La truite arc-en-ciel carnivore (Oncorhynchus mykiss) est considérée comme une espèce pauvre utilisatrice de glucides alimentaires. Des études récentes ont montré qu'une hypométhylation globale de l'ADN hépatique induite par un régime alimentaire riche en glucides et pauvre en protéines pourrait être impliquée dans l'établissement / le maintien du de ce phénotype chez la truite, mais le détail des mécanismes sous-jacents reste inconnu. La thèse vise à étudier les mécanismes épigénétiques sous-jacents à ce phénotype de faible utilisation des glucides alimentaire chez la truite et à examiner si le métabolisme du glucose et l’épigénome chez les juvéniles peuvent être programmés par un stimulus hypoxique précoce. Nous avons d’abord identifié tous les gènes paralogues liés aux voies de méthylation / déméthylation de l’ADN (dnmt, tet et tdg) dans le génome de la truite, clarifié leurs histoires évolutives et analysé leurs profils d’expression au cours de la gamétogenèse et de l’embryogenèse chez la truite. Nous avons ensuite étudiés plus en détail les processus et les mécanismes potentiellement à l’origine de l'hypométhylation de l'ADN hépatique global constatée chez la truite après un régime riche en glucides et pauvre en protéines. Les résultats ont montré pour la première fois qu'une diminution du taux deprotéines et une augmentation du taux de glucides dans l’aliment induisent de manière indépendante et en interaction une hypométhylation hépatique globale chez la truite, qui semble établie par le biais d'une voie de déméthylation active. Nous avons également constaté qu’une forte hyperglycémie induite par une injection de glucose induit une hypométhylation globale de l’ADN au niveau des sites CmCGG dans le foie de la truite. Les mécanismes détaillés de ces processus de déméthylation restent à élucider. Enfin, grâce à la stratégie de programmation métabolique, nous avons pour la première fois confirmé que l’utilisation d’un stimulus non nutritionnel au début de la vie, l’hypoxie, pouvait moduler de façon persistante la transcription des gènes liés au métabolisme du glucose chez la truite juvénile sans nuire aux performances de croissance. De plus, selon sa nature chronique ou aigue, l’hypoxie, a tendance à induire des effets de programmation opposés sur les gènes codants pour les transporteurs au glucose notamment dans le foie et le muscle de la truite juvénile. Dans son ensemble, la thèse met en avant notre compréhension du rôle du méthylome dans la contribution à la faible capacité d'utilisation des glucides alimentaires chez la truiteLa thèse met aussi en lumière le potentiel d'utilisation de l'hypoxie comme stimulus pour programmer le métabolisme du glucose, l'épigénome et l'utilisation des glucides alimentaires chez la truite arc-en-ciel. / The carnivorous rainbow trout (Oncorhynchus mykiss) is considered as a poor user of dietary carbohydrates. Recent studies showed that a high-carbohydrate/low protein diet inducing hepatic global DNA hypomethylation could be involved in the establishment/maintenance of the poor dietary carbohydrates utilisation phenotype in trout, but the detail mechanisms remain unclear. The present thesis aimed at investigating the epigenetic mechanisms underlying this poor dietary carbohydrate utilisation phenotype in trout, and exploring if the glucose metabolism and the epigenome in juveniles can be programmed through a hypoxic stimulus during early life. We first identified all the paralogous genes related to DNA methylation/demethylation pathways (dnmt, tet and tdg) in trout genome, clarified their molecular evolution histories and monitored their transcriptional expression patterns during gametogenesis and embryogenesis in trout. Besides, we investigate further the causes, processes and potential mechanisms about the hepatic global DNA hypomethylation in trout after feeding a high carbohydrate/low protein diet. Results for the first time demonstrated that a decrease in protein content and an increase in carbohydrate content in the diet can independently as well as interactively induce hepatic global hypomethylation in trout. This global loss of methylation is probably established through an active demethylation pathway. We also found that a strong hyperglycaemia induced by glucose injection induces global CmCGG hypomethylation in the liver of trout. The detailed mechanisms of these demethylation processes remain to be elucidated. Finally, through metabolic programming strategy, we confirmed for the first time that using a non-nutritional stimulus, hypoxia, during early life stage persistently modulates the transcription of glucose metabolism-related genes in juvenile trout without negative effects on growth performance. Moreover, acute and chronic hypoxia tended to induce opposite programming effects on glucose-transporter encoding genes in both liver and muscle of juvenile trout. Together, the present thesis brings forward our understandings about the roles of epigenetics in contributing to the low ability to use dietary carbohydrates in trout, and sheds light on the potential of using hypoxia as the stimulus in metabolic programming strategy to tailor the glucose metabolism, the epigenome and dietary carbohydrate utilisation in rainbow trout.

