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Cette dernière décennie a vu augmenter le nombre d’études portant sur la caractérisation des protéines JUMONJI (JMJ) et montrant leur rôle prépondérant dans la régulation des gènes et le développement des organismes. Ces protéines sont capables de déméthyler certains résidus des queues des histones et ont été organisées en groupes phylogénétiques en fonction de la conservation de leur domaine catalytique. Pour chaque clade entre un et trois substrats spécifiques ont pu être identifiés. De la sous famille KDM3, dont le résidu cible est H3K9, seul un membre, IBM1, a été caractérisé chez Arabidopsis. Cette étude montre que la mutation de JMJ24, un autre membre de ce groupe, entraine une augmentation de la taille des racines, cotylédons et organes floraux, suggérant un rôle dans le contrôle du développement à différents stades. De plus, l’analyse de l’expression tissulaire indique que JMJ24 est exprimé dans le phloème, en cohérence avec l’effet pléiotropique de sa mutation. Enfin, nos données suggèrent une interaction entre JMJ24 et d’autres protéines JMJ, telles JMJ14 et IBM1, mais aussi une interaction avec les protéines DCL, impliquées dans la régulation des gènes et des éléments transposables. / Numerous studies over the last decade have reported the characterization of the JUMONJI (JMJ) proteins, showing their critical importance in regulating genes and organism’s development. These proteins are able to demethylate a subset of histone tail residues and were clustered into distinct groups using a phylogenetic analysis based on their catalytic domain conservation. Furthermore, modification of one to three specific residues has been attributed to each JMJ group. Within the KDM3 subfamily, of which target is the H3K9 residue, only one member, IBM1, was first characterized in Arabidopsis. In this report, we showed that the mutation of JMJ24, another member of this subfamily, resulted in an increase of the root length, cotyledon and floral organ size, suggesting that JMJ24 functions is needed at different developmental stage. In addition, the analysis of the tissue-specific expression of JMJ24 indicated that the gene is expressed within the phloem of all organs, correlating with the pleiotropic effect of the gene mutation. Last, our data also suggested that JMJ24 interacts with other JMJ protein like JMJ14 and IBM1, but also with the DCL proteins knowing to be involved in genes and transposable elements regulation.

Neutrophil extracellular traps (NETs), chromatin released by activated neutrophils, were first described for their antimicrobial properties. NETs have a backbone of DNA and histones lined with microbicidal proteins such as neutrophil elastase. NET release has pathological consequences, particularly within blood vessels where NETs can trap red blood cells and platelets, thus contributing to thrombosis (Chapter 1-Overview). NET formation (NETosis) is an active and coordinated biological process involving many enzymatic components. One enzyme in particular, peptidylarginine deiminase 4 (PAD4), citrullinates histones and is required for chromatin decondensation during NETosis. Neutrophils from PAD4-deficient mice are unable to form NETs. We obtained these mice from our collaborator Dr. Yanming Wang, and thus were able to compare PAD4-/- mice to wild-type (WT) mice in mouse models where NETs are formed. These studies have allowed for investigation of the biological relevance of PAD4 and NETs in vivo in thrombotic and/or inflammatory disease. This dissertation focuses on mouse models of deep vein thrombosis and of sepsis. In venous stenosis, thrombosis is initiated by restricting blood flow in the inferior vena cava (IVC). Here, PAD4-/- mice were greatly protected from thrombus formation (Chapter 2). Leukocyte rolling and platelet plug formation in response to vessel injury were unaffected, indicating that endothelial and platelet activation occurred normally in these mice. The mice did not exhibit any defects in hemostasis, and could be induced to produce deep vein thrombi by infusion of WT neutrophils that formed NETs as a part of the thrombus scaffold. Because there is potential to develop anti-NET therapies in thrombosis, I investigated if NET-deficiency would render mice immunocompromised (Chapter 3). PAD4-/- mice had similar mortality in the cecal ligation puncture model, and they were protected from shock in an LPS sepsis model where NETs are released in the absence of live bacteria. Therapies aimed at NET prevention or destruction would likely be beneficial without compromising host immunity. Thus, in summary, studying PAD4-deficient mice has revealed the impact of NETs in thrombotic/inflammatory disease and identified PAD4 as an attractive therapeutic target.

