Bromodomains (BRDs) are protein modules which bind to acetylated lysines on histones and transcriptional regulating proteins. BRD-containing proteins are involved in a large variety of critical cellular processes and their misregulation, or mutation of the genes encoding for them, has been linked to pathogenesis in humans. The generation of chemical probes (potent, selective and cell permeable small molecules) in cellular experiments to investigate the biological role of the BRDs is thus desirable. A chemical probe for the CREB (cyclic-AMP response element binding protein) binding-protein (CBP) and E1A binding protein (p300) BRDs was developed, starting from a low molecular weight, weak and non-selective dimethylisoxazole benzimidazole compound. Parallel synthesis was used to optimise the initial hit into a weak, but selective CBP inhibitor. Further modification of the two N-1 and C-2 moieties of the benzimidazole scaffold, led to highly potent and selective CBP inhibitors. Structure-guided design was then applied to optimise the selectivity of the series for CBP over the first domain of bromodomain-containing protein 4 BRD4(1). A strategy to reduce the flexibility of the N-1 and C-2 ethylene linker groups through the incorporation of conformational constraints led to inhibitors with increased selectivity. The optimal compound was highly potent for the CBP and p300 BRDs (K_d 21 nM and 32 nM, respectively) and selective over BRD4(1) (40-fold and 27-fold, respectively). On-target cellular activity was observed in a fluorescence recovery after photobleaching (FRAP) assay (0.1 μM), a p53 reporter gene assay (IC₅₀ 1.5 μM) and a Förster resonance energy transfer (FRET) assay (5 μM). A weak indolizine bromodomain-containing protein 9 (BRD9) inhibitor was used as the starting point for the development of a BRD9/BRD7 chemical probe. Analogues were synthesised via [3+2] cycloadditions. An optimised compound was found to be highly potent (68 nM) and selective over BRD4(1) (34-fold). On-target cellular activity was observed in a FRAP assay (5 μM). Efforts were made to improve the cellular activity through the introduction of an ionisable centre to aid solubility. A selection of piperazine analogues were shown to be potent and selective, and these compounds warrant further investigation of their selectivity and cellular activity. Overall, the work has led to the first potent and selective inhibitors of the CBP/p300 and BRD9 BRDs. It also highlights the role of structural analysis in the development of inhibitors that modulate protein-protein interactions.

Mammalian genomes are characterised by global and pervasive DNA methylation and this modification is generally thought to be inhibitory to transcription. An exception to this widespread DNA modification are genomic elements called CpG islands (CGI), contiguous regions of non-methylated DNA which encompass the transcription start site of two thirds of mammalian genes. Although CGIs represent the most prominent feature of mammalian promoters, the contribution of these elements to promoter function remains unclear. Work in this study shows that the histone lysine demethylase KDM2B (FBXL10/ JHDM1B) is a nuclear protein which binds specifically to non-methylated CpG dinucleotides and associates with CGI elements genome-wide through its zinc-finger CxxC (ZF-CxxC) DNA binding domain. Furthermore, in mouse embryonic stem cells, biochemical investigation revealed that KDM2B associates with Polycomb group E3 ubiquitin ligase RING1B to form a variant Polycomb repressive complex 1 (PRC1) characterized by the PCGF1 subunit. Considering that KDM2B has clear DNA-binding activity and that CGIs were reported to function as nucleation sites for polycomb repressive complexes, a potential role for KDM2B in mediating PRC1 recruitment to target genes was investigated. Stable depletion studies indicated that KDM2B is required for the normal targeting of RING1B to CGIs and the regulation of expression of a subset of Polycomb-occupied genes. By taking advantage of a genetic ablation system in which the DNA binding domain of KDM2B can be conditionally deleted, results in this thesis reveal that the ability of KDM2B to recognize non-methylated DNA is essential for polycomb domain formation and normal embryonic development. Finally, through the use of a de novo targeting assay, an unexpected PRC2 recruitment pathway was discovered which is dependent on PRC1-mediated H2AK119ub1 deposition. Together this work uncovers a novel mechanism linking KDM2B-dependent recognition of non-methylated DNA with recruitment of Polycomb proteins and provides the framework on which to further investigate the contribution of CGIs to formation of polycomb domains.

