructs were designed. These include 5' deletions MC1, MC2, MC4, MC7 and MC8 and internal deletions MC5, MC11, MC12 and MC13. The frequency with which silencing was encountered in populations of the deletion mutants was used to characterize the effects of various promoter elements. Deletion of the region from -406 to -208 (compared MC11 to YXB, and MC13 to MC1) revealed that region contains a negative element. Among 36 independent transformants, 33% with MC11 expressed GUS and 85% with MC13 showed GUS expression. Comparing MC7 transgenic plants to MC1 revealed that the region ?888 to ?729 is another negative regulatory element, and comparing MC11 to MC12, the proportion of expression of transgenic plants indicated the region ?729 to ?406 is a positive regulatory element.

Epigenetic mechanisms are essential for normal cell development. Alteration in those normal processes leads to malignant cell transformation and with this to cancer development. Use of inhibitors that alter the epigenetics of DNA methylation and histone post translational modifications has lead to the exploration of the epigenetic mechanism involved in silencing of tumor suppressor genes in cancer, including acute myeloid leukemia (AML). Moreover, combinations of inhibitors that target various epigenetic enzymes have being recognized to be more effective in the re-activation of tumor suppressor genes than individual drug treatments. Here, we reported that p15, p21 and E-cadherin genes are more effectively re-expressed using a combination of DNA methyltransferase and histone methyltransferase inhibitors in AML cell lines. Re-expression of hypermethylated p15 and E-cadherin genes required reduced levels of promoter histone 3 lysine 9 (H3K9) methylation rather than inhibition of DNA methylation itself. Moreover, induction of p21 expression was associated with changes in promoter histone 3 lysine 9 methylation (H3K9Me) by achieving inhibition of the histone methyltransferase, SUV39H1, activity. Altogether, our results highlight the potential of combining epigenetic drugs in the re-activation of epigenetically silenced tumor suppressor genes and the need for evaluating histone methyltransferases as therapeutic targets for treatment of acute myeloid malignancies.

Aberrant DNA methylation in gene promoters causes gene silencing and is a common event in prostate cancer development and progression. While commonly identified methylated genes have been analyzed for their potential clinical utility in a variety of cancers, few studies have attempted a genome-wide methylation approach to discover new and possibly improved biomarkers for prostate cancer. In order to identify DNA methylation changes associated with aggressive prostate cancer, we performed a genome-wide analysis of 40 prostate cancers using Agilent human CpG island microarrays. Methylation profiles of candidate genes were validated using quantitative MethyLight technology in an independent series of 219 radical prostatectomies and compared to clinicopathological parameters. The effects of methylation on expression of HOXD3 and HOXD8 and the possible role of HOXD8 in progression of PCa were also investigated. We discovered previously unidentified methylation in the HOXD cluster of genes, namely HOXD3 and HOXD8, as well as TGFβ2 and GENE X as potential prognostic biomarkers. Furthermore, unsupervised clustering of samples by methylation signature indicated ERG oncogene expression as significantly different between clusters. Within the independent cohort, we observed strong correlations between Gleason score (GS) and HOXD3 as well as GENE X, while HOXD3 and HOXD8 methylation were associated with ERG expresson. TGFβ2 was an independent predictor of disease recurrence using Cox multivariate regression analysis. In gene expression studies, both HOXD3 and HOXD8 were elevated in cancers with poor prognosis, while DNA methylation did not correlate with expression levels. Both genes were found to contain alternative transcription start sites, explaining the poor correlation between methylation and expression. Finally, knockdown of HOXD8 expression did not have any effect on viable cells or cell motility in an in vitro model. These results indicate that a panel of novel DNA methylation markers distinguish indolent prostate cancers from aggressive ones, and that expression of HOXD3 and HOXD8 is regulated by mechanisms including, but not dependent on, DNA methylation.

Early diagnosis represents an effective way to improve patient prognosis in cancer. New opportunities for cancer diagnosis and screening may arise from identification of cancer-specific epigenetic alterations in the cell-free circulating DNA (cirDNA). This study investigated biomarkers at the level of DNA methylation in the plasma cirDNA of individuals affected with prostate cancer or colorectal cancer. A methylation-sensitive restriction enzyme-based method was used to enrich methylated DNA fractions, which were interrogated on CpG island and human genome tiling microarrays. A number of genes and non-coding loci exhibited differential methylation between prostate cancer patients and controls. The candidate loci identified from these microarray experiments underwent verification by bisulfite modification coupled with pyrosequencing. Our results suggest that microarray-based studies of DNA methylation in the cirDNA can be a promising avenue for the identification of epigenetic biomarkers in cancer.

Early diagnosis represents an effective way to improve patient prognosis in cancer. New opportunities for cancer diagnosis and screening may arise from identification of cancer-specific epigenetic alterations in the cell-free circulating DNA (cirDNA). This study investigated biomarkers at the level of DNA methylation in the plasma cirDNA of individuals affected with prostate cancer or colorectal cancer. A methylation-sensitive restriction enzyme-based method was used to enrich methylated DNA fractions, which were interrogated on CpG island and human genome tiling microarrays. A number of genes and non-coding loci exhibited differential methylation between prostate cancer patients and controls. The candidate loci identified from these microarray experiments underwent verification by bisulfite modification coupled with pyrosequencing. Our results suggest that microarray-based studies of DNA methylation in the cirDNA can be a promising avenue for the identification of epigenetic biomarkers in cancer.

