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Le retard de croissance intra-utérin (RCIU) est une complication fréquente de la grossesse qui se traduit à la naissance par un poids et/ou une taille inférieurs au dixième percentile par rapport à l’âge gestationnel. Il représente un problème majeur de santé publique avec une augmentation de la morbi-mortalité néonatale et un risque accru de développer des maladies cardiovasculaires et un diabète à l’âge adulte. Le RCIU humain est une pathologie complexe et multifactorielle avec une physiopathologie incomplètement élucidée dans près de 30 à 40% des cas. L’objectif de ce travail a été de progresser sur la physiopathologie du RCIU humain et de proposer des gènes candidats impliqués. J’ai analysé les perturbations d’expression génique dans des placentas issus de grossesses avec et sans RCIU, participé à la caractérisation d’un modèle animal de rate gestante soumise à un régime hypoprotidique, mis au point et validé un modèle cellulaire de cellules JEG-3 transformées par surexpression du gène PDX1 et/ou déplétées en acides aminés constituant un modèle important des effets du RCIU. J’ai étudié le rôle et l’implication de plusieurs gènes candidats : IMP3, les Cullines et PDX1. J’ai enfin participé à l’identification de nouveaux gènes soumis à empreinte dans le placenta. Ce travail de thèse a confirmé le rôle essentiel de l’apoptose et des régulations épigénétiques, plus particulièrement les modifications de profil de méthylation de l’ADN, dans le RCIU. L’analyse transcriptomique des placentas humains de grossesse avec RCIU a confirmé le rôle essentiel de 3 gènes : la leptine, IGFBP1 et RBP4. Enfin l’utilisation de notre modèle cellulaire a permis de confirmer l’implication de PDX1 dans le RCIU, PDX1 contrebalance les effets transcriptomiques de la déplétion en acides aminés sur les cellules JEG en culture. En extrapolant ces résultats à la pathologie humaine, on peut émettre l’hypothèse que l’induction placentaire de PDX1 en situation de RCIU permettrait de contrebalancer ou de limiter les effets délétères du RCIU pour la survie du fœtus. La leptine, IGFBP, RBP4 et PDX1 sont de bons candidats à la fois pour l’origine du RCIU et également pour la prédiction de l’évolution possible vers un syndrome métabolique chez les adultes anciens RCIU. Des études complémentaires sur le rôle et l’implication de ces 4 gènes dans la physiopathologie du RCIU ainsi que dans la programmation fœtale des pathologies cardiovasculaires, de l’obésité et de l’intolérance glucidique à l’âge adulte restent nécessaires. / Intra-uterine growth restriction (IUGR) is a frequent complication of pregnancy that leads to a baby with a birth weight and/or size below the 10th percentile for a given gestational age. IUGR represents a major public health problem associated with neonatal increased morbidity and mortality and an increased risk to develop cardiovascular pathologies and diabetes in adulthood. Human IUGR is a complex and multi-factorial pathology with an incompletely characterised physiopathology in up to 30 to 40% of cases. The goal of this work was to improve the knowledge on the physiopathology of IUGR and to propose implicated candidate genes.I analysed modulations of gene expression in placentas from pregnancies with and without IUGR, participated to the characterisation of an IUGR animal model of female rat fed with a hypoprotidic diet during gestation, set up and validated a cellular model of JEG-3 cells transformed by over-expression of the PDX1 gene and/or depletion of amino acids to provide a handy model of IUGR effects. I studied the role and implication of candidate genes: IMP3, the Cullin family and PDX1. Finally I participated to the identification of new imprinted genes in the human placenta. This thesis work confirmed the essential role of apoptosis and epigenetic regulations, in particular modifications of DNA methylation profiles, in IUGR. Transcriptomic analysis of human placentas from IUGR and control pregnancies confirmed the crucial role of 3 genes: leptin, IGFBP1 and RBP4. Finally, the use of our cellular model strengthened the role of PDX1 in IUGR, where it counterbalances the transcriptomic effects of amino acid depletion in cultured JEG-3 cells. By extrapolating these results to the human pathology, we can suggest the hypothesis that the placental induction of PDX1 in IUGR could counterbalance or limit the deleterious effects of IUGR on the foetus’ survival. Leptin, IGFBP1, RBP4 and PDX1 are good candidates to explain the origin of IUGR as well as to predict the potential evolution towards a metabolic syndrome in adults that suffered from IUGR. Complementary studies on the role and function of these 4 genes in IUGR physiopathology and also in foetal programming of cardiovascular pathologies, obesity and glucidic intolerance in adulthood remain necessary.

