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La pollution atmosphérique et les évènements météorologiques sont reconnus pour avoir des graves conséquences sur la santé telles que la mortalité cardiovasculaire et respiratoire, et par conséquent représentent un enjeu majeur de santé publique. Des travaux plus récents ont suggéré un effet des expositions à ces facteurs environnementaux durant la période intra-utérine. La vie fœtale est déterminante pour le bon développement de l’enfant. L’exposition maternelle aux facteurs environnementaux pendant la grossesse pourrait avoir des conséquences sur les issues de grossesse et la santé à court ou long terme. Par ailleurs, les mécanismes biologiques qui pourraient expliquer l’effet des expositions environnementales sur les issues de grossesse indésirables sont à l’heure actuelle très peu connus. Les objectifs de la thèse étaient : 1) d’étudier les associations entre d’une part la température et l’humidité relative pendant la grossesse et d’autre part le poids de naissance, la durée de gestation et la prématurité ; 2) d’étudier les associations entre l’exposition maternelle aux polluant de l’air et aux évènements météorologiques pendant la grossesse et la méthylation de l’ADN placentaire en utilisant A) une approche agnostique et B) une approche avec a priori basée sur l’intégration de connaissances biologiques. Le premier objectif s’est appuyé les données issues de deux cohortes mère-enfant, EDEN (avec un recrutement à Poitiers et Nancy en 2003-2006) et PELAGIE (Bretagne, 2002-2006) soit au total 5185 couples mère-enfant ; le deuxième objectif s’est appuyé sur un échantillon de 668 femmes de la cohorte EDEN chez qui le placenta a été prélevé et le niveau de méthylation de son ADN caractérisé. Les données quotidiennes de température et d’humidité ont été obtenues de Météo-France et l’exposition à la pollution atmosphérique a été estimée par un modèle de dispersion atmosphérique. Différentes fenêtres d’exposition au cours de la grossesse ont été considérées. Des échantillons de placenta centraux ont été collectés à l’accouchement et la méthylation de l’ADN a été analysée en utilisant la puce Illumina 450K. Pour répondre au premier objectif, des modèles de régression linéaire et des modèles de Cox ont été réalisés. Pour répondre au second objectif, des modèles de régression linéaire robuste, notamment à l’échelle du génome entier, ont été réalisés en appliquant des méthodes de correction de tests multiples telles que Bonferroni et Benjamini-Hochberg. Nos résultats suggèrent un effet délétère de la température et l’humidité sur le poids de naissance et ne mettent pas en évidence d’association entre pollution atmosphérique et issues de grossesse. L’exposition aux polluants atmosphériques (NO2 et PM10) pendant la grossesse était associée à une diminution de la méthylation de l’ADN placentaire pour les gènes ADORA2B et ADARB2 ; le premier gène étant connu pour être potentiellement impliqué dans la pré-éclampsie et l’hypoxie de la femme enceinte et le deuxième étant potentiellement impliqué dans les troubles métaboliques chez l'adulte tels que la circonférence abdominale et l'IMC. Les résultats des approches agnostique et avec a priori étaient cohérents pour le gène ADORA2B. Nous n’avons pas identifié d’association entre les conditions météorologiques et la méthylation de l’ADN placentaire. A notre connaissance, nous sommes les premiers à avoir étudié l’association entre la méthylation de l’ADN dans le tissu placentaire et l’exposition prénatale aux polluants de l’air et aux conditions météorologiques en utilisant des données de l’épigénome entier. Ce travail de thèse ouvre de nouvelles voies possibles concernant les mécanismes d’actions des polluants environnementaux et fournit des pistes quant aux effets à long terme possibles de ces expositions. / Nowadays, air pollution and weather conditions represent a major public health issue. It is recognized that they may have serious consequences for health especially in the most sensitive populations such as pregnant women. More recent studies have suggested an effect of exposure to these environmental factors during the fetal period. Fetal life is a critical period for the healthy development of the child. Maternal exposure to environmental exposures during pregnancy could have serious consequences on pregnancy outcomes and short- and long- term health. Furthermore, the biological mechanisms that could explain the effect of environmental exposures on adverse pregnancy outcomes are largely unknown up to now. The objectives of the thesis were: 1) to study the association between maternal exposure to temperature and relative humidity during pregnancy and birth weight, gestational duration and preterm birth; 2) to study the association between maternal exposure to air pollutants and meteorological conditions during pregnancy and placental DNA methylation using A) an agnostic approach and B) a priori approach based on integration of biological knowledge. The first part of this work relied on data from two mother-child cohorts EDEN (Poitiers and Nancy, 2003-2006) and PELAGIE (Britain, 2002-2006) corresponding to 5185 mother-infant pairs analyzed; and the second part relied on a sample of the EDEN cohort for which methylation data were available (n = 668). Daily data of