Long terminal repeat (LTR) retrotransposons account for approximately 10% of mouse and 8% of human genomes and may play a role in modifying gene expression. Many species harbor retrotransposon families encompassing both autonomous and non-autonomous members. Specifically, the mouse Early Transposon (ETn) family members lack all retroviral genes but are transcriptionally and retrotranspositionally active, causing over 20 known insertional germline mutations. ETns owe their retrotransposition potential to proteins encoded by structurally intact MusD retrotransposons with whom they share LTRs. ETn elements are transcribed at a much higher level than MusD retrotransposons in embryos and undifferentiated cells, suggesting their evasion of host restriction mechanisms. However, mechanisms responsible for the replicative success of non-autonomous retrotransposon subfamilies over their coding-competent relatives are poorly understood. In the first stage of my research, I analyzed regulatory sequences in an ETn LTR responsible for its high promoter activity in the undifferentiated cell line P19. I found that three GC-boxes that may function as Sp1/Sp3 binding sites act synergistically and are indispensable for undifferentiated cell-specific promoter activity of the LTR. Sp1 binding partners may be responsible for the restricted ETn expression. Moreover, I have shown that unlike many retroviruses, ETn elements possess multiple transcription initiation sites and that they have amplified via intracellular retrotransposition in the P19 teratocarcinoma cell line. In the next step of my research, I performed analysis of epigenetic mechanisms as a means of ERV suppression. Specifically, I showed that in embryonic stem cells, autonomous MusD retrotransposons are epigenetically suppressed to a greater degree than non-autonomous ETn retrotransposons, illustrated by a higher level of DNA methylation and a lower level of active histone modifications. I hypothesize that MusD elements may be silenced by DNA methylation and repressive chromatin spreading into the LTR from the CpG-rich internal retroviral sequence absent in ETn elements. I propose that internal structure largely devoid of high CG content enables ETn elements to evade host-imposed transcriptional repression, contributing to their high mutagenic activity in the mouse germline. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Understanding the specific mechanisms regulating chemotherapeutic drug anti-cancer activities will uncover novel strategies to enhance the efficacy of these drugs in clinical settings. Activating Transcription Factor 3 (ATF3) is a stress inducible gene whose expression has been associated with survival outcomes in cancer models. This study characterizes the chemotherapeutic drugs, cisplatin and Histone Deacetylase Inhibitor (HDACi), M344 as novel inducers of ATF3 expression. Cisplatin is a DNA damaging agent widely used in various tumour types including lung, head and neck, and ovarian carcinomas. The HDAC inhibitor, SAHA, has recently been approved as a single agent in the treatment of subcutaneous T-cell lymphoma and HDACis themselves show potential for synergistic anti-cancer effects when used in combination with established chemotherapeutic drugs, including cisplatin. This study evaluates the mechanisms by which cisplatin and HDACi induce ATF3, as well as the role ATF3 plays as a mediator of cisplatin-induced cytotoxicity and the enhanced cytotoxicity between HDACi and cisplatin in combination. In this study, we demonstrate that cytotoxic doses of cisplatin and carboplatin consistently induced ATF3 expression in a panel of human tumour derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response (ISR) independent mechanism, all previously implicated in stress mediated ATF3 induction. Analysis of MAPKinase pathway involvement in ATF3 induction by cisplatin revealed a MAPKinase dependent mechanism. Cisplatin treatment, in combination with specific inhibitors to each MAPKinase pathway (JNK, ERK and p38) resulted in decreased ATF3 induction at the protein level. MAPKinase pathway inhibition led to decreased ATF3 mRNA expression and a reduction in the cytotoxic effects of cisplatin as measured by MTT cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific shRNAs also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3 -/- MEFs were shown to be less sensitive to cisplatin induced cytotoxicity as compared with ATF3+/+ MEFs. Taken together, we identified cisplatin as a MAPKinase pathway dependent inducer of ATF3 whose expression regulates in part cisplatin’s cytotoxic effects. Furthermore, we demonstrated that the HDAC inhibitor M344 was also an inducer of ATF3 expression at the protein and mRNA level in the same human derived cancer cell lines. Combination treatment with M344 and cisplatin lead to increased induction of ATF3 compared with cisplatin alone. Utilizing the MTT cell viability assay, M344 treatment was also shown to enhance the cytotoxic effects of cisplatin in these cancer cell lines. Unlike cisplatin, the mechanism of ATF3 induction by M344 was found to be independent of MAPKinase pathways. Utilizing ATF4 heterozygote (+/-) and knock out (-/-) mouse embryonic fibroblast (MEF) M334 induction of ATF3 was shown to depend on the presence of ATF4, a known regulator of ATF3 expression as part of the ISR pathway. HDACi treatment did not affect the level of histone acetylation associated with the ATF3 promoter as determined through Chromatin immunoprecipitation (ChIP) analysis, suggesting that ATF3 induction was not a direct effect of HDACi mediated histone acetylation. We also demonstrated that ATF3 regulates the enhanced cytotoxicity of M344 in combination with cisplatin as evidenced by attenuation of cytotoxicity in shRNAs targeting ATF3 expressing cells. This study identifies the pro-apoptotic factor, ATF3 as a novel target of M344, as well as a mediator of the co-operative effects of cisplatin and M344 induced tumour cell cytotoxicity.

Adult skeletal muscles can regenerate after injury due to the presence of satellite cells, a quiescent population of myogenic progenitor cells characterized by expressing the transcription factor Pax7. Once activated, satellite cells repair the muscle damage and replenish the stem cell niche due to the coordinated function of several transcription factors including Pax7 and the myogenic regulatory factors (MRFs). MRFs are skeletal muscle-specific transcription factors that can convert non-muscle cells into the myogenic lineage. MRFs are known to cooperate with other transcription factors in regulating the complex transcriptional network driving myogenic differentiation of muscle progenitors. The Six4 transcription factor emerges as a strong candidate for cooperating with MRFs. Six4 is expressed in skeletal muscles; the lack of a muscle development phenotype in Six4-null mice has been attributed to compensation by other Six family members. However, this did not exclude a critical role for Six4 during muscle development as Six1;Six4 double mutant mice show a more severe muscle phenotype than Six1 mutant mice. Nevertheless, the role of Six4 during adult muscle regeneration has never been addressed. I combined a partial loss-of-function of Six4 with high-throughput approaches to address the role of Six4 during adult skeletal muscle regeneration. I observed an important function of Six4 during muscle regeneration in vivo and in in vitro cell models. Using RNA interference assays against Six4 in tibialis anterior muscle regeneration after cardiotoxin-induced muscle damage, I observed for the first time that Six4 plays a role in proper muscle regeneration. The ability of the MRF MyoD, a central regulator of skeletal myogenesis, to convert a non-muscle cell model into the myogenic lineage was impaired with attenuated Six4 expression. I employed genome-wide approaches by combining ChIP-sequencing with gene expression profiling and identified a set of muscle genes coordinately regulated by both Six4 and MyoD. Throughout the genome, the cooperation between Six4 and MyoD was associated with binding of the H3K27me3 demethylase Utx and depletion of the H3K27me3 repressive chromatin mark. Together, these results reveal an important role for Six4 during adult muscle regeneration, and suggest a widespread mechanism of cooperation between Six4 and MyoD that correlates with modifying the epigenetic landscape of the regulatory regions of a large set of genes needed for efficient myogenesis.

