La différenciation et le développement requièrent une régulation fine de l’expression desgènes, médiée en partie par les modifications épigénétiques. Parmi les modificationsd’histones, la chromatine bivalente, signature chromatinienne atypique associant lesmarques permissive H3K4me2/3 et répressive H3K27me3, est de par sa plasticité, pressentiepour jouer un rôle décisionnel dans l’acquisition d’une identité cellulaire. Pour étudier le rôlede la chromatine bivalente au cours du développement, nous avons choisi d’utiliserl’empreinte parentale. Ce cadre développemental bien caractérisé, conduit à l’expression decertains gènes à partir d’un seul des deux allèles selon son origine parentale. La méthylationdifférentielle de l’ADN d’une région clé, appelée ICR (Imprinting Control Region), bienqu’absolument requise pour l’expression mono-allélique de ces gènes, n’est pas suffisantepour rendre compte de la complexité du profil d’expression de ces gènes suggérantl’implication d’autres mécanismes. Sur 15 ICR méthylés sur l’allèle maternel, nous avonsprécisément mis en évidence que la chromatine bivalente est présente par défaut sur l’allèlenon-méthylé lorsque celui-ci est transcriptionnellement inactif, quel que soit le stadedéveloppemental ou le tissu étudié, participant ainsi à la régulation fine de l’expressiontissu-spécifique à partir de ces régions. Dans leur ensemble, nos données révèlent que lachromatine bivalente joue un rôle moins dynamique que pressentie. Ainsi, au niveau del’empreinte parentale, sa fonction principale serait de protéger l’allèle non-méthylé des ICRcontre l’acquisition de méthylation tout en aidant à le maintenir réprimé dans certainstissus. Nous proposons que la chromatine bivalente joue un rôle similaire sur l’ensemble desîlots CpG du génome, contribuant ainsi à la protection de l’identité cellulaire. Afin decompléter cette première étude, j’ai étudié la régulation de l’expression d’un candidat de larégulation de la dynamique de la chromatine bivalente, l’histone déméthylase pourH3K27me3, JMJD3. Les résultats obtenus suggèrent que l’induction d’expression observéeau cours de la différenciation neurale s’appuie sur une dynamique de la structuretridimensionnelle de la chromatine qui pourrait elle-même être régulée par la transcriptiond’un eARN (enhancer ARN) et l’hydroxyméthylation. Ce modèle souligne un mode derégulation complexe de ce nouvel acteur épigénétique, impliquant des régionsintragéniques, et pourrait notamment permettre de comprendre les mécanismes impliquésdans sa dérégulation dans les cancers. / Fine-tuned regulation of gene expression is required for cell fate determination anddevelopment. Epigenetics modifications are well documented to be instrumental in thisprocess. Among them, bivalent chromatin, an unusual chromatin signature, which associatesthe permissive mark H3K4me2/3 and the repressive mark H3K27me3, is believed to arbitrategene expression during cell commitment. To study its precise role in development, we haveundertaken to study bivalency in the context of genomic imprinting. This well-defineddevelopmental frame is a process restricting expression of some genes to one parental alleleonly. The constitutive differential DNA methylation at the key region called ICR (ImprintingControl Region), is absolutely required but not sufficient to explain the complexity of themono-allelic expression pattern of imprinted genes, indicating that other mechanisms couldbe involved. Specifically, on 15 maternally methylated ICR, we showed that bivalentchromatin is acquired by default on the unmethylated allele of ICR when it istranscriptionally inactive whatever the developmental stage or the tissue studied and thuscontribute to tissue-specific expression from these regions. Altogether, our results revealthat chromatin bivalency is much less dynamic than proposed. In the context of genomicimprinting, it seems to plays more a safeguard function at ICR by protecting theunmethylated allele against DNA methylation acquisition while keeping it silent in a subsetof tissues. To complete this study, I studied the regulation of JMJD3, a histone demethylasefor H3K27me3, candidate to regulate bivalency dynamic. Our results suggest that theinduction of Jmjd3 expression observed during neural differentiation rely on the dynamic ofthe tridimensional architecture at the locus which could be regulated by the transcription ofan eRNA (enhancer RNA) and by hydroxymethylation. This model highlight a complex way ofregulation for this new epigenetics actor, involving intragenic regions and could help tounderstand how Jmjd3 expression is deregulated in a pathological context such as in cancer.