temperature and humidity were obtained from Météo-France and pollution data were obtained using a dispersion model. Their exposure was averaged over different periods during pregnancy. Central placenta samples were collected at delivery and DNA methylation was analyzed using Illumina 450K chip. For the first objective, linear regression models and Cox models were used. For the second objective, robust linear regression models, especially across the genome-wide, were used and correction methods for multiple testing such as Bonferroni and Benjamini-Hochberg were applied. Our results suggest a deleterious effect of temperature and relative humidity on birth weight and did not show an association between air pollution and pregnancy outcomes. Exposure to air pollutants (NO2 and PM10) during pregnancy was associated with a decrease in placental DNA methylation for ADORA2B and ADARB2 genes; the first gene is known to be potentially involved in preeclampsia and hypoxia of the pregnant woman and the second being potentially involved in metabolic disorders in adults such as abdominal circumference and BMI. The results of agnostic and a priori approaches were consistent for ADORA2B gene. We did not found association between weather conditions and placental DNA methylation. To our knowledge, we are the first to study the association between DNA methylation in the placental tissue and prenatal exposure to air pollutants and weather conditions using data from the entire epigenome. This work opens up new possible pathways regarding mechanisms of action of environmental pollutants and provides pointers as to the possible long-term effects of these exposures.

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Bivalent genes are frequently associated with developmental and lineage specification processes. Resolving their bivalency enables fast changes in their expression, which potentially can trigger cell fate decisions. Here, we provide a theoretical model of bivalency that allows for predictions on the occurrence, stability and regulatory capacity of this prominent modification state. We suggest that bivalency enables balanced gene expression heterogeneity that constitutes a prerequisite of robust lineage priming in somatic stem cells. Moreover, we demonstrate that interactions between the histone and DNA methylation machineries together with the proliferation activity control the stability of the bivalent state and can turn it into an unmodified state. We suggest that deregulation of these interactions underlies cell transformation processes as associated with acute myeloid leukemia (AML) and provide a model of AML blast formation following deregulation of the Ten-eleven Translocation (TET) pathway

Endocrine disrupting chemicals (EDCs) are ubiquitous and their adverse impact on nature, wildlife and humans is extensively researched. We are constantly exposed to EDCs, such as the widespread and extensively researched Bisphenol A, as well as its substitute Bisphenol F, which is coming into wider use, even though it is much less is researched and limited information is available about its endocrine effects. Realistically, we are exposed to mixtures rather than single substances. In the Swedish Environmental Longitudinal, Mother and Child, Asthma and allergy (SELMA) study, the co-exposure of EDCs was assessed. Based on the SELMA cohort data, a study identified a mixture of EDCs, Mixture N1, which is associated with delayed language development. In recent years, it has been hypothesized that epigenetic alterations are one of the underlying mechanisms for the effect of EDC exposures. For example, EDC induced changes in DNA Methylation of the promoter region of a gene might lead to altered gene expression, which can result in adverse health effects. Several studies already indicate an impact of the formerly introduced chemicals/mixtures on the DNA methylation on genes such as NR3C1 and GRIN2B in animals. However, limited research is available on the impact on NR3C1 and GRIN2B in the early human brain, which is of interest since both genes are crucial for the development of the brain and altered gene expression often leads to adverse effects. This study aimed to investigate the impact of BPA, BPF and Mixture N1 on NR3C1 and GRIN2B in the developing human brain as well as establish a protocol for differentiation of human stem cells into neuroprogenitor cells that express GRIN2B and NR3C1. In the end stem cells were differentiated in vitro into neural progenitor cells (NPCs) using the protocol of Hosseini et al. (2020). During the differentiation, the cells were exposed to different concentrations of the former mentioned chemicals. Afterwards, RNA and DNA were extracted, followed by a qPCR and bisulfite-pyrosequencing to investigate the changes in gene expression and DNA methylation of NR3C1 and GRIN2B. This study established the differentiation protocol but revealed no significant results regarding the chemical exposure. However, some chemical exposures showed a clear tendency towards an impact of the chemicals on the gene expression and the DNA methylation. Furthermore, a negative correlation between DNA methylation at 2 CpG sites and gene expression in NR3C1 could be observed. In conclusion, the DNA methylation at promoter region in NR3C1 is important for the gene expression.