De nombreuses bacteries pathogènes sont capables d'affecter les programmes transcriptionnels de la cellule hôte pendant l'infection. Cependant, les mécanismes contrôlant ce processus restent largement méconnus. En investigant les effets de la Listerai monocytogenes sur les modifications des histones de l'hôte, nous avons mis en évidence un nouveau mecanisme de régulation de transcription nécessaire pour la répression de certains gènes, pendant l'infection. Lors de l'infection par L. monocytogenes, le facteur de virulence sécrété, InlB, se lie au récepteur c-Met et active la signalisation par les intermédiaires PI3K et Akt. cette plateforme de signalisation est nécessaire pour la relocalisation de la deacetylase d'histone, SIRT2, au noyau et l'association à la chromatine.En caractérisant me mécanisme gouvernant la relocalisation nucléaire de SIRT2 lors de l'infection, nous avons démontrés que SIRT2 subit une modification post-traductionnelle. SIRT2 est déphosphorylée à un nouveau site de phosphorylation localisé à la partie terminale de la protéine. SIRT2 est recrutée au site de démarrage de la transcription des gènes réprimés lors de l'infection menant à la deacetylation de H3K18 et la répression transcriptionnelle. Nous avons mis en évidence que SIRT2 est détournée par L. monocytogenes et provoque une croissance des bactéries intracellulaires. Ces résultats démontrent un clef de SIRT2 en provoquant la deacetylation de H3K18 mors de l'infection et dévoilent un nouveau mécanisme imposée par les bactéries pathogènes dans le but de reprogrammer la cellule hôte. / Bacterial pathogens dramatically affect host cell transcription programs for their own profit, however the underlying mechanism in most cases remain elusive. While investigating the effects of listeria monocytogenes on histone modifications, we discovered a new transcription regulatory machanism by which the expression of genes is repressed, during infection. Upon infection by L. monocytogenes, the secret virulence factor, InlB, binds the c-Met receptor and activates signaling through PI3K/Akt. This signaling platform is necessary for causing the relocalization of the histone deacetylase, SIRT2, to the nucleus and associating to chromatin.In characterizing the mechanism governing SIRT2 nuclear relocazing during infection, our results have demonstrated that SIRT2 undergoes a post-translational modification. SIRT2 undergoes dephosphorylation at a novel N-terminal phospho-site. SIRT2 is recruiter to the transcription star sites of genes repressed during inection leading to H3K18 deacetylation and transcriptional repression.finnaly, my results demonstrate that SIRT2 is hijacked by L monocytogenes and promotes an increase in intracellular bacteria. Together, these data uncover a key role for SIRT2 mediated H3K18 deacetylation during infection and characterize a novel mechanisme imposed by a pathogenic bacteriomto reprogram the host cell.

Lactate has been long thought of as a dead-end waste product of glycolysis. In the brain, recent evidence has revealed a key role of L-lactate creating a paradigm-shift in our understanding of the neuronal energy metabolism. The Astrocyte neuron lactate shuttle (ANLS) model, has shown L-Lactate as the main energy substrate delivered by astrocytes to neurons to sustain neuronal oxidative metabolism. This metabolic coupling is an essential mechanism for long-term memory formation. Experimental evidence indicates that the role of lactate in cognitive function is not limited to being a neuronal metabolic substrate, but rather it is also an important signaling molecule for synaptic plasticity. One of the new emerging roles of lactate is its effect on gene expression levels; however, our current understanding of the mechanism of lactate effect on gene expression is rudimentary. Here, I investigate the role of lactate as an epigenetic modulator in neuronal cultures. First, I explored the effect of lactate on the transcriptome and methylome of the neuronal cells using primary neuronal cell culture models. Our results reveal a significant role for lactate in inducing neuronal cell differentiation. Following, I characterized a neuroblastoma cell line as our neuronal differentiation cell model and assessed its metabolic features relative to other immortal cell lines. Further, using the cell line in vitro model, I looked into the metabolic reprograming that occurs in parallel with the first indications of differentiation, focusing on lactate production rates. Subsequently, I investigated the role of lactate in differentiation through transcriptomic analysis. We show that lactate induced histone acetylation and promoted expression of dopaminergic markers, with a stronger effect of D-lactate over L-lactate. Further studies to establish potential linkages between those two pathways will enhance our understanding of the effect of lactate.

