Marked by a defect in the production of the Beta (β)-globin chain that make-up hemoglobin, Beta (β)-thalassemia is the most prevalent form of inherited single-gene disorders in the world. To understand the molecular mechanisms that govern the expression of the β-globin polypeptide encoded by the β-globin locus, we examined closely the enzymes involved in the epigenetic regulation of gene expression through histone 3 lysine 9 mono and di-methylation (H3K9 me1/2). G9a-like protein (GLP), a mammalian methyltransferase involved in the establishment and maintenance of H3K9 me1/2 mark at euchromatin, regions was found to be critical for the full activation of the adult β-globin genes in vivo during Murine erythroleukemia cell line (MEL) differentiation. Though it was initially hypothesized that GLP binding to H3K9 me1/2 mark through its Ankyrin domain was key to its activating function, we found that Flag- GLP ankyrin mutants E817R and W791A unable to bind to the methyl mark, are able to activate β-globin genes as well as their wild-type counterpart. Additionally, this study found that the embryonic gene εγ, known to be re-activated after G9a KD at the mRNA level, was effectively transcribed at the protein level using Triton Urea Acetic acid (TAU) western blots, thereby identifying potential therapeutic applications for treatment for β-thalassemia patients.

Marked by a defect in the production of the Beta (β)-globin chain that make-up hemoglobin, Beta (β)-thalassemia is the most prevalent form of inherited single-gene disorders in the world. To understand the molecular mechanisms that govern the expression of the β-globin polypeptide encoded by the β-globin locus, we examined closely the enzymes involved in the epigenetic regulation of gene expression through histone 3 lysine 9 mono and di-methylation (H3K9 me1/2). G9a-like protein (GLP), a mammalian methyltransferase involved in the establishment and maintenance of H3K9 me1/2 mark at euchromatin, regions was found to be critical for the full activation of the adult β-globin genes in vivo during Murine erythroleukemia cell line (MEL) differentiation. Though it was initially hypothesized that GLP binding to H3K9 me1/2 mark through its Ankyrin domain was key to its activating function, we found that Flag- GLP ankyrin mutants E817R and W791A unable to bind to the methyl mark, are able to activate β-globin genes as well as their wild-type counterpart. Additionally, this study found that the embryonic gene εγ, known to be re-activated after G9a KD at the mRNA level, was effectively transcribed at the protein level using Triton Urea Acetic acid (TAU) western blots, thereby identifying potential therapeutic applications for treatment for β-thalassemia patients.

The N-terminal tails of histones are subject to many chemical modifications that are involved in a variety of biological functions. Histone methylation is a major epigenetic modification found in both single and multicellular organisms and is directly involved in the regulation of gene expression. Methylation of lysine 9 of histone 3 (H3K9) has been shown to have diverse functions depending on the number of methyl groups added; H3K9me1 marks the active promoters, while H3K9me2 and H3K9me3 are present within inactive gene promoters and pericentric heterochromatin. G9a, also known as euchromatic histone-lysine N-methyltransferase 2 (Ehmt2), is a histone methylase that catalyses addition of mono- and dimethyl groups to H3K9 in euchromatic regions of the genome to silence genes. Therefore, it is a vital component of the gene expression regulation machinery. In mouse embryonic stem (ES) cells, G9a forms a stable heterodimer with the G9a-like protein (GLP or Ehmt1), which is further stabilised by the C2H2-type zinc finger protein, widely interspaced zinc finger protein (WIZ). These three proteins form the core G9a complex, which is essential for mouse development. Lack of any G9a complex member leads to embryonic lethality at E9.5 with severe growth defects. The ankyrin repeat domain of G9a/GLP can bind to H3K9me1/2 with high affinity in vitro (Collins et al. 2008). This enables the self-recruitment of the G9a complex to sites with H3K9me1/2 and maintenance of the mark. However, the initial recruitment of the G9a complex to sites lacking H3K9me1/2 mark during differentiation is poorly understood. Neither G9a nor GLP has a DNA/RNA binding domain, so recruitment of the G9a complex to specific sites must be mediated by other binding partners of the G9a complex. Using mass spectrometry, I was able to identify a number of zinc finger proteins as binding partners of G9a. Among these, WIZ was identified in stoichiometric amounts to G9a and GLP, and is a potential DNA binding protein similar to other C2H2-type zinc fingers. The aim of this study was to determine the role of WIZ in the recruitment of the G9a complex to specific sites. I showed that knockdown of WIZ had no significant effect on the chromatin binding of G9a in undifferentiated mouse ES cells, which indicates WIZ is dispensable in the maintenance of H3K9me2. However, I observed a 30% decrease in the G9a levels upon WIZ knockdown, which shows that WIZ might have a role in stabilising G9a. Using recombinant WIZ zinc finger pairs, I was able to show that the 3rd and 4th zinc finger of WIZ bind DNA in vitro. Furthermore, using the systematic evolution of ligands exponential enrichment (SELEX) approach I demonstrated that the zinc fingers of WIZ preferentially bind to G-rich double-stranded DNA sequences. Binding site analysis with synthetic DNA indicated that WIZ ZF3-4 require two binding sites that are a certain distance apart from each other for efficient binding. In addition, ZF3-4 binds ssDNA with higher affinity than dsDNA, and binding to ssDNA is sequence-independent. This study shows for the first time that mouse WIZ zinc finger pairs can bind DNA and RNA in vitro. Therefore, sequence-specific recruitment of G9a might be mediated by WIZ during differentiation. Furthermore, DNA binding preference of WIZ might suggest that WIZ-mediated recruitment of G9a to establish H3K9me2 could occur at the R-loops where G-rich DNA forms a hybrid with newly transcribed RNA or at the G-rich repetitive sequences.