M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Obesity has steadily increased and represents a major public health problem worldwide, reducing quality of life and causing a range of health problems. Obesity has emerged as the fifth leading risk of global deaths. Annually, 2.8 million adults die as a result of being overweight or obese. The increase of obesity remains inexplicable in terms of genetic susceptibility to obesity. The genetic loci identified by genome-wide association studies (GWASs) explains about 2% of the heritability for obesity. Perhaps other factors such as epigenetics may be involved in the increase of obesity and may offer solutions for the management of obesity. Epigenetics is defined as a heritable change in gene expression without altering the genome sequences. It may help in providing a logical explanation between the genome and environment which shapes obesity risk and may help to explain the "missing heritability". Epigenetics may affect two mechanisms, namely: i) DNA methylation,and ii) histone modifications. DNA methylation might give scientists a link to the rise in obesity.The study aimed to investigate the effect of sugars used as sweeteners in sugar-sweetened beverages (SSB) on DNA methylation in a Caco-2 cell line in vitro. Four major objectives were pursued in the study which were to:(1) stimulate the Caco-2 cells with varying concentrations of sugar sweeteners and assess the morphological changes of the cells; (2) evaluate the cytotoxicity of different concentrations of the sugar sweetener on the Caco-2 cell line using the Alamar blue and LDH assay; (3) obtain genomic DNA from the treated Caco-2 cell line and perform bisulfite conversion and rest; and (4) amplify the WT1, MEG3, TNFRSF9, ATP10A, and CD44 obesity-associated genes and ascertain their degree of methylation. Caco-2 cells were stimulated with sugar sweeteners at varying concentrations (low, medium and high) for an incubation period of 62 days,and images of the cells were captured for morphological characterisation. The incubation condition entailed cells plated in a 12 or 96 well plate, incubated in a humidified 5% CO2 incubator at 37 °C and there is nutrient renewal every three days.Alamar blue, a cell proliferation colourimetric assay and lactate dehydrogenase assays (LDH), a homogenous membrane fluorimetric assay were used for the cytotoxicity studies. The results of the characterisation showed that different concentrations of sugar sweeteners affected the morphology of the cells as the incubation period progressed. The cytotoxicity results of both LDH and Alamar blue depicted low concentration of sweeteners that had low-to-moderate toxicity and the medium and high concentration of the sweeteners had a moderate to high toxicity on the Caco-2 cells. DNA from the Caco-2 cells was extracted. Techniques used to study DNA methylation such as bisulfite conversion, PCR amplification and restriction enzymes that have differential sensitivity to 5-methyl-cytosine were performed. The quality of DNA extracted was good. The bisulfite conversion was conducted andno amplification was observed, as a contingency plan Normal PCR was performed to amplify the CpG islands, and there was amplification. In conclusion, the study showed that a low concentration of a sugar sweetener (fructose: glucose) used in beverages had low toxicity to the Caco-2 cell line and prolonged exposure of the low concentration might have an adverse effect on the cells' morphology. At medium concentrations, the sugar sweetener used in beverages had medium toxicity to Caco-2 cells; prolonged exposure may lead to morphological changes. These findings indicated that control of dietary glucose intake is an important strategy in combating the development of obesity and type-2 diabetes. DNA methylation could not be established.

Malgré la stabilité historiquement associée à la méthylation de l’ADN, cette modification épigénétique subit des changements rapides importants dans les types cellulaires plus plastiques. Par exemple, l’infection de cellules dendritiques humaines est associée à des milliers de changements dans le paysage de méthylation, principalement des pertes de méthylation dans les amplificateurs. Cette déméthylation est corrélée à l’apparition de marques actives d’histones et à l’activation des gènes à proximité. Dans la présente thèse, le rôle plus précis de cette déméthylation active a été investigué. Une étude temporelle de l’infection de cellules dendritiques par Mycobacterium tuberculosis a révélé que les changements d’expression génique et la liaison des facteurs de trancription classiquement activés en réponse à une infection bactérienne, comme les familles de NF-B et AP-1, étaient préalables aux changements de méthylation, suggérant un rôle minimal de la déméthylation dans la réponse à l’infection. D’autres résultats allaient dans le même sens, soit en inhibant TET2, une enzyme participant à la déméthylation active de l’ADN dans les cellules dendritiques via sa conversion en intermédiaires. En effet, l’impact d’une telle inhibition sur la réponse transcriptionnelle à l’infection par Salmonella typhimurium était minime. Cependant, l’inhibition de TET2 entraîne un niveau basal d’expression de cytokines pro-inflammatoires plus élevé, ce qui m’entraîne à proposer que la déméthylation puisse participer au retour à la normale post-infection. Elle pourrait également avoir une fonction dans la mémoire