Les travaux épidémiologiques et expérimentaux effectués à ce jour sur le cancer du sein ont montré que les oestrogènes - comme l’eostradiole (E2) - et leur récépteur (ER) - un facteur de transcription les liants - sont fortement impliqués dans au moins 70% des cas de cancer du sein. Cette implication est d’autant plus visible que les patients, suite à une thérapie anti-oestrogénique, ont tendance à développer une résistance endocrinienne au traitement. Pendant longtemps, l’ER a été étudié en tant que facteur indépendant liant directement une séquence ADN spécifique sur le génome. Aujourd’hui le paradigme a profondément changé. Il est bien connu que ER s’associe avec de nombreux autres facteurs de transcription et protéines régulant la chromatine afin de réguler l’expression des gènes. Cependant, nos connaissances concernant la fonction de modifications épigénétiques suite à l’activation de ER - notamment la méthylation de l’ADN et l’acétylation des histones - sont encore limitées. Dans cette étude, j’ai mis en place un protocole de culture cellulaire adapté à l’étude de la privation et à la re-stimulation d’E2 stricto sensu. Dans un premier temps, ce protocole a été évalué à l’aide de la toute dernière technologie de puce permettant la lecture du méthylome et couvrant la liste complète des éléments amplificateurs. Dans un deuxième temps, j’ai mesuré le transcriptome et les profiles d’acétylation de l’histone H3 (H3K27ac) afin de déterminer la capacité de ER à réguler l’expression des gènes J’ai découvert que, suite à la privation de E2, les niveaux de méthylation de l’ADN et de H3K27ac changent et que ces changements s’accentuent avec le temps, en particulier au niveau des éléments amplificateurs. Une analyse d’enrichissement des facteurs de transcription et des séquences de liaison spécifiques a révélé que les facteurs de transcriptions des familles AP-1 et FOX sont des intermédiaires favorisants la liaison de ER aux éléments amplificateurs. Finalement, la re-stimulation des cellules par de l’E2 a montré que la majorité des changements épigénétiques observé sont réversibles mais que certains éléments amplificateurs restent hyperméthylés et déacétylés. Ceci pourrait indiquer que les traitements anti-oestrogéniques sont efficaces mais pourrait également indiquer un marqueur de résistance endocrinienne. Cette étude apporte des informations nouvelles quant aux effets de l’inhibition et l’activation de ER sur la méthylation de l’ADN et l’acétylation de l’histone H3 à l’échelle du génome et renforce l’importance du rôle d’autres facteurs au niveau des amplificateurs / Previous epidemiological and experimental studies have strongly implicated estrogens in breast cancer risk and Estrogen Receptor (ER), the transcription factor to which estrogen binds, is considered as the major molecular driver of around 70% breast cancers. The importance of the deregulated estrogen signalling is further highlighted by increasing evidence that current chemopreventive and therapeutic strategies that target hormonally responsive breast cancers frequently result in the development of resistance to anti-estrogens and metastatic progression, highlighting the need for understanding the molecular underlying mechanisms. While until recently, ER was believed to act as a stand-alone transcription factor, which can directly bind its motifs in DNA, it is now accepted that ER activity is a complex and dynamic process that requires highly concerted actions of a dozen transcriptional cofactors and various chromatin regulators at DNA. Recent studies focused on characterising ER-associated cofactors and their role in opening the chromatin provided a remarkable insight into transcriptional regulation mediated by ER. However DNA methylation and histone acetylation are poorly understood in the context of ER’s dynamic binding. In this thesis, I combined a cell culture protocol adapted for studying estradiol (E2) deprivation and re-stimulation in stricto sensu in ER-positive breast cancer cells with the latest methylation array, that allowed a genome-wide interrogation of DNA methylation (including a comprehensive panel of enhancers). I further investigated histone acetylation (ChIP-seq) and transcriptome (RNA-seq) after E2 deprivation and re-stimulation to better characterise the ability of ER to coordinate gene regulation. I found that E2 deprivation and re-stimulation result in time-dependent DNA methylation changes and in histone acetylation across diverse genomic regions, many of which overlap with enhancers. Further enrichment analysis of transcription factor (TF) binding and motif occurrence highlights the importance of ER tethering mainly through two partner TF families, AP-1 and FOX, in the proximity of enhancers that are differentially methylated and acetylated. This is the first study that comprehensively characterized DNA methylation at enhancers in response to inhibition and activation of ER signalling. The transcriptome and genome occupancy data further reinforced the notion that ER activity may orchestrate a broad transcriptional programme through regulating a limited panel of critical enhancers. Finally, the E2 re-stimulation experiments revealed that although the majority of the observed epigenetic changes induced by E2 deprivation could be largely reversed when the cells were re-stimulated we show that DNA hypermethylation and H3K27 acetylation at enhancers as well as several gene expression changes are selectively retained. The partial reversibility can be interpreted as a sign of treatment efficiency but also as a mechanism by which ER activity may contribute to endocrine resistance. This study provides entirely new information that constitutes a major advance in our understanding of the events by which ER and its cofactors mediate changes in DNA methylation and chromatin states at enhancers. These findings should open new avenues for studying role of the deregulated estrogen signalling in the mechanism underlying the “roots” of endocrine resistance that commonly develops in response to anti-estrogen therapy