En France comme dans la majorité des pays développés, le cancer de la prostate est le plus fréquent chez l’homme. Il est clairement établi que les altérations génétiques et épigénétiques sont des événements communs dans les cancers de la prostate, se traduisant par l’expression aberrante de gènes critiques. La méthylation des histones participe à la régulation de l’expression des gènes dans la cellule. La marque épigénétique H3K27me3 est associée à la répression génique et se trouve dérégulée dans les cancers de la prostate. Ses niveaux sont déterminés par l’équilibre entre les activités de la méthyltransférase d’histone EZH2 et de la déméthylase d’histone JMJD3. Afin de comprendre le mécanisme de dépôt de H3K27me3 dans la tumorigenèse prostatique, le travail de cette thèse s’est orienté sur l’évaluation simultanée de l’impact de JMJD3 et de EZH2. Dans un premier temps, les niveaux d’expression de JMJD3 et de EZH2 ont été montrés augmentés simultanément dans le cancer de la prostate. Cette augmentation est corrélée à un enrichissement de ces deux protéines sur le promoteur des gènes RARβ2, ERα, RGMA, AR et PGR. Dans un deuxième temps, une analyse transcriptomique a permis d’identifier une signature génique corrélée avec le niveau d’agressivité de la tumeur. L’utilisation des « épidrogues » GSK-J4 et DZNeP ciblant JMJD3 et EZH2 permettent de moduler l’expression de ces gènes. L’ensemble de ces résultats caractérise JMJD3 et EZH2 comme des facteurs clés dans le processus de tumorigenèse prostatique. Le panel de gènes identifié devrait permettre de développer de potentiels marqueurs de diagnostic mais également de pronostic dans le cancer de la prostate et sa modulation par les « épidrogues » permettra de développer de nouvelles stratégies thérapeutiques. / In France like in majority of developed countries, prostate cancer is the most common cancer in men. It has been clearly established that genetic and epigenetic alterations are common events in prostate cancer resulting in aberrant gene expression. Histone methylation are involved in gene expression of cells. The H3K27me3 epigenetic mark is a repressive mark and it is deregulated in prostate cancer. H3K27me3 levels are determined by the balance between histone methyltransferase EZH2 and histone demethylase JMJD3 activities. In order to understand the mechanism of H3K27me3 deposition in prostatic tumorigenesis, this thesis focused on the simultaneous assessment of the impact of JMJD3 and EZH2.Firstly, expression levels of JMJD3 and EZH2 were shown to be simultaneously increased in prostate cancer. The increase is correlated to both protein enrichments on RARβ2, ERα, RGMA, AR and PGR gene promotors. Secondly, transcriptomic analysis identified gene signature correlated with tumor aggressiveness. The utilization of GSK-J4 and DZNeP epidrugs targeting JMJD3 and EZH2 allowed us to modulate gene expressionOur results characterized JMJD3 and EZH2 as key factors in prostatic tumorigenesis process. The identified gene panel would be able to develop potential diagnostic and prognostic markers in prostate cancer and their modulation by epidrugs would make new therapeutic strategies.