L'empreinte génomique est un mécanisme épigénétique qui conduit à l'expression d'un seul des deux allèles parentaux pour une centaine de gènes autosomaux chez les mammifères. La majorité des gènes soumis à l'empreinte est regroupée en clusters et tous ces gènes sont sous le contrôle de séquences discrètes appelées ICR (Imprinting Control Region). Les ICRs sont marquées épigénétiquement par une méthylation d'ADN et des modifications des histones alléliques. La méthylation d'ADN au niveau de ces ICRs est un facteur clé de l'empreinte et va être établie dans les lignées germinales suivant le sexe de l'embryon. Après fécondation, le nouvel embryon portera les empreintes paternelles et maternelles, ces empreintes devront alors être maintenues pendant tout le développement et interprétés dans le but de conduire à l'expression allélique des gènes soumis à l'empreinte. Cependant, la méthylation d'ADN ne peut expliquer à elle seule tous les aspects de l'empreinte génomique. Ainsi, d'autres marques épigénétiques doivent agir dans le maintien et la lecture de ces empreintes. Nous avons mis en évidence dans un premier temps que le contrôle de l'expression allélique dans le cerveau de Grb10 repose sur la résolution d'un domaine bivalent allélique spécifiquement dans le cerveau. Ces résultats mettent en avant pour la première fois un domaine bivalent dans le contrôle de l'expression des gènes soumis à l'empreinte et propose un nouveau mécanisme dans l'expression tissu spécifique de ces gènes. D'autre part, bien que des études en cellules ES aient démontré un rôle de G9a dans le maintien des empreintes au cours du développement embryonnaire, nos données suggèrent que G9a ne serait pas essentielle a ce maintien dans un contexte in vivo. / Genomic imprinting is a developmental mechanism which leads to parent-of-origin-specific expression for about one hundred genes in mammals. Most of imprinted genes are clustered and all are under control of sequence of few kilobases called Imprinting Control Region or ICR. ICRs are epigenetically marked by allelic DNA methylation and histone modifications. DNA methylation on ICRs is a key factor which is established in germ cells according to the sex of the embryo. After fecundation, the new embryo will harbored both paternal and maternal imprints which have to be maintained during the development and read to lead to allelic expression of imprinted genes. However, allelic DNA methylation alone cannot explain every aspect of genomic imprinting. Thus, there should be other epigenetic marks which act in the maintaining and reading of the imprints.Our data first indicate that bivalent chromatin, in combination with neuronal factors, controls the paternal expression of Grb10 in brain, the bivalent domain being resolved upon neural commitment, during the developmental window in which paternal expression is activated. This finding highlights a novel mechanism to control tissue-specific imprinting. On an other hand, although previous studies in ES cells show a role for G9a in the maintaining of imprints during embryonic development, our data suggest that G9a would not be essential in an in vivo model.