Methyl-CpG-binding protein 2 (MeCP2 is) is an epigenetic regulator that binds to methylated DNA. Initially identified as transcriptional repressor, MeCP2 also binds to different proteins functioning as gene activator. Importantly, MecCP2 gene mutations and changes in MeCP2 levels are associated to several forms of mental retardation and autism-related disorders; including Rett, a neurodevelopmental disorder affecting primarily girls. While brain MeCP2 was considered to be exclusively neuronal, this regulator is also present in glia. We found that oligodendrocytes, the myelinating cells of the central nervous system (CNS), express particularly high MeCP2 levels at a developmental stage that precedes their final maturation. Moreover, downregulation of MeCP2 levels by treatment of immature oligodendrocytes with small interference RNA (siRNA), reduced the expression of 14 kDa myelin basic protein (MBP) and MOG, two markers of mature oligodendrocytes. These observations raise the possibility that oligodendrocytes have a direct participation in Rett syndrome and other autism-related disorders.

An understanding of the human fetal to adult hemoglobin switch offers the potential to ameliorate β-type globin gene disorders such as sickle cell anemia and β-thalassemia through activation of the fetal γ-globin gene. Chromatin modifying complexes, including MBD2-NuRD and GATA-1/FOG-1/NuRD play a role in γ-globin gene silencing, and Mi2β (CHD4) is a critical component of NuRD complexes. In the studies presented in Chapter 2, we observed that the absence of MBD2 in a sickle cell mouse model leads to a decrease in the number of sickled cells observed in the peripheral blood, and significantly increases survival in these mice. Although further studies will be necessary to fully understand the effect of MBD2 knockout in sickle cell disease mice, absence of MBD2 appears to partially ameliorate the sickle cell anemia phenotype in vivo. In the studies presented in Chapter 3, we observed that knockdown of Mi2β relieves γ-globin gene silencing in β-YAC transgenic murine CID hematopoietic cells and in CD34+ progenitor derived human primary adult erythroid cells. We show that independent of MBD2-NuRD and GATA-1/FOG-1/NuRD, Mi2β binds directly to and positively regulates both the KLF1 and BCL11A genes, which encode transcription factors critical for γ-globin gene silencing during β-type globin gene switching. Remarkably, less than 50% knockdown of Mi2β is sufficient to significantly induce γ-globin gene expression without disrupting erythroid differentiation of primary human CD34+ progenitors. These results indicate that Mi2β is a potential target for therapeutic induction of fetal hemoglobin.

A targeted approach to the development of antifolate therapies has been sought for many years. Central to the success of such development is an understanding of the molecular mechanisms dictating the sensitivity of cells to antifolates and the fundamental differences of these processes between normal and neoplastic phenotypes. This dissertation addressed transcriptional mechanisms and cell-signaling events responsible for the efficacy of antifolate therapies. Transcriptional processes and cell signaling pathways are often aberrant in neoplastic tissues, providing a potential point of distinction between a normal and neoplastic cellular state. Folylpolyglutamate synthetase (FPGS) catalyzes the formation of poly-γ-glutamate derivatives of folates and antifolates, which permits intracellular retention and accumulation of these compounds. The mouse fpgs gene uses two distant promoters to produce functionally distinct isozymes in a tissue-specific pattern. We questioned how the two promoters were differentially controlled. An analysis of DNA methylation and histone post-translational modifications across the length of the mouse fpgs gene showed that epigenetic mechanisms contributed to the tissue-specific control of the upstream (P1), but not the downstream (P2) fpgs promoter. RNAPII complexes and general transcription factors were present over P1 only when P1 was transcribed, but these components were present over P2 in most tissues, and promoter-proximal pausing was evident in brain. Clear promoter occlusion was found over P2 in liver. These studies concluded that tissue-specific coordination of dual promoters required multiple interacting controls. The mammalian target of rapamycin (mTOR) controls protein translation initiation, and is central to a cell-signaling pathway rich in tumor suppressor and oncogenic proteins. mTOR dysregulation is a common feature of several human cancers and inhibition of this protein has been sought as an ideal cancer drug target. We have determined that antifolates inhibiting the two folate-dependent steps of purine synthesis (GART or AICART) activate AMP-dependent protein kinase (AMPK) and inhibit mTOR. The mechanism of AMPK stimulation appears to be mediated by either nucleotide depletion (GART inhibitors), or ZMP accumulation (AICART inhibitors). These studies discovered a new mechanism for antifolates that surprisingly defines them as molecularly targeted therapeutics.

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.