Cardiovascular disease continues to be the leading cause of morbidity and mortality worldwide with an estimated 17 million annual deaths. A majority of cases are attributed to disease affecting the vascular system including arterial, venous and lymphatic vessels. Despite progress in understanding the molecular bases of vascular development and disease, the role of chromatin modifying enzymes in vascular processes remains ill defined. Here we show that the histone-modifying enzyme Hdac3 is a critical regulator of lymphatic vascular development. Endothelial specific loss of Hdac3 in mice affects the development of lymphovenous and lymphatic valves resulting in aberrant blood lymph separation, lymphedema and complete lethality. We demonstrate that Hdac3 functions in a flow responsive manner to regulate the expression of Gata2, a transcription factor essential for lymphatic valve development. In response to flow, transcription factors Tal1, Ets1/2 and Gata2 recruit Hdac3 to an evolutionarily conserved intragenic enhancer of Gata2 gene. In turn, Hdac3 recruits p300, a histone acetyl transferase, to render activation of the Gata2 enhancer, and thus promotes Gata2 transcription. Together, our findings demonstrate the molecular basis by which cell extrinsic and intrinsic cues cooperate to regulate lymphatic development.

La schistosomiase est la seconde endémie parasitaire mondiale après le paludisme puisqu’en 2016, environ 200 millions de personnes ont été traitées pour cette parasitose. Plusieurs espèces de schistosomes peuvent être la cause de cette maladie dont Schistosoma mansoni, responsable de la schistosomiase intestinale. Son cycle de vie est complexe et comprend deux hôtes : un hôte définitif vertébré, l’Humain et un hôte intermédiaire qui est un mollusque d’eau douce. Actuellement, un seul médicament, le praziquantel, est utilisé contre toutes les espèces de schistosomes, mais son utilisation de façon massive et répétée à favoriser l’émergence de souches parasitaire tolérantes et/ou résistantes. La nécessité de trouver de nouveaux médicaments et de nouveaux traitements est donc devenue impérative.Les lysines désacétylases ou KDAC(s) constituent des cibles thérapeutiques intéressantes, notamment parce que ce sont des enzymes impliquées dans des processus cellulaires essentiels tels que la régulation de l'expression des gènes et du cycle cellulaire, ou encore la différenciation cellulaire. À ce jour, des inhibiteurs de KDAC(s) sont déjà approuvés dans le traitement du cancer et d’autres sont en essais cliniques.Chez S. mansoni, trois KDAC(s) de classe I ont été identifiées: HDAC 1, 3 et 8. D'autre part, l'utilisation d'inhibiteurs de KDAC(s) a démontré qu'il était possible d'induire l'apoptose et la mort des parasites en culture. Des études réalisées sur la protéine HDAC8 humaine et SmHDAC8 ont montré qu'il existait des différences significatives au niveau de la poche catalytique entre ces deux protéines. Ces données soulignent l’intérêt de développer des inhibiteurs sélectifs de SmHDAC8. Il est devenu, néanmoins essentiel de déterminer le rôle de SmHDAC8 dans la biologie du parasite et notamment ses partenaires protéiques. De ce fait, la première partie de ce travail de thèse s’est focalisé sur la mise en évidence de l’interactome de l’enzyme parasitaire SmHDAC8. Par l’utilisation du système en double hybride chez la levure et de la co-immunoprécipitation couplée à la spectrométrie de masse, nous avons identifié plusieurs partenaires de SmHDAC8 qui sont impliqués dans des processus essentiels à la cellule tel que la régulation de la transcription et de la traduction, le cycle cellulaire, le métabolisme, la réparation de l’ADN, la protéolyse ou encore le transport des protéines. Parmi ces interactants, nous avons également retrouvé la GTPase SmRho1 suggérant que l’enzyme SmHDAC8 serait impliqué dans la modulation de l’organisation du cytosquelette.Dans une seconde partie, nous nous sommes donc intéresser à l’interaction entre SmHDAC8 et la SmRho1. Nous avons initialement démontré que cette interaction