La transition épithélio-Mésenchymateuse (TEM) est un processus de plasticité cellulaire qui existe dans le développement embryonnaire et qui permet la formation des tissus et organes. Dans la cancérogénèse, ce processus est réactivé par des facteurs de transcription dont l’action implique très probablement un remodelage de la chromatine. La cartographie exacte de ces changements épigénétiques est peu connue à l’échelle du génome entier, même si il y a eu quelques études antérieures explorant les changements de quelques loci de façon bien ciblée. Ce mémoire traite du remodelage épigénétique médié par le facteur de transcription Twist1 dans un modèle de lignée mammaire immortalisée. L’architecture de ce remodelage a été cartographiée grâce à l’utilisation des techniques de haut-Débit pour analyser la méthylation de l’ADN (DREAM) et les modifications des histones (ChIPseq). Nos résultats montrent un changement majeur du méthylome pendant la TEM avec une hyperméthylation focale et une hypométhylation globale des corps des gènes prédominant au niveau des « domaines partiellement méthylés »; ces domaines sont déjà connus dans le développement pour gagner de façon concomitante à leur hypométhylation des marques d’histone répressives. Nous avons aussi observé un remodelage des domaines de l’histone répressive H3K27me3 avec une réduction de leur taille, et surtout le quasi doublement du nombre de gènes bivalents qui accompagne la transition. Le couplage de la méthylation de l’ADN avec le profil des microRNA nous a permis d’identifier le miR-203 comme l’unique microRNA régulé par méthylation de l’ADN durant la TEM; nous avons aussi montré que l’extinction épigénétique du miR-203 est requise pour la TEM et l’acquistion des propriétés de cellules souches. Enfin, nous avons réalisé une caractérisation génétique et/ou épigénétique de deux cancers rares, les carcinomes fibrolamellaires du foie et les carcinomes du rein à translocation. Pour les carcinomes fibrolamellaires du foie, nous avons décrit la nature endocrine de cette tumeur et établi une signature épigénétique basée sur la méthylation de l’ADN pouvant servir à différencier les formes histologiques appelées « pures » des formes « mixtes ». Pour les cancers du rein à translocation, nous avons montré les bases génétiques et épigénétiques de la différence entre les formes pédiatriques et adultes, avec la découverte fréquente du gain du bras chromosomique 17q dans les formes adultes. Nous avons aussi identifié une mutation récurrente dans le gène qui remodèle la chromatine INO80D appartenant à la famille INO80. En conclusion, ce travail explore le rôle de l’étude de l’épigénome pour comprendre la reprogrammation pendant les processus physiologiques comme la TEM d’une part et le cancer d’autre part. / The epithelial-Mesenchymal transition (EMT) is a process of cellular plasticity that exists in embryonic development and which allows the formation of tissues and organs. In carcinogenesis, the process is reactivated by transcription factors whose action probably involves chromatin remodeling. The exact mapping of these epigenetic changes is poorly understood genome-Wide, although there have been some previous studies exploring changes in so few well-Targeted loci. This thesis deals with the epigenetic remodeling mediated by the transcription factor Twist1 in a model of human mammary immortalized cell line. The architecture of this remodeling has been mapped through the use of high-Throughput techniques to analyze DNA methylation (DREAM) and histone modifications (ChIPseq). Our results suggest a major change in the EMT methylome with focal hypermethylation and gene body hypomethylation predominantly within "partially methylated domains"; these areas are already known in development to gain repressive histone marks concomitantly with DNA hypomethylation. We also observed landscape remodeling of repressive histone mark H3K27me3 with a reduction in domains size, and especially the almost doubling of the number of bivalent genes. The coupling of DNA methylation with the profile of microRNA has allowed us to identify miR-203 as single microRNA regulated by DNA methylation during EMT; we have also shown that epigenetic suppression of miR-203 is