Introduction: The epigenetic modifications can significantly affect and alter the gene activity by regulating their expression, having direct impact on various processes in human body. Epigenetic processes are involved in ethiopathogenesis of many diseases. From this point of view, MHC genes are very important as they were linked to many autoimmune disorders, for example type 1 diabetes mellitus. In general autoimmune diseases appear to be connected to certain MHC class II genes. Aims: The aim of this thesis is to determine the relationship between expression levels and histone modifications present in the promoter area of MHC class II gene, DQA1. Moreover, we also analyze and compare the DQA1 gene mRNA expression depending on the QAP promoter allele. Methods: We isolated both nucleic acids (DNA and RNA) and leukocytes from peripheral blood samples collected from voluntary donors. DNA was utilized for genotypization of individuals. RNA was subjected to reverse transcription and the quantitative PCR was performed in order to determine the level of expression. Leukocytes were used for chromatin immunoprecipitation, which was evaluated using quantitative PCR. Results: The expression level of QAP allele 3.1 was found to be higher than for the rest of the alleles Allele 4.1A showed, on the other hand,...

La reproduction sexuée implique une différentiation extrême des gamètes qui s’accompagne de profonds remaniements des chromosomes parentaux. Au moment de la fécondation, ces chromosomes doivent être rendus compétents pour la formation du premier noyau zygotique. Au cours de ma thèse, j’ai étudié l’importance fonctionnelle de plusieurs voies moléculaires paternelles et maternelles participant à cette étape chez la drosophile. Le complexe HIRA est impliqué dans l’assemblage de nucléosomes dans le pronoyau mâle à la fécondation. J’ai décrit le rôle de HIRA et de son partenaire Yemanucléine-α dans cette voie. J’ai caractérisé plus finement ce complexe en étudiant son rôle somatique dans l’assemblage des nucléosomes et son implication dans la stabilité de l’hétérochromatine, améliorant notre compréhension des besoins biologiques qui conditionnent sa conservation et son évolution. Je me suis aussi intéressé à diverses situations affectant l’intégrité des chromosomes parentaux à la fécondation. (1) J’ai décrit les conséquences catastrophiques pour la méiose femelle de l’expression naturelle d’un transposon à travers l’étude d’un cas de dysgénésie hybride. (2) J’ai contribué à montrer que la protéine K81 est essentielle pour la protection des télomères dans les chromosomes paternels au cours de la spermatogénèse. (3) J’ai participé à caractériser les conséquences pour les chromosomes paternels de l’incompatibilité cytoplasmique induite par la bactérie Wolbachia. Ensemble, ces travaux soulignent les particularités des chromosomes parentaux à la fécondation et aident à cerner l’importance des voies maternelles et paternelles dans leur intégration dans le premier noyau du zygote / Sexual reproduction involves dramatic gamete differentiation and profound parental chromosomes remodelling. At fertilization, these chromosomes need to be rendered competent for the formation of the fist zygotic nucleus. I have studied the functional relevance of several paternal and maternal molecular pathways that participate during this process in Drosophila. The HIRA complex is required for nucleosome assembly in the male pronucleus at fertilization. I have further described the rôle of HIRA and its obligatory partner Yemanuclein-α during this step. I have characterized the somatic roles of this complex during nucleosome assembly and its involvment in heterochromatin stability, which gives us a better understanding of the biological needs that drive its conservation and evolution. I have also focused on several situations where parental chromosomes integrity at fertilization is compromised. (1) I have described a meiotic catastrophe associated with the natural expression of a transposon in the female germline during hybrid dysgenesis. (2) I have contributed to show that K81 is an essential protein for telomere protection in paternal chromosomes during spermiogenesis. (3) I have participated in the characterization of the chromosomal abnormalities associated with cytoplasmic incompatibility induced by Wolbachia. Together, these results underscore the specificities of parental chromosomes at fertilization and shed light into the importance of maternal and paternal pathways for their integration in the first zygotic nucleus

