Lors de la phase haploïde de la spermatogenèse (spermiogenèse) des mammifères, un important remodelage de la chromatine est nécessaire à la compaction de l'ADN. Au cours de ce remodelage, les histones sont remplacées successivement par les protéines de transition, puis finalement par les protamines. Ce processus implique une succession encore peu connue de modifications post-traductionnelles des histones telles que l'acétylation et la méthylation.Lors de récents travaux, notre laboratoire a montré des évidences suggérant que l'hyperacétylation de l'histone H4 (H4h) semble impliquée dans ce remaniement de la chromatine et fournirait un contexte favorable à l'apparition de cassures de l'ADN. Puisque le contexte chromatinien d'une spermatide diffère de celui d'une cellule somatique, la mise au point de techniques pouvant établir la distribution de H4h dans cette cellule germinale haploïde serait un atout précieux pour établir l'association de cette modification post-traductionnelle à la formation des cassures. Ce mémoire présente ainsi une démarche bipartite visant la mise au point d'approches microscopiques de même que le développement d'une approche d'immunoprécipitation de la chromatine pouvant s'appliquer à la chromatine particulière des spermatides. Grâce à la mise au point de la technique d'immunoprécipitation de la chromatine combinée à l'utilisation de biopuces d'ADN (ChIP-on-chip), nous tentons d'établir la cartographie de H4Ac sur une portion du chromosome X utilisé en guise de sentinelle. Avec la cartographie simultanée de [gamma]-H2AX (H2AFX) en tant que marqueur des cassures bicaténaires de l'ADN, nous tentons de vérifier, au niveau moléculaire, l'hypothèse d'une relation entre l'hyperacétylation des histones et l'apparition des cassures dans les spermatides allongeantes. La compréhension de la formation des cassures est importante puisque la réparation de l'ADN, dans cette cellule haploïde, ne peut compter sur la recombinaison homologue; l'instabilité génétique associée à ce phénomène pourrait fournir l'étiologie d'anomalies génétiques idiopathiques associées au développement de l'embryon. A l'aide d'anticorps couplés à des billes d'or, la microscopie électronique nous permet d'obtenir les premiers résultats d'une double détection de l'histone H4 hyperacétylée et des cassures de l'ADN au sein des noyaux des cellules germinales de la spermatogenèse. Les travaux d'immunoprécipitation de la chromatine dévoilent une répartition hétérogène de l'hyperacétylation de l'histone H4 au niveau du chromosome X, suggérant que l'hyperacétylation du génome lors de la spermiogenèse est progressive. Nous démontrons aussi qu'il existe une interaction entre cette chromatine et le variant d'histone [gamma]-H2AX. Les différences entre les patrons de distribution des deux histones ne permettent pas d'établir si l'hyperacétylation de la chromatine serait à l'origine du dépôt de [gamma]-H2AX, par le biais des cassures double brin.

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Eukaryote DNA is organised into the more compact nucleosome by wrapping 147bp of DNA around a histone octamer core. The N-terminal tails of the histones protrude through the DNA and can be modified by a variety of enzymes. Acetylation of Histone 4 Lysine 16 (H4K16ac) is an important modification associated with an increase in transcription, and in flies is an important component of the doseage compensation system. It is also unique amongst histone modifications in that it has been directly associated with chromatin decompaction. H4K16ac has been linked to development through its Histone Acetyltransferase, MOF. Deletion of MOF in mice leads to mass chromatin defects, and embryonic lethality prior to the blastocyst stage. I set out to understand the role of H4K16ac in differentiating Embryonic Stem cells (ES cells) and chromatin compaction in vivo. I generated a ChIP-seq profile for H4K16ac in undifferentiated ES cells, and after 3 days of retinoic acid (RA) differentiation. This revealed an association of H4K16ac with the promoters of transcribed genes in pluripotent ES cells, followed by loss H4K16ac on ES cell specific genes and gain of the modification on differentiation specific genes. There were some silent genes in ES cells, however, which were acetylated on their promoters. Through this study I also found that H4K16ac and MOF mark active enhancers in ES cells, along with H3K4me1 and H3K27Ac and p300. H4K16ac did not mark a known regulatory region in limb cells, and it is possible that it marks active enhancers only of ES cells. Furthermore, I looked at the compaction state large regions (>100kb) which lost H4K16ac upon differentiation by FISH, to determine if loss of H4K16ac could predict compaction. The regions selected showed no change in compaction state between UD and D3 cells, meaning that loss of H4K16ac does not directly lead to chromatin compaction in vivo. However loss of H4K16ac may be necessary for any subsequent compaction, or the change in compaction may take place at nucleosomal level. Finally, I attempted both to overexpress and reduce the level of MOF in ES cells. I was unable to manipulate the level of MOF in this cell type in either direction; expression of endogenous MOF was silenced after very little time, and stable MOF shRNA cell lines showed no reduction in levels of MOF. Therefore, potentially, dosage of MOF/H4K16ac in this cell type is critical. This study may help to understand the significance of H4K16ac in ES cell differentiation and chromatin compaction.