La méthylation de l'ADN est un processus vital pour le développement des mammifères. Sa distribution anormale,notamment au niveau des régions répétées du génome, est une signature pathologique. La découverte de maladies héréditaires touchant la stabilité du génome a permis des avancées considérables dans l'identification des acteurs et des mécanismes. Nous avons choisi d'étudier le syndrome ICF (Immunodéficience, instabilité Centromérique et anomalies Faciales), première maladie génétique identifiée avec des défauts de la méthylation de l’ADN, liés à une instabilité chromosomique. Lorsque j'ai commencé ma thèse, des mutations dans les gènes DNMT3B et ZBTB24 avaient été décrites comme causes génétiques du syndrome. Cependant, d'autres causes génétiques restaient inconnues. Nos travaux ont permis d'identifier deux nouveaux gènes, CDCA7 et HELLS, dont les mutations sont responsables du syndrome. J'ai montré que leur perte de fonction dans les cellules somatiques entraîne un défaut de méthylation des répétitions centromériques, suggérant leur rôle dans le maintien de la méthylation de l'ADN. Par conséquent, l'étude de l'étiologie d'une maladie génétique rare a permis d'identifier de nouveaux « gardiens » de la stabilité du génome, avec des fonctions jusqu'alors insoupçonnées dans les processus de méthylation de l'ADN et dans le développement. Au cours de mon doctorat, j'ai établi des cartes de méthylation des cellules de patients ICF afin d'identifier les cibles communes et distinctes de ces facteurs, ainsi que leurs caractéristiques génomiques et épigénomiques. Contrairement aux mutations de DNMT3B,celles de ZBTB24, CDCA7 et HELLS affectent la méthylation dans des régions pauvres en CpG, dans des régions intergéniques et dans des répétitions d'ADN intercalées. Plus généralement, ce sont les régions d'hétérochromatine qui sont les plus touchées et en particulier des clusters des gènes codants et non codants, dont certains sont exprimés de manière monoallélique. Pour mieux caractériser le rôle de ZBTB24 dans le développement et la méthylation de l'ADN,nous avons généré un modèle murin mutant qui nous a permis de monter que ZBTB24 était essentielle pour le développement embryonnaire précoce. De plus, ZBTB24 jouerait un rôle dans l'établissement de la méthylation des séquences répétées de l'ADN, à la fois en tandem ou intercalé. Fait intéressant, ZBTB24 semble être également impliqué dans l'établissement de la marque répressive H3K9me3, suggérant un rôle de la protéine dans le "dialogue" entre la méthylation de l'ADN et celle des histones. Dans l'ensemble, mon travail met l'accent sur la façon dont la méthylation de l'ADN et les marques d'hétérochromatine sont établies et maintenues à des gènes uniques et des répétitions de l'ADN, et fournit de nouveaux acteurs et mécanismes à considérer dans les études sur le maintien de la stabilité du génome. / DNA methylation is an essential process for the development of mammals. Its abnormal distribution, particularly at the level of the repeated regions of the genome, is a pathological signature. The discovery of hereditary diseases affecting DNA methylation and the stability of the genome allowed a considerable progress in the identification of their actors and mechanisms. We chose to study the ICF (Immunodeficiency, Centromeric Instability and Facial Abnormalities) syndrome, the first genetic disorder identified with defects in the distribution of DNA methylation, linked to chromosomal instability. When I started my PhD, mutations in two genes had been described to cause the ICF syndrome: DNMT3B and ZBTB24. However, the genetic origin of a subset of ICF patients remained unknown. We identified mutations in CDCA7 and HELLS as causative of the ICF syndrome. I showed that their loss of function in somatic cells results in the loss of DNA methylation at centromeric repeats, strongly suggestive of a role DNA methylation maintenance. Hence, the study of the aetiology of a genetic disease provided new candidate “guardians” of DNA repeats and genome stability, with virtually unknown functions but with exciting potential roles in the DNA methylation machinery and in development. During my PhD, I established methylation maps in ICF patients cells to identify common and distinct targets of these factors, as well as their genomic and epigenomic characteristics. In contrast to DNMT3B mutations, those in ZBTB24, CDCA7 and HELLS affect methylation at CpG-poor regions in intergenic genomic locations and at interspersed DNA repeats, and more generally, at genomic locations with heterochromatic features. Their integrity is required for the methylated status of coding and non-coding clusters of genes, some of which are expressed in a monoallelic manner. To better characterize the role of ZBTB24 in development and DNA methylation pathways, we generated a mouse model carrying mutations in ZBTB24. We showed that ZBTB24 is essential for early development, while it seemed to be dispensable for in vitro differentiation of murine ES cells. We implicated ZBTB24 in the establishment of DNA methylation at DNA repeats, both in tandem or interspersed, in differentiating ES cells. Interestingly, ZBTB24 seems to be also implicated in the establishment of the repressive mark H3K9me3 suggesting that ZBTB24 may indirectly control DNA methylation through an interplay with histone marks. As a whole, our work sheds light on how DNA methylation and heterochromatin marks are established and maintained at unique genes and DNA repeats, and provides new actors and mechanisms to consider in studies of the maintenance of genome stability.