both required for EMT and acquisition of stem cell properties. Finally, we performed a genetic and/or epigenetic characterization of two rare cancers, named fibrolamellar hepatocellular carcinomas and translocation renal cell carcinomas. In fibrolamellar hepatocellular carcinoma, we described the endocrine nature of this tumor and established a signature based on DNA methylation which can be used to distinguish histological forms called "pure" from "mixed" fibrolamellar hepatocellular carcinomas. Regarding translocation renal cell carcinomas, we established the genetic and epigenetic basis of differences between pediatric and adult forms, characterized by frequent gain of 17q gain chromosomal arm in adults. We also identified recurrent mutations in the chromatin remodeling gene INO80D which belongs to INO80 family. In conclusion, this work explores the impact of analyzing the epigenome to understand reprogramming during physiological processes such as EMT and cancer.

Hutchinson-Gilford Progeria Syndrome is described as a series of mutations within the lamin A gene leading to the accumulation of progerin in the nucleus, contributing to premature aging and affecting the epigenetic control. Epigenetic control, such as DNA methylation, relies on DNA methyltransferase enzymes. In human cells, heat shock (HS) leads to the formation of nuclear stress bodies (nSBs); ribonucleoprotein aggregates of Sat III RNA and RNA-binding proteins. The objectives of this study were to determine if epigenetic status induces varying responses to HS and assess the variability of nuclear proteins in similar conditions. Results show epigenetic modifications do not prevent a stress response; however the extent may be affected. In addition the functions of most nuclear antigens were not affected. It is most likely the sum of interactions at the inner nuclear membrane and nuclear lamina interface that result in nuclear strength pertaining to lamin A.

Pubertal timing displays wide, normally distributed variation in a healthy population of sexually maturing adolescents. However, like many complex traits, factors contributing to the variation are not well understood. Epigenetic regulation may contribute to some of the population variation. The role that epigenetics, specifically DNA methylation and histone acetylation, may play in regulating pubertal timing was investigated in C57BL/6 female mice: investigating whether population variation in pubertal timing among inbred mice could be explained by environmental factors; whether perturbing the epigenome using a histone deacetylase inhibitor or methyl-donor would alter pubertal timing; and examining genome-wide methylation patterns in hypothalami of early versus late maturing mice. Results demonstrate that measurable micro-environmental factors have only negligible effects on pubertal timing; pubertal timing was significantly altered by administration of epigenetic modifying agents; differences in methylation patterns are subtle. This initial evidence supports the involvement of epigenetic mechanisms in regulating pubertal timing.

Pubertal timing displays wide, normally distributed variation in a healthy population of sexually maturing adolescents. However, like many complex traits, factors contributing to the variation are not well understood. Epigenetic regulation may contribute to some of the population variation. The role that epigenetics, specifically DNA methylation and histone acetylation, may play in regulating pubertal timing was investigated in C57BL/6 female mice: investigating whether population variation in pubertal timing among inbred mice could be explained by environmental factors; whether perturbing the epigenome using a histone deacetylase inhibitor or methyl-donor would alter pubertal timing; and examining genome-wide methylation patterns in hypothalami of early versus late maturing mice. Results demonstrate that measurable micro-environmental factors have only negligible effects on pubertal timing; pubertal timing was significantly altered by administration of epigenetic modifying agents; differences in methylation patterns are subtle. This initial evidence supports the involvement of epigenetic mechanisms in regulating pubertal timing.