ARS2 is a member of the nuclear cap-binding complex (CBC) that is critical for a number of RNA processing pathways. The emerging model is that ARS2 acts as a master regulator of RNAPII transcript maturation by bringing capped RNA substrates together with the appropriate processing machinery. ARS2 is essential for early mammalian development but it remains unclear precisely how ARS2 functions in stem and progenitor cell maintenance and differentiation. The purpose of this study was to answer basic questions about the localization and function of ARS2 in muscle progenitor cells. Here I describe the localization of ARS2 in proliferating myoblasts and post-mitotic differentiating myotubes and show that disruption of ARS2 expression levels by knockdown or overexpression results in impaired myogenic differentiation. I also discovered a new isoform of ARS2 that is localized exclusively in the cytoplasm and found preliminary evidence that ARS2 is required for nonsense-mediated decay (NMD). This study includes the first evidence that an ARS2 isoform is expressed in the cytoplasm and opens the door for the discovery of new ARS2 functions beyond its reported roles in the nucleus. / Graduate

The N-terminal tails of histone proteins protrude from the nucleosome core and are extensively post-translationally modified. These modifications are proposed to affect many DNA-based processes such as transcription, DNA replication and repair. Post-translational modifications on histone tails do not act independently but are subject to crosstalk. One example of crosstalk is on histone H3 between lysine 14 (H3K14) and trimethylated lysine 4 (H3K4me3), a modification found at the 5’ end of most active or poised genes. In this work, Western blots and chromatin immunoprecipitation (ChIP) experiments show that different amino acid substitutions at histone H3 position 14 cause varying degrees of H3K4me3 loss, indicating that H3K14 is not essential for H3K4me3 but acts as a modulator of H3K4me3 levels. A neighbouring residue, H3P16 is also important for H3K4me3 and may operate in concert with H3K14 to control H3K4me3. These crosstalk pathways have gene-specific effects and the levels of H3K4me3 are influenced to different extents on genes that fall into functionally distinct classes. A model is proposed to explain how H3K14/H3P16 may exert these varying effects on H3K4me3 at individual genes. In addition to its ability to regulate H3K4me3, H3K14 also influences the levels of two modifications on H3K18, acetylation and monomethylation. A ChIP-sequencing experiment has shown that H3K18me1, a previously uncharacterised modification in S. cerevisiae, is widely distributed throughout the genome and correlates strongly with histone H3 levels. The potential for a functional acetyl/methyl switch at H3K18 is explored. Together, these data indicate that, with gene-specific effects, crosstalk between histone modifications may be even more complex than originally thought.

Barbituric acids have been historically classified as compounds that act on the central nervous system, and as such provide therapeutic uses as anxiolytics, sedatives, hypnotics, and anticonvulsants. Recent investigations of barbituric acid derivatives have provided scientists with information that barbituric acids may have applications in antibacterial, anti-chlamydial, anti-viral, as well as anti-cancer treatments. Additionally, recent literature accounts have indicated that barbituric acid derivatives may also act as immune modulators. The recent explorations of barbiturates and their potential anti-cancer and immune modulating properties are the subject of this work. Novel synthetic approaches to the development of new barbituric acid derivatives were explored thoroughly, and the mechanisms of these novel syntheses were detailed by experiment and spectroscopic characterizations. In many cases the reaction procedures were designed for large scale, efficient syntheses, that are directly applicable to pharmaceutical production of these potentially valuable therapeutic compounds. Several new products unique to barbituric acid reactions were characterized spectroscopically. Barbituric acid derivatives were the subject of biological evaluation, and the results are reported in this work. Overall, unique synthetic approaches to the production of novel barbituric acid derivatives were accomplished to create several new classes of barbiturates with potential applications in cancer treatment.