DNA damage can cause genome instability, which may lead to human cancer. The most common form of DNA damage is DNA base damage, which is efficiently repaired by DNA base excision repair (BER). Thus BER is the major DNA repair pathway that maintains the stability of the genome. On the other hand, BER mediates DNA demethylation that can occur on the promoter region of important tumor suppressor genes such as Breast Cancer 1 (BRCA1) gene that is also involved in prevention and development of cancer. In this study, employing cell-based and in vitro biochemical approaches along with bisulfite DNA sequencing, we initially discovered that an oxidized nucleotide, 5’,2-cyclo-2-deoxyadenosine in DNA duplex can either cause misinsertion by DNA polymerase β (pol β) during pol β-mediated BER or inhibit lesion bypass of pol β resulting in DNA strand breaks. We then explored how a T/G mismatch resulting from active DNA demethylation can affect genome integrity during BER and found that pol β can extend the mismatched T to cause mutation. We found that AP endonuclease 1 (APE1) can use its 3'-5' exonuclease to remove the mismatched T before pol β can extend the nucleotide preventing a C to T mutation. The results demonstrate that the 3'-5' exonuclease activity of APE1 can serve as a proofreader for pol β to prevent mutation. We further explored the effects of exposure of environmental toxicants, bromate and chromate on the DNA methylation pattern on the promoter region of BRCA1 gene with bisulfite DNA sequencing. We found that bromate and chromate induced demethylation of 5-methylcytosines (5mC) at the CpG sites as well as created additional methylation at several unmethylated CpG sites at BRCA1 gene in human embryonic kidney (HEK) 293 cells. We further demonstrated that the demethylation was mediated by pol β nucleotide misinsertion and an interaction between pol β and DNA methyltransferase 1 (DNMT1) suggesting a cross-talk between BER and DNA methyltransferases. We suggest that DNA base damage and BER govern the interactions among the environment, the genome and epigenome, modulating the stability of the genome and epigenome and disease development.

In forensic sciences, the serological methods used to determine which body fluid was collected from the crime scene are merely presumptive or labor intensive since they rely on protein detection or on microscopic identification of cells. Given that certain forensic cases may need the precise identification of a body fluid to determine criminal contact, such is the example of a suspected sexual assault of a minor; certainty in the body fluid of origin may depict a precise picture of the events. The identification of loci that show differences in methylation according to the tissue of origin can aid forensic analysts in determining the origin of a DNA sample. The process of DNA methylation occurs naturally in the genome of living organisms and consists in the presence of a methyl group on the carbon 5 of a cytosine, which is typically followed by a guanine (CpG). Analyzing patterns of DNA methylation in body fluids collected from a crime scene is preferential to the analysis of proteins or mRNA since the same extracted DNA used for STR typing can be used for DNA methylation analysis. We have validated and identified loci able to discriminate blood, saliva, semen and vaginal epithelia. In the current study, we have also established the minimum amount of DNA able to provide reliable results using methodologies such as pyrosequencing and high-resolution melt (HRM) analysis for the different markers identified. Lastly, we performed an alternative bioinformatic analysis of data collected using an array that studied methylation in over 450,000 individual cytosines on the human genome. We were able to sort the locations that showed potentially higher methylation differences between body fluids and investigated over 100 of them using HRM analysis. The results of that study, allowed the identification of three new loci able to distinguish blood and two new loci able to distinguish saliva and vaginal epithelia, respectively. The use of DNA methylation patterns to aid forensic investigations started with a publication in 2010, therefore each small contribution such as this work may, similarly to what occured in the biochemistry field, result in the discovery of a method able to put the technology in the hands of forensic analysts.