Fetal growth potential reflects a complex regulatory system delivered by genetic and environmental factors acting directly on the fetus or through the placenta. Compromise of this potential, as seen in intrauterine growth restriction (IUGR), is associated with increased perinatal mortality and short and long term morbidity. The expression of several genes has been shown to be disturbed in placentas of fetuses with growth restriction. However, the primary causes for these changes have not yet been elucidated. I proposed that epigenetic mechanisms, specifically DNA methylation, may be involved in placental development leading to modulation of the expression of specific genes, and that their altered regulation will impact fetal development and growth. My primary objective was to identify DNA methylation variation in placenta, in association with variation of gene expression and with poor fetal growth. I used a global genomic screening approach, with 24 selected placental samples, from newborns considered IUGR or normal controls, to identify candidate target genomic regions carrying epigenetic alterations. Candidate regions were followed up, by expression analysis of corresponding regulated genes, for associations with altered expression and by targeted methylation analysis in an expanded cohort of 170 samples, for associations with birthweight percentile. I analyzed methylation variation at imprinting centers (IC), gene promoters and CpG islands. In two genome-wide case control screening studies using distinct commercial microarray platforms I identified approximately 68 differentially methylated autosomal candidate genomic regions overlapping gene promoters. Hypomethylated CpGs mapping to gene promoters were found to be more abundant in placentas of growth restricted newborns than in controls. One of the most interesting candidates, WNT2, was analyzed in an extended sample cohort and showed an association of high promoter methylation to low expression as well as low birthweight percentile. This gene is involved in a pathway that diverts cells from programmed apoptosis. It is highly expressed in placenta, and in mice, targeted biallelic inactivation of Wnt2 has been shown to cause poor growth and perinatal death in 50% of the affected pups. These findings support the hypothesis that dysregulation of epigenetic mechanisms are involved in abnormal placental development and can impact fetal growth.

Fetal growth potential reflects a complex regulatory system delivered by genetic and environmental factors acting directly on the fetus or through the placenta. Compromise of this potential, as seen in intrauterine growth restriction (IUGR), is associated with increased perinatal mortality and short and long term morbidity. The expression of several genes has been shown to be disturbed in placentas of fetuses with growth restriction. However, the primary causes for these changes have not yet been elucidated. I proposed that epigenetic mechanisms, specifically DNA methylation, may be involved in placental development leading to modulation of the expression of specific genes, and that their altered regulation will impact fetal development and growth. My primary objective was to identify DNA methylation variation in placenta, in association with variation of gene expression and with poor fetal growth. I used a global genomic screening approach, with 24 selected placental samples, from newborns considered IUGR or normal controls, to identify candidate target genomic regions carrying epigenetic alterations. Candidate regions were followed up, by expression analysis of corresponding regulated genes, for associations with altered expression and by targeted methylation analysis in an expanded cohort of 170 samples, for associations with birthweight percentile. I analyzed methylation variation at imprinting centers (IC), gene promoters and CpG islands. In two genome-wide case control screening studies using distinct commercial microarray platforms I identified approximately 68 differentially methylated autosomal candidate genomic regions overlapping gene promoters. Hypomethylated CpGs mapping to gene promoters were found to be more abundant in placentas of growth restricted newborns than in controls. One of the most interesting candidates, WNT2, was analyzed in an extended sample cohort and showed an association of high promoter methylation to low expression as well as low birthweight percentile. This gene is involved in a pathway that diverts cells from programmed apoptosis. It is highly expressed in placenta, and in mice, targeted biallelic inactivation of Wnt2 has been shown to cause poor growth and perinatal death in 50% of the affected pups. These findings support the hypothesis that dysregulation of epigenetic mechanisms are involved in abnormal placental development and can impact fetal growth.

Epigenetic mechanisms are essential for normal cell development. Alteration in those normal processes leads to malignant cell transformation and with this to cancer development. Use of inhibitors that alter the epigenetics of DNA methylation and histone post translational modifications has lead to the exploration of the epigenetic mechanism involved in silencing of tumor suppressor genes in cancer, including acute myeloid leukemia (AML). Moreover, combinations of inhibitors that target various epigenetic enzymes have being recognized to be more effective in the re-activation of tumor suppressor genes than individual drug treatments. Here, we reported that p15, p21 and E-cadherin genes are more effectively re-expressed using a combination of DNA methyltransferase and histone methyltransferase inhibitors in AML cell lines. Re-expression of hypermethylated p15 and E-cadherin genes required reduced levels of promoter histone 3 lysine 9 (H3K9) methylation rather than inhibition of DNA methylation itself. Moreover, induction of p21 expression was associated with changes in promoter histone 3 lysine 9 methylation (H3K9Me) by achieving inhibition of the histone methyltransferase, SUV39H1, activity. Altogether, our results highlight the potential of combining epigenetic drugs in the re-activation of epigenetically silenced tumor suppressor genes and the need for evaluating histone methyltransferases as therapeutic targets for treatment of acute myeloid malignancies.