Colorectal tumors are hierarchically organized and governed by populations of self-renewing cancer stem cells, representing one of the deadliest types of cancers worldwide. Emergence of a cancer stem-like phenotype depends on epigenetic reprogramming, associated with profound transcriptional changes. As described for pluripotent reprogramming, epigenetic modifiers play a key role in developing and maintaining cancer stem cells by establishing embryonic stem-like transcriptional programs, thus altering the balance between self-renewal and differentiation. Through my work, I have identified overexpression of histone methyltransferase G9a as a risk factor for colorectal cancer, associated with shorter relapse-free survival. Moreover, using human transformed pluripotent cells as a surrogate model for cancer stem cells, I demonstrate that G9a activity is essential for the maintenance of an embryonic stem-like transcriptional signature that is required to promote self-renewal, tumorigenicity and an undifferentiated state. Such a role was also applicable to colorectal cancer, where inhibitors of G9a histone methyltransferase function induced intestinal differentiation while restricting tumor-initiating activity in patient-derived colorectal tumor samples. By integrating transcriptome profiling with G9a/H3K9me2 loci co-occupancy, the canonical Wnt pathway, epithelial-to-mesenchyme transition and extracellular matrix organization were identified as potential targets of such a chromatin regulation mechanism in colorectal cancer stem cells. Considering such novel insights on the role of G9a as a driver of the cancer stem cell phenotype, as well as a promoter of self-renewal, tumorigenicity and an undifferentiated state, I established and executed a multi-step drug screening pipeline to identify new repurposed drugs that selectively alter G9a functions in human CSCs. This pipeline revealed 3 new drug candidates that inhibit H3K9me2 deposition and impair human CSCs in culture. Future in-depth characterization of those candidates will represent an important step toward the development of novel CSC-targeting therapeutics.

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Chez les eucaryotes, l’expression des gènes dépend en partie du degré de compaction de la chromatine. La structure chromatinienne est régulée par des marques dites épigénétiques,telles que les modifications post-traductionnelles des protéines structurelles de la chromatine, les histones. Ainsi, la méthylation de la lysine 9 de l’histone H3 (H3K9) sur le promoteur des gènes est essentiellement associée à la répression de la transcription. H3K9 est méthylée par différentes enzymes appelées lysine méthyltransférases (KMTs). L’objectif principal de mon projet de thèse a été de mieux comprendre le rôle de principales KMTs de H3K9, que sontG9a, GLP, Suv39h1 et SETDB1, dans la régulation de l’équilibre entre prolifération et différenciation terminale. Pour cela, j’ai utilisé le modèle de différenciation terminale de cellules du muscle squelettique. En effet, durant la différenciation terminale, les myoblastes arrêtent de proliférer et fusionnent entre eux pour former de longues cellules multi nucléées que sont les myotubes. Ce processus implique, d’une part, l’expression des gènes de différenciation musculaire et, d’autre part, la répression irréversible des gènes associés à la prolifération cellulaire. L’introduction bibliographique de ce travail de thèse est séparée en trois chapitres. Le premier chapitre porte sur la chromatine et ses modifications post-traductionnelles. Le second s’attache à décrire les rôles de la méthylation de H3K9 et, en particulier, des quatre KMTs sur lesquelles j’ai travaillé durant ma thèse : G9a, GLP, SETDB1 et Suv39h1. Dans le troisième chapitre, je présente le modèle de la différenciation terminale du muscle squelettique. Dans la partie "Résultats", je décris deux des principales études que j’ai menées durant ma thèse. La première porte sur les rôles antagonistes de G9a et GLP. La seconde porte sur le rôle de SETDB1 durant la différenciation musculaire. Les résultats que j’ai obtenus sont discutés dans cette partie. Je conclus ce manuscrit en discutant mes résultats de manière plus générale et en proposant des perspectives à long terme. Enfin, une annexe présentera les autres articles de recherche auxquels j’ai participé pendant ma thèse. / In eukaryotes, gene expression partly relies on chromatin compaction degree. Chromatin status is controlled by epigenetic marks, such as histones (chromatin structural proteins) posttranslational modifications. As an example, histone H3 lysine 9 (H3K9) methylation on gene promoters is mainly associated with transcriptional repression. H3K9 is methylated by several enzymes called lysine methyltransferases (KMTs). The aim of my thesis project was to understand the role of the H3K9 KMTs, G9a, GLP, Suv39h1 and SETDB1 in regulating the balance between proliferation and terminal differentiation. For this purpose, I used skeletal muscle terminal differentiation as model. Upon muscle terminal differentiation, myoblasts exit, in an irreversible way, from the cell cycle and under go differentiation where cells fusion and form myotubes. During this process, cell cycle genes are permanently silenced and muscle specific genes are activated. Thesis introduction is divided into three chapters. The first chapter focuses on chromatin and post-translational modifications. The second chapter describes H3K9 methylation characteristics and the role of the four KMTs that I studied during my thesis project: G9a,GLP, Suv39h1 and SETDB1. In the third chapter, the skeletal muscle terminal differentiation model is described in details. Results section reports my two major studies outcomes and their discussion. The first concerns the antagonistic roles of G9a and GLP regarding the muscle terminal differentiation and the second focuses on the role of SETDB1 during muscle differentiation. Finally, I conclude this manuscript by a plainer discussion followed by long term perspectives and an appendix presents other research articles, in which I collaborated during my PhD.