Les cellules souches pluripotentes sont capables de donner naissance à tous les types cellulaires constituant un organisme, ce qui leur confère un fort intérêt thérapeutique. A partir de l’embryon de souris on peut en dériver deux types : les cellules souches embryonnaires (ES) au stade blastocyste et les cellules souches d’épiblaste (EpiSC) au stade œuf cylindre. Ces deux types de cellules partagent leurs propriétés pluripotentes mais se distinguent par de nombreux aspects comme leurs conditions de culture et les gènes qu’elles expriment. Nous avons montré que la reprogrammation par clonage par transfert de noyau permet d’obtenir des EpiSC présentant un méthylome et un transcriptome similaires à ceux des EpiSC issues d’embryons après fécondation. Nous avons également caractérisé le profil de méthylation de l’ADN des EpiSC, et montré une tendance à l’hyperméthylation des promoteurs des EpiSC par-rapport aux cellules ES et à l’épiblaste. De plus, l’absence de méthylation empêche la conversion des cellules ES en EpiSC. Les EpiSC semblent donc dépendre fortement de la méthylation de l’ADN pour réguler l’expression de leurs gènes, ce qui les distingue des cellules ES. / Pluripotent stem cells are of great therapeutic interest because of their capability to give rise to all the cells composing an organism. We can derive two types of these stem cells from the mouse embryo: embryonic stem cells (ESCs) from the blastocyst and epiblast stem cells (EpiSCs) from the egg cylinder stage. These two cell types share their pluripotent properties but are distinct on several features, like their culture conditions and gene expression. We showed that reprogramming using cloning by nuclear transfer allows the obtention of EpiSCs with a methylome and a transcriptome similar to those of EpiSCs derived from embryo after fertilisation. We also characterised the DNA methylation pattern of EpiSCs and showed their tendency to present a hypermethylation at their promoters compared to ESCs and epiblast. We also observed that the absence of DNA methylation blocks the conversion of ESCs into EpiSCs. As a conclusion, it seems that EpiSCs are strongly dependant of DNA methylation to regulate gene expression, which distinguishes them from ESCs.

UHRF1 est une protéine nucléaire responsable du maintien et de la régulation de l'épigénome des cellules. Elle favorise la prolifération cellulaire et est surexprimée dans la plupart des cancers. TIP60, l'un des partenaires le plus important d’UHRF1, est impliqué dans le remodelage de la chromatine et la régulation transcriptionnelle grâce à son activité acétyltransférase. Ensemble, les deux protéines régulent la stabilité et l'activité d'autres protéines telles que la DNMT1 et la p53. Le but de cette étude était d'explorer le mécanisme d'interaction entre UHRF1 et TIP60 en visualisant cette interaction dans les cellules. La microscopie par imagerie à temps de vie de fluorescence et d'autres techniques de biologie moléculaire ont été utilisées. Les résultats ont montré que UHRF1 interagit directement avec le domaine MYST de TIP60 et cette interaction se produit dans la phase S du cycle cellulaire. Les deux protéines ont également montré une réponse similaire aux dommages à l'ADN, ce qui prédit une cohérence dans leur fonction dans le mécanisme de réparation de l'ADN. La surexpression de TIP60 a également induit la baisse du niveau d’UHRF1 et de DNMT1 ainsi qu’une induction d'apoptose dans les cellules ce qui suggère un rôle de TIP60 dans la régulation des fonctions oncogéniques d’UHRF1. / UHRF1 is a nuclear protein maintaining and regulating the epigenome of cells. Its promotes proliferation and is found upregulated in most of cancers. TIP60 is one of the important interacting partner of UHRF1 and is involved in chromatin remodeling and transcriptional regulation through its acetyltransferase activity. Together they regulate the stability and activity of other proteins such as DNMT1 and p53. The aim of this thesis was to explore the mechanism of interaction between UHRF1 and TIP60 by visualizing this interaction in cells. Fluorescent lifetime imaging microscopy and other molecular biology techniques were employed for this purpose. Results of this study showed that UHRF1 interacts directly to the MYST domain of TIP60 and this interaction prevails in the S-phase of cell cycle. Both proteins also showed a similar response to DNA damage predicting a coherence in their function in DNA repair mechanism. Overexpression of TIP60 also downregulated UHRF1 and DNMT1 and induced apoptosis in cells suggesting a role of TIP60 in regulation of oncogenic functions of UHRF1.