Indiana University-Purdue University Indianapolis (IUPUI) / Multiple myeloma (MM) is the most frequent cancer to involve the skeleton, with over 80% of myeloma patients developing lytic bone disease (MMBD). Importantly, MM-associated bone lesions rarely heal even when patients are in complete remission. Bone marrow stromal cells (BMSCs) isolated from MM patients have a distinct genetic profile and an impaired osteoblast (OB) differentiation capacity when compared to BMSCs from healthy donors. Utilizing an in vivo model of MMBD and patient samples, we showed that BMSCs from tumor-bearing bones failed to differentiate into OBs weeks after removal of MM cells. Both Runx2 and Osterix, the master transcription factors for OB differentiation, remained suppressed in these BMSCs. However, the molecular mechanisms for MM-induced long-term OB suppression are poorly understood. We characterized both Runx2 and Osterix promoters in murine pre-osteoblast MC4 cells by chromatin immunoprecipitation (ChIP). The transcriptional start sites (TSSs) of Runx2 and Osterix in untreated MC4 cells were co-occupied by transcriptionally active histone 3 lysine 4 tri-methylation (H3K4me3) and transcriptionally repressive histone 3 lysine 27 tri-methylation (H3K27me3), termed the “bivalent domain”. These bivalent domains became transcriptionally silent with increasing H3K27me3 levels when MC4 cells were co-cultured with MM cells or treated with TNF-α, an inflammatory cytokine increased in MM bone marrow microenvironment. The increasing H3K27me3 levels induced by MM cells or TNF-α were associated with the downregulation of the H3K27 demethylase JMJD3 in MC4 cells and murine BMSCs. Knockdown of JMJD3 in MC4 cells was sufficient to inhibit OB differentiation. Further, ectopic overexpression of JMJD3 in MC4 cells partially rescued the suppression of osteoblast differentiation induced by TNFa. We also found that pre-incubation of MC4 cells with the NF-kB inhibitor quinazoline (QNZ) before TNF-a treatment prevented the downregulation of JMJD3. In agreement with our in vitro findings, BMSCs from MM patients had persistently decreased JMJD3 expression compared to healthy BMSCs. Our findings together demonstrate that decreased JMJD3 expression in BMSCs contributes to the long-term OB suppression in MMBD by remodeling histone landscapes at the Runx2 and Osterix TSSs. Thus, developing strategies to restore JMJD3 expression in BMSCs should increase bone formation and possibly decrease tumor burden in MM.

Covalent modifications of histone tails play essential roles in mediating chromatin structure and epigenetic regulation. JmjD3 is a JumonjiC domain containing histone demethylase, belongs to the KDM6 subfamily, and catalyses the removal of methyl groups on methylated lysine 27 on histone 3 (H3K27), a critical mark to promote polycomb mediated repression and gene silencing. The importance of JmjD3 has been implicated in development, cancer biology and immunology. In this thesis, I report the recombinant production of active human JmjD3, development of two in vitro screening assays, a cell-based assay, and structural determination of JmjD3 in complex with the inhibitor 8-hydroxy-5-carboxyquinoline (8HQ). A highly selective and potent small molecule inhibitor GSK-J1 was subsequently identified. The inhibitor is active in HeLa cells and promotes a dose-dependent increase of global H3K27 methylation. The inhibitor GSK-J1 was used in two different cell assay systems related to inflammation and differentiation, to understand how H3K27 demethylation controls cellular functions. By inhibiting H3K27me3 demethylation, it is demonstrated that tumor necrosis factor (TNF) and other pro-inflammatory cytokines are regulated by H3K27 demethylase inhibition in M1- type macrophages derived from healthy volunteers and rheumatoid arthritis patients. It is also shown that inhibition of H3K27me3 demethylation abrogates cellular fusion of M2- type macrophages. During RANKL induced osteoclast differentiation, JmjD3 is up-regulated and promotes the expression of the key transcription factor NFATc1. By inhibiting JmjD3, NFATc1 expression is reduced and osteoclastogenesis is inhibited. This mechanism demonstrates a novel anti-resorptive principle of potential utility in conditions of excess bone resorption such as osteoporosis, bone erosion in inflammatory arthritis or cancer of the bone. These experiments further resolve the ambiguity between scaffold and catalytic function associ- ated with the H3K27 demethylase in these biological systems, and demonstrate that its enzymatic activity is crucial for epigenetic regulation of macrophage and osteoclast function.