La méiose est une étape de la différenciation germinale qui permet la formation des gamètes. Elle est composée de deux divisions successives. La ségrégation des chromosomes homologues à la première division nécessite des connexions entre homologues, mises en place par des événements de crossing-over (CO). Les CO augmentent également la diversité génétique, et leur fréquence et leur distribution sont étroitement régulées. Ils sont générés par un mécanisme de formation et réparation de cassures double brins de l'ADN (CDBs), catalysées par la protéine SPO11 et préférentiellement localisées dans des régions de 1-2 kb appelées points chauds de recombinaison méiotique. Une question majeure est de comprendre comment sont régulés ces CO, ce qui détermine leur fréquence et leur distribution, car toute altération de cette régulation peut conduire à des anomalies chromosomiques graves.Dans ce travail de thèse, pour la première fois chez les mammifères, nous avons montré que des modifications de la chromatine sont associées à l'initiation de la recombinaison méiotique (formation des CDBs par SPO11). Ces résultats ont été obtenus par des analyses d'immunoprécipitation de chromatine (ChIP) sur des spermatocytes purifiés ou non, isolés de différentes lignées de souris. Une des modifications associées à l'activité de deux points chauds testés est la triméthylation de la lysine 4 de l'histone H3 (H3K4Me3). Une analyse fonctionnelle et temporelle de cette modification a permis de montrer qu'elle ne dépend pas de SPO11 et apparaît avant la formation de CDBs. Nous avons montré ici que c'est la protéine PRDM9, récemment identifiée comme un déterminant majeur des points chauds de recombinaison chez les mammifères et possédant une activité méthyltransférase, qui appose H3K4Me3. Nous proposons un modèle où H3K4Me3 et d'autres caractéristiques inconnues constitueraient un substrat pour la machinerie d'initiation et recruteraient SPO11 en des points précis du génome, qui deviendront des points chauds. / Meiosis is a specialized cell division to produce haploid gametes from a diploid cell. It segregates parental genomes by two successive divisions. The faithful segregation of homologous chromosomes is achieved during the first unique division via formation of crossovers (COs). COs establish physical connections between homologs by the reciprocal exchange of genetic material and require the formation and subsequent repair of SPO11-dependent DNA double-strand breaks (DSBs). Studies in many organisms revealed that COs are distributed in highly localized regions (1-2Kb) of genomes called recombination hotspots. The mechanisms of COs regulation are elusive and a main question in the field is to understand how the frequency and distribution of CO are regulated, because either absence or defects of recombination can lead to aneuploidy or reduced fertility. In the present study, for the very first time in mammals, we investigate whether recombination hotspots are associated with any chromatin modifications. We performed chromatin immunoprecipitation (ChIP) on spermatocytes isolated from different mice strains harbouring either active or inactive hotspots. Comparison of hot and cold spots revealed that a specific histone modification i.e. trimethylation of the lysine 4 of histone H3 (H3K4Me3) is enriched at two tested hotspots in mice. Temporal and functional analysis show that H3K4Me3 is not dependent on SPO11 and appears before DSBs formation. Furthermore, we demonstrate here that H3K4Me3 is methylated via the histone methyltransferase activity of PRDM9, recently identified as a major determinant of recombination hotspots in mammals. We propose a model that H3K4Me3 and other unknown chromatin features may specify recruitment of SPO11 initiation machinery to initiate meiotic recombination at the hotspots.