Les océans couvrent plus de 70% de la surface de la Terre (planète bleue) et la productivité primaire nette (PPN) marine est équivalente à celle terrestre. Alors qu‟il ne représente que 1% de la biomasse totale d‟organismes photosynthétiques de la planète, le phytoplancton est responsable d‟environ 45% de la PPN globale. Le terme phytoplancton décrit un assemblage polyphylétique comprenant des eucaryotes et procaryotes photosynthétiques dérivant avec les courants. Dans les océans contemporains, les diatomées constituent un groupe d‟eucaryotes unicellulaires autotrophes très abondant, responsable de 40% de la PPN marine. Les diatomées appartiennent à la lignée des straménopiles qui sont issus d‟un évènement d‟endosymbiose entre une algue rouge et un hôte hétérotrophe. Elles sont classifiées en deux groupes majeurs : les centriques qui son apparues il y a environ 200 millions d‟années (Ma), et les pennées qui ont évolué il y a environ 90 Ma. Deux génomes de diatomées ont récemment été séquencés : celui de la diatomée centrique Thalassiosira pseudonana (32 Mb), et celui de la diatomée pennée Phaeodactylum tricornutum (27 Mb). Mon sujet de doctorat s‟est focalisé sur l‟étude de différents aspects de la régulation de l‟expression génique ainsi que sur la dynamique et l‟évolution de ces génomes. L‟expression des gènes est régulée à différents niveaux: trancriptionel, post-transcriptionel, et épigénétique. Dans le cadre de mon doctorat, une étude de la régulation transcriptionelle chez les diatomées a été effectuée et comprend l‟identification et l‟analyse in silico des facteurs de transcription (FT). Cela a permis par exemple d‟établir qu‟une classe spécifique de FT, les Heat Shock Factors, sont particulièrement abondants chez les diatomées par rapport aux autres eucaryotes. L‟analyse de la représentation des FT identifiés dans différentes librairies d‟EST élaborées à partir de cultures ayant subi divers stress a permis de détecter certaines spécificités d‟expression. L‟évolution des génomes eucaryotes est largement impactée par les effets directs et secondaires des éléments transposables (ET) qui sont des éléments génétiques mobiles se trouvant dans le génome de la plupart des organismes. Dans le but d‟étudier la dynamique des génomes de diatomées, la recherche de différents types d‟ET a permis d‟établir qu‟une certaine classe, les rétrotransposons de type Copia, est la plus abondante dans ces génomes et constitue un part significativement plus importante du génome de P. tricornutum (5,8%) par rapport à T. pseudonana (1%). D‟autre part, des analyses phylogénitiques ont montré que les rétrotransposons de type copia forment deux classes distinctes et éloignées de la lignée Copia. L‟analyse de leurs niveaux d‟expression a montré que la transcription de deux éléments s‟active en réponse à des stress spécifiques comme la limitation en nitrate dans le milieu de culture. Cette activation est accompagnée par un hypométhylation de l‟ADN et l‟analyse de profils d‟insertions chez différents écotypes de P. tricornutum ainsi que l‟étude d‟autres phénomènes suggèrent que les rétrotransposons de type Copia ont joué un rôle important dans l‟évolution des diatomées. Mon grand intérêt pour les ET m‟a ensuite amené à chercher à les caractériser dans d‟autres génomes récemment séquencés tels celui de l‟algue brune Ectocarpus siliculosus. La recherche in silico de différents gènes codant des protéines capables d‟introduire ou de stabiliser des états épigénétiques telle que la modification des histones et la méthylation de l‟ADN a montré leur présence chez P. tricornutum ainsi que leurs particularités. La présence de certaines modifications d‟histones spécifiques d‟une conformation compacte ou ouverte de la chromatine dans le proteome de P. tricornutum a été montrée. De plus, la mise au point de la technique d‟immunoprécipitation de la chromatine chez P. tricornutum a permis d‟établir que les nucléosomes enrobés d‟éléments transposables étaient marqués par des modifications spécifiques. D‟autres expériences ont permis d‟établir que l‟ADN de différents types d‟éléments transposables est marqué par la méthylation de cytosines chez P. tricornutum. Une expérience permettant l‟analyse du profil de méthylation à l‟échelle de génome en utilisant une puce à ADN a été lancée et permettra de découvrir si certains gènes portent aussi des traces de méthylation. Enfin, les ARN interférents constituent un troisième mode de régulation de l‟expression se situant à l‟interface de la régulation transcriptionelle, post-transcriptionelle et épigénétique. Les mécanismes d‟interférences chez les diatomées ont été étudié par la recherche in silico d‟enzymes clés impliquées dans ce processus ainsi qu‟en établissant expérimentalement un lien direct avec la méthylation de l‟ADN.