La méthylation de l’ADN est une modification épigénétique qui prend place durant le développement embryonnaire sur le génome des Mammifères. Durant ma thèse, j’ai déterminé les cinétiques de mise en place de la méthylation de l’ADN sur le génome murin au cours de l’embryogénèse précoce. J’ai identifié les rôles spécifiques et redondants des ADN méthyltransférases DNMT3a et DNMT3b dans ce processus. J’ai également étudié le rôle de deux facteurs dans la mise en place de la méthylation de l’ADN dans l’embryon. Premièrement, j’ai déterminé que l’enzyme G9a joue un rôle essentiel pour la répression et le recrutement de la méthylation de l’ADN à des sites spécifiques du génome, incluant en particulier des promoteurs à ilots CpG de gènes méiotiques. Deuxièmement, l’étude du facteur E2F6 m’a permis de montrer que cette protéine est elle aussi impliquée dans le recrutement de la méthylation de l’ADN, et ce à des promoteurs de gènes méiotiques distincts de ceux régulés par G9a. / DNA methylation is an epigenetic modification which is established during embryonic development on the mammalian genome. In my thesis, I determined the kinetics of DNA methylation acquisition on the mouse genome during early embryogenesis, and determined the specific and redundant roles of the DNA methyltransferases DNMT3a and DNMT3b in this process. I also studied the roles of two factors involved in setting up DNA methylation in embryos. First, I determined that the G9a enzyme plays an essential role for the in vivo repression and DNA methylation of specific genomic sites, including in particular the CpG island promoters of germline genes. Second, the study of the E2F6 factor allowed me to show that this protein is also involved in recruiting DNA methylation at a set of germline gene promoters than are distinct from those regulated by G9a.

Chez l'homme, l'épissage alternatif (EA) affecte presque tous les gènes permettant de générer de vastes répertoires d'ARN et de protéines. L'épissage est un processus hautement régulé qui s'effectue principalement lorsque l'ARN est en cours de synthèse sur la chromatine. Beaucoup d'études suggèrent que la chromatine et ses marques épigénétiques influencent les décisions d'épissage au locus correspondant. A l'inverse, d'autres données laissent penser que l'épissage peut moduler les marques épigénétiques. Au cours de ma thèse, j'ai étudié différentes voies de couplage entre l'épissage et la chromatine. D'une part, j'ai exploré l'impact de la méthylation de l'ADN sur la régulation de l'épissage. J'ai montré que les enzymes qui méthylent l'ADN ont un effet global sur l'épissage d'exons enrichis en méthylation. Mes données suggèrent que les protéines qui lient la méthylation de l'ADN sont impliquées dans cette régulation. D'autre part, j'ai exploré les conséquences de l'EA sur la régulation de la chromatine en étudiant son impact de deux histones-methyltransferases (HMTase) : G9A et SUV39H2 dont les gènes génèrent des transcrits alternatifs. Tous les transcrits variants codent pour des protéines. La conservation des variants d'épissage de G9A dans des espèces et l'absence de différences dans leur activité HMTase, nous amènent à proposer que l'EA est associé à une fonction non liée aux histones. A l'inverse, les isoformes de SUV39H2 exhibent des activités HMTases différentes et régulent l'expression de gènes cibles différents. Ensemble, nos résultats apportent de nouvelles connexions dans le couplage épissage-chromatine et supporte un modèle où ces derniers s'auto-influencent. / In humans, alternative splicing affects almost all genes in the genome and generates extensive repertoires of RNAs and proteins. Splicing is a highly regulated process which occurs primarily when the RNA is being synthesized on chromatin. Many studies suggest that chromatin and epigenetic marks influence splicing choices to the corresponding locus. Conversely, other data suggest that splicing can modulate epigenetic marks. During my thesis, I studied different ways of crosstalk between splicing and chromatin. First, I investigated the effect of DNA methylation on splicing regulation. I have shown that the enzymes that methylate DNA have an overall effect on the splicing of exons with enriched methylation. My data suggest that proteins which bind to methylated DNA are involved in this regulation. On the other hand, I explored the impact of alternative splicing on chromatin regulation studying its impact on the expression and activity of both histone methyltransferases (HMTase): SUV39H2 and G9A. G9A and SUV39H2 generate variants transcripts whose expression is regulated according to tissues. All variants transcripts encode proteins. Conservation of G9A splice variants in species and no differences in their HMTase activity, lead us to propose that G9A alternative splicing is associated with a non-histone function. Conversely, SUV39H2 isoforms exhibit different HMTases activities, and regulate the expression of different target genes. All our results provide new connections in chromatin - splicing coupling and support a model in which they harbor self-influence.