Hutchinson-Gilford Progeria Syndrome is described as a series of mutations within the lamin A gene leading to the accumulation of progerin in the nucleus, contributing to premature aging and affecting the epigenetic control. Epigenetic control, such as DNA methylation, relies on DNA methyltransferase enzymes. In human cells, heat shock (HS) leads to the formation of nuclear stress bodies (nSBs); ribonucleoprotein aggregates of Sat III RNA and RNA-binding proteins. The objectives of this study were to determine if epigenetic status induces varying responses to HS and assess the variability of nuclear proteins in similar conditions. Results show epigenetic modifications do not prevent a stress response; however the extent may be affected. In addition the functions of most nuclear antigens were not affected. It is most likely the sum of interactions at the inner nuclear membrane and nuclear lamina interface that result in nuclear strength pertaining to lamin A.

Las enfermedades neurodegenerativas, como la enfermedad de Alzheimer (EA) y la enfermedad de Parkinson (EP), representan un grave problema de salud pública, sobre todo en los países occidentales, donde el envejecimiento creciente de la población augura un incremento sustancial de la prevalencia de estas patologías. A pesar de que ciertos tratamientos proporcionan una disminución de las manifestaciones clínicas, el avance del proceso neurodegenerativo es irreversible. La identificación de los mecanismos, como la interacción entre factores genéticos y medio-ambientales, implicados en la etiología y evolución de estas patologías es de importancia capital. En el presente trabajo de tesis se explora el papel de la metilación del ADN genómico y el mosaicismo genético en enfermedades neurodegenerativas. El análisis del perfil de metilación del ADN se realizó empleando dos arrays de metilación: “HumanMethylation” (27K y 450K, IlIumina), cuyas sondas distribuidas estratégicamente por todo el genoma, permiten detectar cuantitativamente el estado de mutilación de unos 27.000 y 450.000 dinucleótidos CpG, respectivamente. La comparación de un total de 60 individuos (28 con enfermedad de Alzheimer, 3 con enfermedad de Parkinson y 29 controles) ha permitido identificar el perfil de metilación del genoma de distintas áreas del sistema nervioso central (SNC) (corteza, amígdala, hipocampo, hipotálamo, protuberancia, sustancia negra y cerebelo), mostrando la existencia de un patrón diferencial entre hombres y mujeres, asociado a la inactivación del cromosoma X, un patrón independiente para cerebelo, y un patrón de metilación de un conjunto de dianas característico de los estadíos 3 y 4 de Braak de la EA. Asimismo, se observaron diferencias significativas de metilación (1.112 CpGs, p<0,0l) en el cerebelo asociadas a la EA, confirmando su implicación en la enfermedad. El análisis del mosaicismo somático del cerebro se realizó empleando el "SurePrint G3 human CGH array 400K" (Agilent). Tomando como área de referencia el cerebelo se detectaron ganancias o pérdidas de material genómico entre áreas del cerebro de un mismo individuo. Dos muestras de corteza, pertenecientes a dos controles, presentaron una ganancia de material genómico en el gen WWOX, mientras que tan solo una muestra mostró una ganancia de material genómico en el gen ADAM5P3A. La elevada frecuencia de variantes en el número de copia en WWOX y su posible implicación en EA llevó a genotipar un mayor número de individuos, aunque ninguno mostró mosaicismo somático. El análisis del estado de metilación de las sondas ubicadas en WWOX permitió observar una disminución significativa de la metilación entre pacientes y controles en 14 sondas (T-student, p<0,05), sugiriendo que la regulación epigenética de WWOX puede estar alterada en la EA. En conjunto, estos resultados muestran la alteración de los perfiles de mutilación del SNC en relación con la EA tardía (estadíos 3 y 4 de Braak). Principalmente, en una de las regiones cuya afectación patológica en la EA ha sido más controvertida, cerebelo. Es especialmente interesante remarcar que la aparición de las lesiones características de cerebelo tienen lugar en estadíos más avanzados, indicando la posibilidad de que la alteraciones epigenéticas observadas podrían corresponder a un evento prematuro en la progresión de la patología. / Neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), represent