The imprinting status of the SLC22A18AS gene, located in the human chromosome segment 11p15.5, was studied using PCR-SSCP analysis and fetal tissues from a battery of 17 abortuses. This gene showed monoallelic expression (genomic imprinting) in different tissues from two abortuses which were heterozygous for an SNP (c.473G>A) in its coding region. This gene was found to be paternally imprinted (maternally expressed) in five tissues namely lung, liver, brain, kidney and placenta from an abortus. The parental origin of the expressed allele could not be determined in the second abortus as both the mother and the abortus were heterozygous for the SNP. Since paternal blood samples from none of the 17 abortuses could be collected for DNA isolation, the mother's genotype was used to find the origin of the expressed allele. In order to understand the mechanism underlying imprinting of this gene, it was important to understand the nature of the epigenetic marks (imprints) on the two alleles of this gene. Since these epigenetic marks are generally observed in promoters or CpG islands associated with the imprinted genes, the promoters of the SLC22A18AS gene was characterized using transient transfection of putative SLC22A18AS promoter fragments cloned in the pGL3-Basic vector in human cells followed by luciferase reporter assay. Since the promoter of a gene lies upstream to the transcription start site (TSS), TSS of this gene was mapped. In silico approach revealed an EST (CB129046) which had an additional 39 bases upstream to the known mRNA sequence. TSS was then identified by the 5’ primer extension analysis. TSS was found to be 166 bases upstream to the 5’ end of this EST. In order to select cell lines for transient transfection of putative promoter constructs for promoter charaterization, RT-PCR analysis was used to see the expression of this gene in the following available cell lines in the lab: HuH7, HepG2, A549, HeLa, LNCaP and PC3. This gene was found to be maximally expressed in HepG2 cells. Expression of this gene was also observed in A549, HeLa, LNCaP and PC3 cells. HuH7, on the other hand, did not show any detectable expression of this gene. Based on the above data, HepG2 and A549 cells were selected for promoter characterization. Seven putative promoter constructs were transiently transfected in these cells and the promoter activity of different constructs was measured by luciferase assay. The assay identified two promoters for the gene: P1 promoter in a region from -855 to -254 bp and P2 promoter in a region from -1441 to -855. In order to see the presence of putative transcription factor binding sites in the upstream region of the gene, the MatInspector Professional program was used. The gene was found to be devoid of TATA and CCAAT boxes. Most of the putative transcription factor binding sites were present in a region from -855 to -254 bp which spans the P1 promoter, including a binding site for the Sp1 transcription factor. In order to see if Sp1 binds to the promoter of this gene, ChIP assay was performed. Sp1 was shown to bind the region harboring the P1 promoter. In order to see if Sp1 has a role in the regulation of this gene, Sp1 constructs were co-transfected with the SLC22A18AS P1 promoter construct in HepG2 and Sp1-null Drosophila SL2 cells. The results showed that the Sp1 has a positive regulation on the SLC22A18AS promoter activity. As stated earlier, epigenetic marks such as differential methylation of CpG dinucleotides in two alleles are associated with promoters of the genes. Since the promoters for SLC22A18AS were characterized, the presence of allele-specific differentially methylated regions (DMRs) associated with the promoters was investigated. In order to differentiate the two alleles in the promoter regions by SNPs, DNA sequence analysis of the promoter regions was performed in a battery of 17 abortuses to search for SNPs. Abortus no. 3 showed heterozygosity for a C to A change at nucleotide position -445 in the P1 promoter region, while abortus no. 2 showed heterozygosities for G to A and A to G changes at nucleotide positions -919 and -1321 respectively in the P2 promoter region. The alleles in the abortus no. 3 were designated as allele C and allele A. The alleles in abortus no. 2 were designated as allele GG and allele AA. Once the two alleles were differentiated by these SNPs, identification of DMRs was performed using sodium bisulfite genomic DNA sequencing. Genomic DNA from the abortus no. 3 was taken for the identification of DMR in the P1 promoter region, while genomic DNA from abortus no. 2 was taken for the identification of DMR in the P2 promoter region. Sodium bisulfite genomic DNA sequencing of the P1 promoter region showed heavy methylation of both the alleles. No DMR was observed in this region. Sodium bisulfite genomic DNA sequencing of the P2 promoter region using DNA from abortus no. 2 did not show any differential methylation of the two alleles. However, like the P1 promoter region, the P2 promoter region was also heavily methylated. In order to see the methylation status of both the promoter regions in human sperms, sperm DNA from an unrelated healthy volunteer was also subjected to sodium bisulfite genomic sequencing. A dense methylation was observed in both the promoter regions of the gene. Heavy methylation of CpG dinucleotides in these regions corroborates the imprinting result for this gene. Since the methylation epigenetic mark is also known to be associated with CpG islands, CpG Plot/CpG Report analysis was used to identify CpG islands in this gene. The analysis showed the presence of two CpG islands, CpG I and CpG II, in the second intron of the gene. As the CpG I island is known to lack methylated CpGs (Ali et al., unpublished result from our lab), a DMR was sought for the CpG II island region. Heterozygosity was ascertained in this region by sequencing DNA from 17 abortuses. However, none