Genomic imprinting marks a subset of autosomal loci expressed in parent of origin-dependent monoallelic expression in a non-Mendelian fashion. To restore totipotency and to reset the imprint according to the sex of the individual, the mark must be erased during germline development. The imprinted Igf2-H19 loci located distally on chromosome 7 in mouse and 11p15.5 in human, share common regulatory elements that regulate differential expression. Where the H19 is silenced when paternally inherited, the Igf2 is silenced when maternally inherited. The differentially methylated 5'-flank of H19 gene, termed imprinting control region (ICR), shown to display a unique chromatin organisation harbours hypersensitive sites in linker regions flanked by positioned nucleosomes on the maternal allele. This unique chromatin conformation functions as a methylation-sensitive and unidirectional chromatin insulator, which later was found to depend on the chromatin insulator protein CTCF. The H19 ICR exhibits default-silencing functions in promoter-proximal positions. The maximal distance between the H19 ICR and the promoter of the reporter gene required for this effect was 1.2 ± 0.3kb which can be compared to the 1.9 kb distance between the endogenous H19 ICR and H19 promoter. Results suggest that the H19 ICR adopts a chromatin conformation that must be separated by a minimal distance from pivotal cis-regulatory elements to avoid adverse effects on neighbouring promoters. Poly(ADP-ribosy)lation represents a novel post-translational epigenetic mark that segregates with exclusively the maternal derived H19 ICR and associated with factors that interact with the CTCF target sites. CTCF is itself poly(ADP-ribosy)lated and the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide relieves the insulator function of the H19 ICR. Designed zinc finger proteins were applied to examine if epigenetic marks provided an obstacle for targeted activation and silencing. The zinc finger protein ZFP809 with activator/repressor domain able to efficiently activate/silence the IGF2 target. Murine hybrid cell lines of human chromosome 11, demonstrated that the ZFP809 overcame the epigenetic marks that repressed maternal IGF2 and paternal H19 allele, respectively. Results suggested that imprinted genes are not normally exposed to strong cis-regulatory elements and that the designed ZFPs can be exploited to develop a therapeutic method for rectifying epigenetic lesions.

An overall coordination between the expressions of genes is required for the proper development of an individual. Although most genes are expressed from both the constituent alleles of the genome, a small subset of autosomal genes are preferentially expressed from only one of the parental alleles, a phenomenon known as genomic imprinting. The imprinted H19 and Igf2 genes are considered paradigms of genomic imprinting as their monoallelic expression pattern is coordinated by a short stretch of sequence located upstream of H19, known as the imprinting control region (ICR). This region shows differential methylation, with hypermethylation specifically on the paternal allele. On the maternal allele this region acts as an insulator and harbours maternal specific hypersensitive sites. The hypersensitive sites were identified as the result of association of the vertebrate insulator protein CTCF with the region. This association was investigated in both an in vitro episomal system and in an in vivo mouse model system by mutating the CTCF target sites at the H19 ICR. The importance of CTCF for the insulator property of the region was confirmed in both instances. In the mouse model, the disruption of the binding was also observed to affect the methylation profile of the ICR, which ultimately resulted in the de-repression of the maternal Igf2 allele. The relevance of multiple CTCF target sites in higher vertebrates for the proper insulator function was investigated using another knock-in mouse model with mutation at a single CTCF target site in the H19 ICR. The investigation confirmed the cooperation between the target sites for the establishment of a functional insulator on the maternal allele. Target sites in the ICR were also analysed for their differential binding affinity for the CTCF protein. The utilisation of the CTCF target sites was examined in different human tumours and cell lines. Methylation analysis conveyed a lack of correlation between the loss of insulator function and methylation status of the ICR with the loss of imprinting (LOI) of IGF2. Investigations also identified a novel mechanism, which neutralised the chromatin insulator function of the H19 ICR without affecting its chromatin conformation. This principle might also help in explaining the loss of IGF2 imprinting observed in some instances. In conclusion, this thesis confirms the importance of CTCF in the formation of an epigenetically regulated chromatin insulator at the ICR, which in turn controls the expression pattern of H19 and Igf2. The studies also confirm the role of CTCF in the maintenance of the methylation profile of the region. Investigations into the loss of IGF2 imprinting in human cancer indicate the involvement of other novel mechanisms besides CTCF in the regulation of insulator function.