Une régulation stricte des marques d'histones est critique durant le développement et l'activité aberrante des enzymes modifiant les histones joue un rôle important dans la tumorigénèse. La lysine-spécifique déméthylase 1 (LSD1) a émergé comme régulateur clef de l'expression des gènes. De nombreuses études ont impliqué LSD1 dans le contrôle de la prolifération, de la différentiation et de la mort cellulaire. LSD1 est également exprimé de manière aberrante dans une grande variété de tumeurs. Cependant, les mécanismes par lesquels LSD1 contrôle l'homéostasie cellulaire et tissulaire in vivo restent à déterminer. Durant ma thèse, j'ai utilisé le modèle de l'aile chez la Drosophile afin d'explorer la manière dont dLSD1 contrôle la croissance cellulaire et tissulaire. Spécifiquement, j'ai découvert que (1) la déplétion de dLSD1 conduit à une réduction significative de la taille de l'aile chez la Drosophile. J'ai montré que cette réduction de taille est due à une réduction du nombre de cellules mais pas de leurs tailles. J'ai démontré que (2) la déplétion de dLSD1 dans le disque imaginal de l'aile - tissu larvaire précurseur de l'aile adulte - affecte la prolifération à travers un nombre réduit de cellules en mitoses et un nombre plus élevé de cellules en phase S. Mes études montrent également que (3) la déplétion de dLSD1 induit des dommages à l'ADN affectant la stabilité du génome et causant une augmentation aberrante de l'apoptose. De manière intéressante, j'ai pu montrer que dLSD1 et le facteur de transcription P53 contrôlent la taille de l'aile de manière synergique, suggèrent que P53, facteur de réponse au stress, puisse être impliqué dans la détection et la réponse aux dommages induits par la déplétion de dLSD1. Afin de mieux comprendre le rôle de dLSD1 dans la croissance de l'aile, j'ai comparé les transcriptomes de disques d'aile mutants pour dLSD1 versus disques d'aile sauvages par RNA-Seq. J'ai montré que (4) dLSD1 contrôle l'expression de multiples réseaux de gènes importants pour la croissance des organes et réprime les transposons. De plus, mes résultats montrent que (5) la déplétion des composants de la voie PIWI-interaction ARNs, régulateurs négatifs de l'expression des transposons, affecte également la taille de l'aile. Finalement, (6) j'ai réalisé un crible génétique afin d'identifier des modulateurs du phénotype de réduction de la taille de l'aile dépendant de dLSD1 et j'ai trouvé que WARTS, un composant de la voie Hippo, restreignant la taille des organes, interagit génétiquement avec dLSD1. En conclusion, ma thèse a démontré l'importance du rôle de dLSD1 dans le contrôle de la taille des organes et que sa déplétion cause l'arrêt du cycle cellulaire, une augmentation de l'apoptose. Ces effets phénotypiques sont probablement dus à une dérégulation de l'expression de réseaux géniques spécifiques et à l'instabilité génomique causée par la dé-répression des transposons. J'ai également identifié de nouvelles interactions génétiques entre dLSD1 et P53 ainsi qu'entre dLSD1 et WARTS. / A strict regulation of histone marks is critical for normal development and aberrant activity of histones modifying enzymes plays a role in tumorigenesis. The lysine-specific-demethylase 1 (LSD1) has emerged as a key regulator of gene expression. Multiple studies have implicated LSD1 in the control of cell proliferation, cell differentiation and cell death. Accordingly, LSD1 is aberrantly expressed in a wide variety of tumors. However, the underlying mechanisms by which LSD1 controls cell and tissue homeostasis in vivo remain to be fully elucidated. During my thesis, I used the Drosophila wing model system to explore how dLSD1 controls cell and tissue growth. Specifically, I found that (1) dLSD1 depletion results in a significant reduction of Drosophila wing size. I show that the size reduction is due to decreased cell number but not cell size. I demonstrated that (2) dLSD1 depletion in wing discs - larval precursor tissue of the wing - affects proliferation through a decreased mitotic cell number and increased S-phase cell number. My studies also show that (3) dLSD1 depletion induces DNA damage thus affecting genome