immunitaire innée en permettant une réponse plus rapide à une seconde infection, un phénomène surtout démontré dans les monocytes et les macrophages, mais mis en évidence dans les cellules dendritiques dans la présente thèse. Ainsi, cette étude ouvre de nombreuses perspectives dans la thérapie épigénétique contre les maladies auto-immunes, le développement de vaccins ciblant le système immunitaire inné, et l’utilisation de la méthylation de l’ADN comme biomarqueur d’infections. / DNA methylation has historically been perceived as a highly stable epigenetic modification. However, rapid and important changes in its landscape occur in cells with higher plasticity. For example, human dendritic cells infection associated with thousands of changes of DNA methylation, majoritarily losses in enhancer regions. This demethylation is correlated with gain of active histone marks and nearby gene upregulation. The present dissertation investigated the precise role of this demethylation. Firstly, a temporal study of dendritic cells infected with Mycobacterium tuberculosis revealed that gene expression changes and the classical immune transcription factors binding, such as members of the NF-B or AP-1 families, occurred prior to DNA methylation changes. This suggests a minimal function of demethylation in response to infection. Results from a TET2 inhibition an enzyme participating to active demethylation by converting 5-methylcytosine to intermediates were concordant with this hypothesis. Indeed, this inhibition has a very small impact on transcriptional response to an infection by Salmonella typhimurium. Nevertheless, the pro-inflammatory cytokine production of TET2-inhibited non-infected cells is higher. I therefore propose that demethylation might play a role in the return to basal state after an infection is cleared. It could also participate to innate immune memory by allowing cells to respond faster to a second infection. This phenomenon has especially been demonstrated in monocytes and macrophages but is also highlighted in dendritic cells in the present work. This dissertation opens numerous perspectives in epigenetic therapy of auto-immune diseases, the development of vaccines targeting the innate immune system and the use of DNA methylation as a biomarker.

Molecular mechanisms of lower-grade (II–III) diffuse gliomas (LGG) are still poorly understood, mainly because of their heterogeneity. They split into astrocytoma- (IDH-A) and oligodendroglioma-like (IDH-O) tumors both carrying mutations(s) at the isocitrate dehydrogenase (IDH) gene and into IDH wild type (IDH-wt) gliomas of glioblastoma resemblance. We generated detailed maps of the transcriptomes and DNA methylomes, revealing that cell functions divided into three major archetypic hallmarks: (i) increased proliferation in IDH-wt and, to a lesser degree, IDH-O; (ii) increased inflammation in IDH-A and IDH-wt; and (iii) the loss of synaptic transmission in all subtypes. Immunogenic properties of IDH-A are diverse, partly resembling signatures observed in grade IV mesenchymal glioblastomas or in grade I pilocytic astrocytomas. We analyzed details of coregulation between gene expression and DNA methylation and of the immunogenic micro-environment presumably driving tumor development and treatment resistance. Our transcriptome and methylome maps support personalized, case-by-case views to decipher the heterogeneity of glioma states in terms of data portraits. Thereby, molecular cartography provides a graphical coordinate system that links gene-level information with glioma subtypes, their phenotypes, and clinical context.

Mutually linked expression and methylation dynamics in the brain govern genome regulation over the whole lifetime with an impact on cognition, psychological disorders, and cancer. We performed a joint study of gene expression and DNA methylation of brain tissue originating from the human prefrontal cortex of individuals across the lifespan to describe changes in cellular programs and their regulation by epigenetic mechanisms. The analysis considers previous knowledge in terms of functional gene signatures and chromatin states derived from independent studies, aging profiles of a battery of chromatin modifying enzymes, and data of gliomas and neuropsychological disorders for a holistic view on the development and aging of the brain. Expression and methylation changes from babies to elderly adults decompose into different modes associated with the serial activation of (brain) developmental, learning, metabolic and inflammatory functions, where methylation in gene promoters mostly represses transcription. Expression of genes encoding methylome modifying enzymes is very diverse reflecting complex regulations during lifetime which also associates with the marked remodeling of chromatin between permissive and restrictive states. Data of brain cancer and psychotic disorders reveal footprints of pathophysiologies related to brain development and aging. Comparison of aging brains with gliomas supports the view that glioblastoma-like and astrocytoma-like tumors exhibit higher cellular plasticity activated in the developing healthy brain while oligodendrogliomas have a more stable differentiation hierarchy more resembling the aged brain. The balance and specific shifts between volatile and stable and between more irreversible and more plastic epigenomic networks govern the development and aging of healthy and diseased brain.