The development of a new individual from the fertilized oocyte can ultimately be seen as the consequence of the establishment and maintenance of specific patterns of gene expression. Although regulation of gene activity occurs at different levels, cellular specialization and differentiation are the results of developmental cues that essentially take place at the transcriptional level. The involvement of epigenetics in this process has become increasingly clear during the last decade. Imprinted genes constitute an excellent example as monoallelic expression seems to reflect differential epigenetic marks on the two alleles. This is the case of the imprinted H19 and Igf2 genes were the monoallelic expression is coordinated through a differentially methylated region (hypermethylated on the paternal allele), known as ICR (imprinted control region). In the mouse the ICR harbours four binding sites for the methylation sensitive insulator protein CTCF. Previous studies with episomal constructs had shown that this region behaved as an insulator and that CTCF is required for the insulator activity of the H19 ICR This thesis establish a clear link between the insulator function and the chromatin structure at the H19 ICR and indicates that the precise allocation of the CTCF target sites in the linker regions can play a critical role in this process. The importance of the CTCF interaction at the ICR was also confirmed in vivo using a mouse model that showed how intact CTCF target sites are needed to manifest insulator activity and methylation protection. We have investigated the role of CTCF and a related protein BORIS in establishing the maternal to paternal imprint transition in chromatin structure at the H19/Igf2 locus in the male germline. This thesis also describe the development of a new technique for the localization of chromatin associated factors and modifications with higher sensitivity and resolution compared to existing approaches.

The genetic information encoded by the DNA sequence, can be expressed in different ways. Genomic imprinting is an epigenetic phenomenon that results in monoallelic expression of imprinted genes in a parent of origin-dependent manner. Imprinted genes are frequently found in clusters and can share common regulatory elements. Most of the imprinted genes are regulated by Imprinting Control Regions (ICRs). H19/Igf2 region is a well known imprinted cluster, which is regulated by insulator function of ICR located upstream of the H19 gene. It has been proposed that the epigenetic control of the insulator function of H19 ICR involves organization of higher order chromatin interactions. In this study we have investigated the role of post-translational modification in regulating insulator protein CTCF (CCCTC-binding factor). The results indicated novel links between poly(ADP-ribosyl)ation and CTCF, which are essential for regulating insulators function. We also studied the higher order chromatin conformation of Igf2/H19 region. The results indicated there are different chromatin structures on the parental alleles. We identified CTCF-dependent loop on the maternal allele which is different from the paternal chromatin and is essential for proper imprinting of Igf2 and H19 genes. The interaction of H19 ICR with Differentially Methylated Regions (DMRs) of Igf2 in a parent-specific manner maintains differential epigenetic marks on maternal and paternal alleles. The results indicate that CTCF occupies specific sites on highly condensed mitotic chromosomes. CTCF-dependent long-range key interaction on the maternal allele is maintained during mitosis, suggesting the possible epigenetic memory of dividing cells. In this study, we developed a new method called Circular Chromosome Conformation Capture (4C) to screen genome-wide interactions with H19 ICR. The results indicated there are wide intra- and inter-chromosomal interactions which are mostly dependent on CTCF-binding site at H19 ICR. These observations suggest new aspects of epigenetic regulation of the H19/Igf2 imprinted region and higher order chromatin structure.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Les techniques d’assistance médicale à la procréation particulièrement l’induction de l’ovulation, la maturation in vitro des ovocytes et la culture embryonnaire prolongée impliquent la manipulation des gamètes ainsi que les embryons à des moments critiques de leur maturation et développement qui sont également des étapes clé du remodelage épigénétique. Par conséquent, elles pourraient interférer avec la reprogrammation épigénétique, en particulier la mise en place de la méthylation des gènes soumis a empreinte au cours de l'ovogenèse, ou son maintien au cours du développement préimplantatoire. Afin d’évaluer ce risque nous avons analysé le profil de méthylation de KvDMR1, qui régule l’expression de KCNQ1OT1, dans des ovocytes humains mûris in vivo ou in vitro, provenant de patientes stimulées ou non. Nos résultats montrent que la mise en place de la méthylation au niveau de KvDMR1 