L'hétérochromatine péricentromérique semble jouer un rôle dans la régulation de l'expression génique et par conséquent dans le potentiel de développement des embryons. Nous avons fait l'hypothèse qu'une marque épigénétique, H3S10P, pourrait être un nouveau marqueur permettant le suivi des régions péricentromériques dans l'embryon préimplantatoire de souris. Par des techniques d'immunofluorescence et d'immuno-FISH couplées à de la microscopie en haute résolution, nous avons montré que la distribution de H3S10P dans les embryons de souris est différente de celle observée dans les cellules somatiques. Durant les stades 1 à 4-cellules, H3S10P est détectée en interphase autour des précurseurs des nucléoles (NPB), où elle colocalise avec les sondes ADN reconnaissant l'hétérochromatine péricentromérique, puis marque les bras chromosomiques sur toute la durée des phases de mitose. Après le stade 4-cellules, la distribution de H3S10P redevient similaire à ce qui est connu dans les cellules somatiques, avec un marquage au niveau des chromocentres seulement en fin d'interphase et sur les chromosomes mitotiques seulement jusqu'à la télophase. Cette cinétique particulière observée semble liée à l'absence de la kinase Aurora B aux stades les plus précoces. Nous avons également comparé la localisation de H3S10P avec celle d'autres marqueurs associés à l'hétérochromatine péricentromérique comme H3K9me3, HP1 β et la double modification H3K9me3S10P et en avons conclu que H3S10P est un meilleur marqueur pour l'hétérochromatine péricentromérique des deux génomes parentaux. Enfin, comme les embryons clonés obtenus par transfert nucléaire à partir de cellules somatiques (SCNT) montrent une redistribution anormale de l'hétérochromatine péricentromérique ainsi qu'un développement altéré, nous avons utilisé H3S10P pour détecter les remaniements de l'hétérochromatine après SCNT. Nos résultats montrent que, contrairement aux autres marqueurs, H3S10P n'est présente que sur la portion de l'hétérochromatine qui est correctement remaniée, tandis que l'hétérochromatine incorrectement reprogrammée conserve la signature épigénétique de la cellule donneuse. / Pericentromeric heterochromatin appears to be involved with gene regulation and therefore with the developmental potential of embryos. We hypothesized that an epigenetic modification, H3S10P, could be a new marker to follow pericentromeric heterochromatin in preimplantation mouse embryos. Using immunofluorescence, immunoFISH and high resolution microscopy, we observed that H3S10P shows a different distribution pattern in mouse embryos than in somatic cells. It is detected early in interphase around the Nucleolar-Precursor Bodies from 1- to 4-cell, in co-localization with the DNA probes for pericentromeric heterochromatin, and is seen in the chromosome arms throughout mitosis. In fact, H3S10P shows a similar kinetic as seen in somatic cells only after the 4-cell stage: being solely observed in the chromocenters during late interphase and on the mitotic chromosomes until telophase. This distribution seems related to the absence of Aurora B kinase in the earlier stages. We have also compared H3S10P to other related pericentromeric heterochromatin markers such as H3K9me3, HP1β and the double modification, H3K9me3S10P, and concluded that H3S10P is a better marker for pericentromeric heterochromatin of both parental origins. Finally, as cloned embryos often show abnormal pericentromeric heterochromatin remodelling and impaired development after Somatic Cell Nuclear Transfer (SCNT), H3S10P was used to track down heterochromatin reprogramming after SCNT. Our results show that H3S10P underlines only the portion of heterochromatin which is remodelled when compared with the other related markers and that the unremodelled portion maintains the epigenetic signature of the donor cell.

La compréhension des mécanismes qui contrôlent l'expression des gènes est un enjeu majeur pour la recherche médicale. Elle nécessite un ensemble d'approches pangénomiques telles que les puces à ADN et plus récemment le séquençage à très haut débit qui génèrent une masse toujours plus grande de données numériques à traiter. Au cours de ma thèse, j'ai développé plusieurs outils informatiques innovants pour faciliter leur exploitation. Ainsi, j'ai créé une librairie R (AgiND) qui vérifie la qualité des données de puces à ADN Agilent et permet de les normaliser. Le nombre croissant d'expériences stockées dans Gene Expression Omnibus a motivé la mise en place du projet TBrowser. Une méthode originale DBF-MCL a été créée pour extraire des signatures transcriptionnelles annotées par l'intégration de diverses sources d'information. Stockées dans une base de données, elles sont accessibles à travers une interface Java, un service web SOAP et une librairie R/Bioconductor (RTools4TB). Enfin, un pipeline d'analyse dédié au ChIP-seq a été implémenté. Tous ces outils ont servi pour l'étude de diverses maladies dans le cadre de collaborations. / Understanding the mechanisms that control gene expression is a major challenge for medical research. This requires using a large set of pangenomic approaches such as those using DNA microarrays and high-throughput sequencing that generate an ever growing mass of digital data. During my thesis, I have developed several computer-based tools to facilitate their processing and analysis. I have created a R library (AgiND) that controls the quality of Agilent DNA microarray data and allows their statistical normalization. The growing number of experiences stored in Gene Expression Omnibus has motivated the development of the TBrowser project. An original method, DBF-MCL, was created to extract annotated transcriptional signatures by integrating various sources of information. Stored in a database, these signatures are accessible using a Java interface, a SOAP web service and a R/Bioconductor library (RTools4TB). Finally, a pipeline dedicated to the ChIP-seq analyses has been implemented. All these tools were used to study various diseases in collaborations.