était bien présente chez le parasite et notamment chez les vers adultes et les schistosomules. L’acétylation de SmRho1 sur la lysine K136 a également été mise en évidence par spectrométrie de masse et nous avons aussi pu observer un effet de l’inhibition de SmHDAC8 sur l’organisation du cytosquelette d’actine chez le parasite.Deux isoformes de SmRho1 (SmRho1.1 et SmRho1.2) ont été identifiées et caractérisées. La technique du double hybride chez la levure et la co-immunoprécipitation en ovocytes de xénope, a permis de démontrer que seule SmRho1.1 pouvait interagir avec SmHDAC8. Enfin, la caractérisation des motifs d'interaction entre SmHDAC8 et SmRho1.1, par co-immunoprécipitation en ovocytes de xénope, suggère que le domaine C-terminal de SmRho1 serait impliqué dans cette interaction. Ces données sont en faveur d’un rôle potentiel de SmHDAC8 dans la modulation du cytosquelette d’actine via son interaction spécifique avec la GTPase SmRho1.1. / Schistosoma mansoni is the major parasitic platyhelminth species causing intestinal schistosomiasis, for which around 200 million people are in need of treatment. The schistosome life cycle is complex and includes two hosts: a definitive mammalian host, mainly humans in the case of S. mansoni, and an intermediate snail host. Currently one drug, praziquantel, is the treatment of choice against all species of schistosomes, but tolerant/resistant strains have been isolated in endemic areas following its extensive use in mass treatment programs, as well as in laboratory studies. The need to find new drugs and new treatments is therefore imperative.Lysine deacetylases (KDACs) form a family of enzymes that are conserved in metazoans. They are attractive therapeutic targets in a variety of pathologies, particularly cancer, because they are involved in the regulation of gene transcription and several KDAC inhibitors have already been approved as drugs. Our previous studies identified and characterized three class I KDACS in Schistosoma mansoni: HDAC 1, 3 and 8. Invalidation of the transcription of SmHDAC8 by RNAi led to the impaired survival of the worms after the infection of mice, showing that it is a valid therapeutic target.The analysis of the 3D structure of SmHDAC8 by X-ray crystallography showed that the catalytic domain structure diverges significantly from that of human HDAC8 and this was exploited to identify selective inhibitors that induce apoptosis and death of the worms and are thus lead compounds for the development of novel anti-schistosomal drugs.The precise biological roles of mammalian or schistosomal HDAC8 are unknown and in order to determine why SmHDAC8 knockdown or inhibition causes apoptosis and death it is essential to study the cellular signaling pathways involving SmHDAC8. In the first part of the work described in this thesis, protein partners of SmHDAC8 were characterized by screening a yeast two-hybrid cDNA library and co-immunoprecipitation/mass spectrometry (MS) analysis. SmHDAC8 partners are involved in different processes, included transcriptional and translational regulation, cell cycle, metabolism, DNA repair, proteolysis or protein transport. Among the partners thus identified the schistosome orthologue of the human RhoAGTPase, suggesting that SmHDAC8 may be involved in the modulation of the organization of the cytoskeleton.The second part of the work focused on the interaction between SmHDAC8 and SmRho1. In adult worms and schistosomula S. mansoni, SmHDAC8 interacts with SmRho1 GTPase which is acetylated on lysine K136. Treatment with an SmHDAC8 inhibitor caused massive disruption of the worm and schistosomula actin cytoskeleton. We have also identified two closely related isoforms of SmRho1 (SmRho1.1 and SmRho1.2). By using two heterologous expression systems (the yeast two hybrid assay and Xenopus oocytes), we have demonstrated a specific interaction between SmHDAC8 and SmRho1.1 involving its C-terminal moiety. Our results show that SmHDAC8 is potentially involved in cytoskeleton organization via its interaction with the SmRho1.1 isoform.