se poursuit au cours de la maturation de l’ovocyte du stade VG au stade MII, in vivo et in vitro et que l’induction ovarienne des patientes génère des ovocytes épigénétiquement immatures. Par ailleurs, l’étude de la méthylation de H19 DMR qui régule l’expression d’Igf2 et H19 dans des embryons d’ICSI, atypiques bloqués en culture prolongée et dans les spermes correspondants met en évidence une hypométhylation de l'allèle paternel et une méthylation de l'allèle maternel dans certains embryons, sans que l'on puisse établir de lien entre les dérégulations de l’empreinte et l’arrêt du développement au stade blastocyste. / Assisted reproductive technologies particularly the induction of ovulation, oocytes in vitro maturation, and prolonged embryo culture require in vitro manipulation of gamete and embryos at critical times of their maturation and development. In consequence, they may interfere with epigenetic reprogramming and affect particularly demethylation and remethylation of imprinted genes. To evaluate such a risk, we have determined the methylation profile of KvDMR1, the region that regulates KCNQ1OT1 imprinted gene, in human oocytes retrieved from stimulated or unstimulated cycles, at different phases of their maturation in vivo or in vitro. Our results show that the timing of establishment of the methylation profile of KvDMR1 covers the maturation phase of oocyte growth, in vivo and in vitro, and that hyperstimulation likely recruits young follicles epigenetically immature. Analysis of the methylation profile of H19DMR (DMR of IGF2/H19) in atypical ICSI embryos and corresponding sperm suggests that imprinting disorders are not responsible of embryo developmental failure prior the blastocyst stage.

Lors de la fécondation, le génome subit des transformations épigénétiques qui vont guider le développement et le phénotype de l’embryon. L'avènement des techniques de reprogrammation cellulaire, permettant la dédifférenciation d'une cellule somatique adulte, ouvre la porte à de nouvelles thérapies régénératives. Par exemple, les procédures de transfert nucléaire de cellules somatique (SCNT) ainsi que la pluripotence par induction (IP) visent à reprogrammer une cellule somatique adulte différentiée à un état pluripotent similaire à celui trouvé durant la fécondation chez l'embryon sans en impacter l'expression génique vitale au fonctionnement cellulaire. Cependant, la reprogrammation partielle est souvent associée à une mauvaise méthylation de séquences géniques responsables de la régulation des empreintes géniques. Ces gènes, étudiés chez la souris, le bovin et l'humain, sont exprimés de manière monoallélique, parent spécifique et sont vitaux pour le développement embryonnaire. Ainsi, nous avons voulu définir le statut épigénétique du gène empreinté H19 chez l'équin, autant chez le gamètes que les embryons dérivés de manière in vivo, SCNT ainsi que les cellules pluripotentes induites (iPSC). Une région contrôle empreinté (ICR) riche en îlots CpG a été observée en amont du promoteur. Couplé avec une analyse de transcrit parent spécifique du gène H19, nous avons confirmé que l'empreinte du gène H19 suit le modèle insulaire décrit chez les autres mammifères étudiés et résiste à la reprogrammation induite par SCNT ou IP. La déméthylation partielle de l'ICR observée chez certains échantillons reprogrammés n'était pas suffisante pour induire une expression biallélique, suggérant un contrôle des empreintes chez les équins durant la reprogrammation. / After fertilization, the animal genome undergoes a complex epigenetic remodeling that dictates the growth and phenotypic signature of the animal. The development of reprogramming methods using adult differentiated cells as the primordial genetic source has opened the door to new regenerative therapies for animals. Somatic cell nuclear transfer (SCNT) and induced pluripotency are two techniques which aim to reprogram a cell from its adult differentiated state to an embryonic-like pluripotency level, without impairing the expression of genes vital for the cellular function. Albeit promising, the mechanisms involved in these techniques remain only moderately understood. Partial reprogramming is frequently associated with irregular methylation of DNA sequences responsible for imprint regulation. These imprinted genes, mostly studied in rodents, cattle and humans, are expressed in a monoallelic parent-specific fashion and are vital for embryo growth. Hence, we aim to define the equine H19 imprinting control region (ICR) in gametes, in vivo and in SCNT derived embryos, as well as in induced pluripotent stem cells (iPSC). A CpG rich ICR was characterized upstream of the promotor using bisulfite treated DNA sequencing. Coupled with parent-specific gene expression analysis, we confirmed that the imprinted gene H19 is resistant to cellular reprogramming, and that partial demethylation of its ICR does not result in biallelic expression, suggesting that equine species have rigorous imprint maintenance during cellular reprogramming.