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.

Aberrant promoter methylation is known to silence tumor-suppressor genes in prostate cancer. Using a quantitative real-time PCR assay(MethyLight), I determined promoter methylation levels of APC, RASSF1A, CYP26A1 and TBX15 in 219 radical prostatectomies diagnosed between 1998-2001, examined their correlation with clinicopathological follow-up data including Gleason Pattern(GP), Gleason Score(GS) and pathological stage, and explored their potential in predicting biochemical recurrence(BR) using univariate and multivariate analyses. I demonstrated that methylation status of all four genes could accurately differentiate normal from cancerous tissues. Quantitative methylation levels of APC and TBX15 correlated strongly with GP, GS, and pathological stage. Both APC and TBX15 methylation levels could significantly predict BR in univariate analysis(p-value=0.028 and 0.003, respectively). The methylation profiles of APC and TBX15 combined could discriminate patients into high, intermediate, and low risk groups of BR(p-value=0.005). My project demonstrated that quantitative increase in promoter methylation levels of APC and TBX15 were associated with PCa progression.

Aberrant promoter methylation is known to silence tumor-suppressor genes in prostate cancer. Using a quantitative real-time PCR assay(MethyLight), I determined promoter methylation levels of APC, RASSF1A, CYP26A1 and TBX15 in 219 radical prostatectomies diagnosed between 1998-2001, examined their correlation with clinicopathological follow-up data including Gleason Pattern(GP), Gleason Score(GS) and pathological stage, and explored their potential in predicting biochemical recurrence(BR) using univariate and multivariate analyses. I demonstrated that methylation status of all four genes could accurately differentiate normal from cancerous tissues. Quantitative methylation levels of APC and TBX15 correlated strongly with GP, GS, and pathological stage. Both APC and TBX15 methylation levels could significantly predict BR in univariate analysis(p-value=0.028 and 0.003, respectively). The methylation profiles of APC and TBX15 combined could discriminate patients into high, intermediate, and low risk groups of BR(p-value=0.005). My project demonstrated that quantitative increase in promoter methylation levels of APC and TBX15 were associated with PCa progression.