During cell specification, temporal and spatially restricted gene expression programs are set up, forming different cell types and ultimately a multicellular organism. In this thesis, we have studied the molecular mechanisms by which sequence specific transcription factors and coactivators regulate RNA polymerase II (Pol II) transcription to establish specific gene expression programs and what epigenetic patterns that follows. We found that the transcription factor Dorsal is responsible for establishing discrete epigenetic patterns in the presumptive mesoderm, neuroectoderm and dorsal ectoderm, during early Drosophila embryo development. In addition, these different chromatin states can be linked to distinct modes of Pol II regulation. Our results provide novel insights into how gene regulatory networks form an epigenetic landscape and how their coordinated actions specify cell identity. CBP/p300 is a widely used co-activator and histone acetyltransferase (HAT) involved in transcriptional activation. We discovered that CBP occupies the genome preferentially together with Dorsal, and has a specific role during development in coordinating the dorsal-ventral axis of the Drosophila embryo. While CBP generally correlates with gene activation we also found CBP in H3K27me3 repressed chromatin. Previous studies have shown that CBP has an important role at transcriptional enhancers. We provide evidence that the regulatory role of CBP does not stop at enhancers, but is extended to many genomic regions. CBP binds to insulators and regulates their activity by acetylating histones to prevent spreading of H3K27me3. We further discovered that CBP has a direct regulatory role at promoters. Using a highly potent CBP inhibitor in combination with ChIP and PRO-seq we found that CBP regulates promoter proximal pausing of Pol II. CBP promotes Pol II recruitment to promoters via a direct interaction with TFIIB, and promotes transcriptional elongation by acetylating the first nucleosome. CBP is regulating Pol II activity of nearly all expressed genes, however, either recruitment or release of Pol II is the rate-limiting step affected by CBP. Taken together, these results reveal mechanistic insights into cell specification and transcriptional control during development. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p><p> </p>

La chromatine sert de support à de multiples processus biologiques, cependant son organisation spatiale diffère fortement selon l'échelle considérée. L'expression des gènes est ainsi coordonnée par des éléments régulateurs dispersés dans le génome mais capables d'interagir entre eux. Chez les métazoaires, des expériences de capture de conformation de chromosome (3C) combinées au séquençage haut-débit (Hi-C) ont permis la découverte de domaines d'association topologique (TAD), à l'échelle de la mégabase. Puisque la résolution du Hi-C reste limitée, nous avons utilisé la 3C-qPCR pour explorer, dans des cellules souches embryonnaires murines, la dynamique chromatinienne à l'intérieur de ces domaines ainsi qu'à leurs bordures. Nous identifions ainsi une modulation des fréquences de contacts, sur quelques centaines de kilobases. Cette modulation est plus ou moins importante en fonction du contenu en gènes des domaines, mais elle semble néanmoins universelle. Des modèles dérivés de la physique des polymères permettent de décrire cette modulation sous la forme d'une hélice statistique, que la chromatine adopterait en moyenne et en l'absence d'interactions spécifiques, à l'intérieur des TAD. Cette hélice reflète certaines contraintes que la chromatine subit à l'échelle supranucléosomale. Elle est très affectée par les bordures, qui bloquent la modulation, mais elle l'est beaucoup moins par le contenu en histone de liaison H1. Par ailleurs, grâce à des résultats de Hi-C à haute résolution, nous montrons que la modulation observée chez les souris n'est pas retrouvée chez la drosophile, où les caractéristiques des TAD semblent avant tout liées au paysage épigénétique local. Pour ces deux organismes, la dynamique chromatinienne à l'intérieur des domaines est donc sous le contrôle de phénomènes différents / The chromatin hosts various biological processes. However, its organization differs considerably depending on the scale. For example, gene expression is coordinated by regulatory elements that are dispersed in the genome but that are able to interact within the tridimensional space of the nucleus. In the Metazoa, chromosome conformation capture (3C) assays combined with high-throughput sequencing (Hi-C) uncovered the existence of topologically associating domains (TADs), at the mégabase scale. Due to the limited resolution of Hi-C, we used the 3C-qPCR method to explore, in murine embryonic stem cells, the chromatin dynamics inside TADs as well as at their borders. We found that contact frequencies undergo a periodic modulation over large genomic distances (few hundred kilobases). This modulation is weaker in gene-deserts than in gene-containing domains but it seems nevertheless to be universal. Using models derived from polymer physics, we show that this modulation can be understood as a fundamental helix shape that chromatin tends to adopt statistically, when no strong locus-specific interaction takes place, within the TADs. This statistical helix reflects some constraints that the chromatin undergoes at the supranucleosomal scale. It is affected by TADs borders, which disrupt the modulation, but linker histone H1 depletion only leads to subtle changes in the helix characteristics. Furthermore, using high-resolution Hi-C data, we found that chromatin dynamics is unconstrained in Drosophila where it seems mainly linked to the local epigenetics landscape. Therefore, distinct genome organization principles govern chromatin dynamics within mouse and Drosophila topologically associating domains.