Les techniques d'assistance médicale à la procréation particulièrement l'induction de l'ovulation, la maturation in vitro des ovocytes et la culture embryonnaire prolongée impliquent la manipulation des gamètes ainsi que les embryons à des moments critiques de leur maturation et développement qui sont également des étapes clé du remodelage épigénétique. Par conséquent, elles pourraient interférer avec la reprogrammation épigénétique, en particulier la mise en place de la méthylation des gènes soumis a empreinte au cours de l'ovogenèse, ou son maintien au cours du développement préimplantatoire. Afin d'évaluer ce risque nous avons analysé le profil de méthylation de KvDMR1, qui régule l'expression de KCNQ1OT1, dans des ovocytes humains mûris in vivo ou in vitro, provenant de patientes stimulées ou non. Nos résultats montrent que la mise en place de la méthylation au niveau de KvDMR1 se poursuit au cours de la maturation de l'ovocyte du stade VG au stade MII, in vivo et in vitro et que l'induction ovarienne des patientes génère des ovocytes épigénétiquement immatures. Par ailleurs, l'étude de la méthylation de H19 DMR qui régule l'expression d'Igf2 et H19 dans des embryons d'ICSI, atypiques bloqués en culture prolongée et dans les spermes correspondants met en évidence une hypométhylation de l'allèle paternel et une méthylation de l'allèle maternel dans certains embryons, sans que l'on puisse établir de lien entre les dérégulations de l'empreinte et l'arrêt du développement au stade blastocyste.

Upon fertilization the paternal and maternal genomes unite, giving rise to the embryo, with its unique genetic code. All cells in the human body are derived from the fertilized ovum: hence they all contain (with a few exceptions) the same genetic composition. However, by selective processes, genes are turned on and off in an adaptable, and cell type-specific, manner. The aim of this thesis is to investigate how signals coming from outside the cell and epigenetic factors residing in the cell nucleus, cooperate to control gene expression. The transforming growth factor-β (TGF-β) superfamily consists of around 30 cytokines, which are essential for accurate gene regulation during embryonic development and adult life. Among these are the ligands TGF-β1 and bone morphogenetic (BMP) -7, which interact with diverse plasma membrane receptors, but signal via partly the same Smad proteins. Smad4 is essential to achieve TGF-β-dependent responses. We observed that by regulating transcription factors such as Id2 and Id3 in a specific manner, TGF-β1 and BMP-7 achieve distinct physiological responses. Moreover, we demonstrate that CTCF, an insulator protein regulating higher order chromatin conformation, is able to direct transcription by recruiting RNA polymerase II to its target sites. This is the first mechanistic explanation of how an insulator protein can direct transcription, and reveals a link between epigenetic modifications and classical regulators of transcription. We also detected that DNA loci occupied by CTCF replicate late. The timing of replication is a crucial determinant of gene activity. Genes replicating early tend to be active, whereas genes replicating late often are silenced. Thus, CTCF can regulate transcription at several levels. Finally, we detected a substantial cross-talk between CTCF and TGF-β signaling. This is the first time that a direct interplay between a signal transduction pathway and the chromatin insulator CTCF is demonstrated.