Post translational modifications in histone proteins are transmissible changes that are not coded for in the DNA sequence itself but have a significant affect in the control of gene expression. Eukaryotic transcription is a regulated process, and acetylation plays a major role in this regulation. Deranged equilibrium of histone acetylation can lead to alteration in chromatin structure and transcriptional dysregulation of genes that are involved in the control of proliferation, cell-cycle progression, differentiation and or apoptosis. Evidence shows that high glucose conditions mimicking diabetes can increase histone acetylation and augment the inflammatory gene expression. Recent advances also highlight the involvement of altered histone acetylation in gastrointestinal carcinogenesis or hyperacetylation in amelioration of experimental colitis. However, the role of histone acetylation under obesity conditions is not yet known. Therefore in the present study, western blot analysis in the liver of Zucker obese versus lean rats was performed to determine the pattern and level of H3 and H4 acetylation (both in nuclear and homogenate fractions) at specific lysine (K) in pathological state of hepatic steatosis The same technique was also applied in the liver of obese rats fed higher amounts of vitamin B6 (OH) versus those fed normal amounts of vitamin B6 (ON) to assess if hyper-acetylation can be a protective response to hepatic steatosis. In both experimental models, it was also of interest to elucidate the expression of anti- and pro- apoptotic factor Bcl-2 and Bax in respect to histone acetylation. It was observed that, in liver homogenate fractions in control animals (LC/OC), there was a higher level of histone H3 acetylation at (K9, K14) and H4 acetylation at K5 in the obese animals. In contrast, the nuclear level of H3 and H4 acetylation at the same lysine residues was considerably higher in the lean and lower in the obese animals. Obese animals contained lower liver preneoplastic lesions as well as liver weight as a result of higher amounts of vitamin B6, had significantly higher H3 acetylation at K9 and K14 and H4 acetylation at K5, in both homogenate and nuclear fractions. However, histone acetylation was not detected for histone H4 at lysine 12 (K12) in either control group (LC/OC) or obese with different B6 diet group (OH/ON). Nevertheless, global histone H3 and H4 acetylation in both homogenate and nuclear fractions, was slightly higher in the lean rats and obese rats fed higher amounts of B6. By using the western blot technique, the level of anti- and pro- apoptotic Bcl-2 and Bax were also evaluated. The moderately higher level expression of anti-apoptotic Bcl2 protein was found in lean animals, whereas the expression of pro-apoptotic Bax was significantly higher in obese animals. Furthermore, anti-apoptotic Bcl2 protein expression was slightly higher in the obese rats fed normal amounts of B6 diet; but, pro-apoptotic Bax was higher in the obese rats fed higher amounts of vitamin B6. This is the first study which shows that hyperacetylation of histones in liver nuclei can be correlated with amelioration of hepatic steatotis. Histone acetylation and B6 rich diet might be involved in the regulation of biological availability of key apoptotic proteins, which, in turn, can possibly modify the severity of the disease.

There are around 20.000 genes in the human genome all of which could potentially be expressed. However, it is obvious that not all of them can be active at the same time. Thus, there is a need for coordination achieved through the regulation of transcription. Transcriptional regulation is a crucial multi-component process involving genetic and epigenetic factors, which determine when and how genes are expressed. The aim of this thesis was to study two of these components, the transcription factors and the DNA sequence elements with which they interact. In papers I and II, we tried to characterize the regulatory role of repeated elements in the regulatory sequences of nitric oxide synthase 2 gene. We found that this type of repeat is able to adopt non B-DNA conformations in vitro and that it binds nuclear factors, in addition to RNA polymerase II. Therefore it is probable that these types of repeats can participate in the regulation of genes. In papers III-V, we intended to analyze the genome-wide binding sites for six transcription factors involved in fatty acid and cholesterol metabolism and the sites of an epigenetic mark in a human liver cell line. For this, we applied the chromatin immunoprecipitation (ChIP) method together with detection on microarrays (ChIP-chip) or by detection with the new generation massively parallel sequencers (ChIP-seq). We found that all of these transcription factors are involved in other liver-specific processes than metabolism, for example cell proliferation. We were also able to define two sets of transcription factors depending on the position of their binding relative to gene promoters. Finally, we demonstrated that the patterns of the epigenetic mark reflect the structure and transcriptional activity of the promoters. In conclusion, this thesis presents experiments, which moves our view from genetics to genomics, from in vitro to in vivo, and from low resolution to high resolution analysis of transcriptional regulation.