a major issue of public health in developing countries where the aging of the population is leading to a progressive increase of its prevalence rates. Currently, several therapeutic strategies help to palliate clinical symptoms, but the neurodegeneration is progressive and irreversible. Identification of underlying mechanisms leading to these disorders is essential to improve patient's life expectancy and quality. In this context, many efforts have been focused on identifying genetics and environment causes of these disorders with little success, highlighting the need to evaluate new mechanisms and factors involved. The present thesis project has explored the implication of new mechanisms, such as DNA methylation and somatic mosaicism in AD and PD. The analysis of DNA methylation was performed with a new methylation array technology: 'HumanMethylation' (27K and 450K, IlIumina), whose probes strategically distributed along the human genome, enables to quantify the methylation state of around 27,000 and 450,000 CpG sites, respectively. The pattern of methylation of 60 subjects (28 AD, 3 PD and 29 unaffected) with four to seven brain regions (cortex, amygdala, hippocampus, hypothalamus, pons, substantia nigra and cerebellum) has been assessed. The study has shown three ma in clusters depending on gender (female/male), brain area (cerebellum vs others) and disease stage (AD3 vs AD4). In addition, a' differential analysis performed in individual CpG sites proved the presence of significant differences associated to AD patient's cerebellum (1112 CpG sites, p<0.01). Somatic mosaicism analysis has been carried out with a 'SurePrint G3 human CGH array 400K' (Agilent) to detect intra-individual genomic gains and losses compared to cerebellum. A total of two cortex samples showed a genomic gain in the WWOX gene, whereas only one sample showed a gain on ADAM5P3A. WWOX has been considered as a potential candidate gene in previous AD studies, and was further analyzed in a larger cohort of human brain samples. Genotyping assays did not confirm the presence of new somatic mosaicism cases, but it was possible to determine the genotype distribution and compared data between samples. A significant hypomethylation of the WWOX promoter region was observed in AD patients compared to controls subjects (T-test, p<0.05) in 14 probes, suggesting a potential regulation of expression by methylation. Overall, these results highlight the implication of epigenetic mechanisms in neurodegenerative disorders, as AD. In particular, it is remarkable the specific pattern of methylation in the cerebellum in intermediate stages of AD, suggesting an overlap with early modifications, which could contribute to unraveling new mechanisms implicated in AD.

<p>Environmental stress has shown to affect both the exposed individuals and the development of their offspring. Generally, it is thought that the stressed organism responds to stress by trying to adapt to it. This thesis investigates possible evolutionary consequences of cross-generational transmissions of stress, where the parent has been stressed but the offspring has not. In two studies we have exposed chicken parents of different breeds to an unpredictable circadian light rhythm, to investigate the influence of genetic background on the transmission of behaviour and patterns of genome-wide gene expression across generations. In Paper I, we can show that the domesticated chicken, by means of epigenetic factors, transmit their behaviours as well as their gene expression profiles to their offspring to a higher extent than their wild ancestor, the red junglefowl. Furthermore, in Paper II, even though the offspring never experienced the stress or had any contact with their stressed parents, they seemed to have adapted to it, which suggests that the parents might have prepared (or pre-adapted) them for living in the unpredictable environment. Additionally, eggs of stressed hens showed increased levels of estradiol that might have affected gene expression of specific immune genes, which were up-regulated in the offspring of stressed parents. It is possible that the traditional distinction between stress responses and evolutionary adaptation may be reevaluated, since our results indicate that they could be parts of the same evolutionary event.</p>

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