of the abortuses showed heterozygosity. It was reasoned that if there is a differential methylation of the two alleles in this region, half of the clones (alleles) should be unmethylated, and the other half should show methylation. Therefore, DNA from abortus no. 3 was randomly chosen for sodium bisulfite genomic sequence anaylsis to identify DMR. The CpG II island showed heavy methylation. However, a DMR was not identified. In order to see the methylation status of the CpG II island in human sperms, sperm DNA from an unrelated healthy volunteer was also subjected to sodium bisulfite genomic sequencing. Almost all the CpG sites showed methylation. The observation of a dense methylation of both the promoters and CpG II island suggested that methylation has a role in the expression of this gene. In order to confirm this observation, A549 and HuH7 cells were treated with a methyltransferase inhibitor, 5-aza-2’-deoxycytidine. 5-Aza-2’-deoxycytidine treatment in HuH7 cells restored the expression of this gene. Further, the expression of this gene was increased in A549 cells following the drug treatment. These results suggested that DNA methylation has a definite role in the modulation of expression of the SLC22A18AS gene. Histone acetylation is another key epigenetic player which is known to have a role in the expression of genes. In order to study the role of the histone acetylation, HuH7 and A549 cells were treated with TSA, a histone deacetylase inhibitor. Treatment of HuH7 and A549 cells with TSA didn’t have any effect on the expression of this gene. On the other hand, the expression of TPA, a gene shown to be regulated by TSA earlier, increased following the TSA treatment in both cell lines. These results suggested that histone acetylation doesn’t have any effect on the expression of this gene. Based on this observation, it was reasoned that histone acetylation is not associated with the imprinting of this gene. Therefore, we did not look for the allele-specific acetylation of histones in this gene. The SLC22A18AS gene has a weak ORF of 253 amino acids as the translation intiation site does not contain a consensus Kozak sequence for efficient translation. In order to determine if it codes for a protein, Western blot analysis was performed using lysates from A549 cells and human fetal liver tissue, and a polyclonal antibody raised in a rabbit against a bacterially expressed SLC22A18AS protein fragment from amino acids 138 to 245. The Western blot result was negative. It was reasoned that this gene might be expressed at a low level and therefore its expression could not be detected by Western blot analysis. Immunoprecipitation was then performed to enrich the SLC22A18AS protein in the lysates followed by Western blot analysis. SLC22A18AS was shown to be expressed as a 30 kDa band in the immunoprecipiates from A549 cell and human fetal liver tissue lysates. The subcellular localization of this gene was studied by immunofluorescence. The fluorescence immunolocalization was performed on A549 cells with anti-SLC22A18AS antibody. The SLC22A18AS protein was found to be localized in the cytoplasm of A549 cells.

Eukaryotic genomes are packed into the confines of the nucleus through a nucleoproteic structure called chromatin. Chromatin is a dynamic structure that can respond to developmental or environmental cues to regulate and orchestrate the functions of the genome. The fundamental unit of chromatin, the nucleosome, consists of a protein octamer, which contains two molecules of each of the core histone proteins (H2A, H2B, H3, H4), around which 147 bp of DNA is wrapped. The post-translational modifications (PTMs) of histones and methylation of the cytosine residues in DNA (DNA methylation) constitute primary epigenomic markers that dynamically alter the interaction of DNA with nucleosomes and participate in the regulation and control access to the underlying DNA. The main objective of my thesis was to understand the spatial and temporal dynamics of chromatin states in Arabidopsis by investigating on a genome-wide scale, patterns of DNA methylation and a set of well-characterized histone post-translational modifications. DNA methylation, a hallmark of epigenetic inactivation and heterochromatin in both plants and mammals, is largely confined to transposable elements and other repeat sequences. I show in this thesis that in Arabidopsis, methylated TE sequences having no or few matching siRNAs, and therefore unlikely to be targeted by the RNA-directed DNA methylation (RdDM) machinery, acquire DNA methylation through spreading from adjacent siRNA-targeted regions. Further, I propose that this spreading of DNA methylation through promoter regions can explain, at least in part, the negative impact of siRNA-targeted TE sequences on neighbouring gene expression. In a second part, I have contributed to integrative analysis of DNA methylation and eleven histone PTMs. I have shown through combinatorial and cluster analysis that the Arabidopsis epigenome shows simple principles of organisation and can be distinguished into four primary types of chromatin that preferentially index active genes, repressed genes, TEs, and intergenic regions. Finally, in a third part, I integrated epigenomics with transcriptome data at three different time points in a developmental window to investigate the temporal dynamics of chromatin states in response to an external stimulus. This used the light-induced transcriptional response as a paradigm to assess the impact of histone H2B monoubiquitination (H2Bub), and showed that this PTM is associated with active transcription and implicated in the selective fine-tuning of gene expression. Taken together, the work presented here contributes significantly to our understanding of the spatial organisation of chromatin states in plants, its dynamic nature and how it can contribute to allow plants to respond to a signal from the environment.