Post translational modifications in histone proteins are transmissible changes that are not coded for in the DNA sequence itself but have a significant affect in the control of gene expression. Eukaryotic transcription is a regulated process, and acetylation plays a major role in this regulation. Deranged equilibrium of histone acetylation can lead to alteration in chromatin structure and transcriptional dysregulation of genes that are involved in the control of proliferation, cell-cycle progression, differentiation and or apoptosis. Evidence shows that high glucose conditions mimicking diabetes can increase histone acetylation and augment the inflammatory gene expression. Recent advances also highlight the involvement of altered histone acetylation in gastrointestinal carcinogenesis or hyperacetylation in amelioration of experimental colitis. However, the role of histone acetylation under obesity conditions is not yet known. Therefore in the present study, western blot analysis in the liver of Zucker obese versus lean rats was performed to determine the pattern and level of H3 and H4 acetylation (both in nuclear and homogenate fractions) at specific lysine (K) in pathological state of hepatic steatosis The same technique was also applied in the liver of obese rats fed higher amounts of vitamin B6 (OH) versus those fed normal amounts of vitamin B6 (ON) to assess if hyper-acetylation can be a protective response to hepatic steatosis. In both experimental models, it was also of interest to elucidate the expression of anti- and pro- apoptotic factor Bcl-2 and Bax in respect to histone acetylation. It was observed that, in liver homogenate fractions in control animals (LC/OC), there was a higher level of histone H3 acetylation at (K9, K14) and H4 acetylation at K5 in the obese animals. In contrast, the nuclear level of H3 and H4 acetylation at the same lysine residues was considerably higher in the lean and lower in the obese animals. Obese animals contained lower liver preneoplastic lesions as well as liver weight as a result of higher amounts of vitamin B6, had significantly higher H3 acetylation at K9 and K14 and H4 acetylation at K5, in both homogenate and nuclear fractions. However, histone acetylation was not detected for histone H4 at lysine 12 (K12) in either control group (LC/OC) or obese with different B6 diet group (OH/ON). Nevertheless, global histone H3 and H4 acetylation in both homogenate and nuclear fractions, was slightly higher in the lean rats and obese rats fed higher amounts of B6. By using the western blot technique, the level of anti- and pro- apoptotic Bcl-2 and Bax were also evaluated. The moderately higher level expression of anti-apoptotic Bcl2 protein was found in lean animals, whereas the expression of pro-apoptotic Bax was significantly higher in obese animals. Furthermore, anti-apoptotic Bcl2 protein expression was slightly higher in the obese rats fed normal amounts of B6 diet; but, pro-apoptotic Bax was higher in the obese rats fed higher amounts of vitamin B6. This is the first study which shows that hyperacetylation of histones in liver nuclei can be correlated with amelioration of hepatic steatotis. Histone acetylation and B6 rich diet might be involved in the regulation of biological availability of key apoptotic proteins, which, in turn, can possibly modify the severity of the disease.

Cancer cells employ a litany of immunosuppressive and immunevasive strategies to avoid detection and elimination by the various arms of the innate and adaptive immune system. Many hematologic and solid tumors progressively develop a specialized microenvironment which promotes tissue restructuring inflammation while masking the immune signature of the tumor cells themselves. Chronic lymphocytic leukemia, a malignancy of mature B lymphocytes must constantly balance on the precipice of immune recognition. A mature antigen presenting cell themselves, CLL clonal growth is dependent on the very interactions which, if effective, could potentially lead to their demise. To circumvent this, CLL clones set up unique signatures which promote immune recognition yet provide diversionary signals to the remaining immune armament resulting in profound immune dysfunction. While the aforementioned immune dysfunction is widespread, the B cell and T cell repertoire are severely impaired during leukemic progression. The lack of healthy B cells due to displacement by malignant B cells results in the obvious loss of an important antigen presenting cell as well as antibody-based immunity. Additionally, deficient interactions with T cells result in anergy and the preponderance of improperly polarized T lymphocytes which are impotent to eliminate both pathogens and leukemic cells. The result of such severe immune dysfunction is chronic infection and progressive disease which is the primary cause of death in CLL patients. Our research was focused on the premise that alleviating immune dysfunction and providing immunotherapeutic tools will significantly benefit CLL therapy. To this end we developed methods to improve the cellular interaction between CLL cells and T cells a critical step towards improving the antigen presentation capacity of the diseased B cell repertoire. We also identified a therapeutic strategy which can revert the anergic or improperly polarized state of T cells already in circulation allowing those cells to more effectively perform the effector functions necessary to fight pathogenic attack and malignant transformation. Finally, we identified a number of novel targets in CLL which could be used in a vaccinate-induce method to license the elimination of CLL cells by the patient's adaptive immune system. To achieve our goals we utilized a relatively new class of drugs called epigenetic modifiers which specifically alter the chromatin structure resulting in novel genetic signatures which are heritable over cellular generations. The unique properties of these drugs allow for the elicitation of suppressed genetic programs which, when properly controlled, have the potential to reassert healthy lymphocyte functions. Our studies provide a comprehensive therapeutic initiative which, by simultaneously alleviating the major causes of immune dysfunction in addition to facilitating the use of novel active immunotherapeutic strategies could potentially impact clinical therapy for CLL.