stability and causing aberrant apoptosis. Interestingly, I show that dLSD1 and the transcription factor P53 synergistically control wing size, suggesting that P53, a stress-response factor, might be involved in sensing the damage induced by dLSD1 depletion. To better understand dLSD1 role in wing growth, I compared the transcriptome of dLSD1-depleted wing discs to wild-type discs by RNA-Seq. I found that (4) dLSD1 controls the expression of multiple gene networks important for organ growth and represses transposons. Additionally, my results show that (5) depletion of PIWI-interacting RNAs pathway components, negative regulators of transposons expression, also affects wing size. Finally, (6) I performed a genetic screen to identify modulators of the dLSD1 wing size phenotype and I found that WARTS, a component of the Hippo pathway, which restricts organ size, genetically interacts with dLSD1. Overall, my work shows that dLSD1 plays an important role in organ size control and that its depletion causes cell cycle arrest and increased apoptosis. These phenotypic effects probably reflect the misregulation of the expression of specific gene networks and the genomic instability caused by de-repression of transposons. I also identified new interplay between dLSD1 and P53 as well as between dLSD1 and WARTS.

L’émergence et le développement des maladies métaboliques est sous le contrôle de multiples facteurs génétiques et environnementaux. Le diabète et la résistance à l’insuline sont des maladies métaboliques caractérisées par des défauts dans la sécrétion de l’insuline ou son utilisation périphérique, ou les deux. L’insuline est l’hormone clé de l’utilisation du glucose, et régule également transcriptionnellement et épigénétiquement l’expression des gènes.En travaillant sur le muscle, l’implication de l’épigénétique dans la régulation de l’expression des gènes de la voie de l’insuline a été mis en évidence. L’hexokinase 2 (HK2) est régulée par l’insuline et participe au métabolisme glucidique. Le rôle de l’épigénétique y est démontré avec l’augmentation de l’acétylation des histones autour du site d’initiation de la transcription (SIT) de HK2 et l’accumulation d’une isoforme permissive des histones, H2A.Z. Ces deux phénomènes sont le signe d’une transcription permissive.Nous avons ensuite étudié le rôle de l’acétylation des histones dans les régulations amenées par l’insuline dans les myotubes L6. Nous avons utilisé le butyrate, un inhibiteur des histones deacetylase (HDACi), dans un contexte d’insulino-résistance induite par une lipotoxicité. Le butyrate a en partie restauré la sensibilité à l’insuline visible au niveau des phosphorylations de la PKB (protein kinase B) et de la MAPK (Mitogen-activated protein kinase), inhibées par le traitement au palmitate. Le butyrate a augmenté l’expression de l’ARNm et de la protéine d’IRS1. La surexpression génique d’IRS1 est épigénétique-dépendante car liée à une augmentation de l’acétylation des histones au SIT d’IRS1.L’ensemble de ces résultats démontre l’existence d’un lien entre les modifications épigénétique et l’action de l’insuline. Cela suggère qu’une intervention pharmacologique sur la machinerie épigénétique pourrait être un moyen d’améliorer le métabolisme, et l’insulino-résistance / Diabetes and insulin resistance are metabolic diseases characterized by altered glucose homeostasis due to defects in insulin secretion, insulin action in peripheral organs, or both. Insulin is the key hormone for glucose utilization and regulates gene expression via transcriptional and epigenetic regulations.We determined the epigenetic implications in the regulation of expression of insulin signaling pathway genes. Hexokinase 2 (HK2) is known to be upregulated by insulin and directs glucose into the glycolytic pathway. In L6 myotubes, we demonstrated that insulin-induced HK2 gene expression rely on epigenetic changes on the HK2 gene, including an increase in histone acetylation around the transcriptional start site (TSS) of the gene and an increase in the incorporation of the histone H2A.Z isoform – a histone variant of transcriptionally active chromatin. Both are epigenetic modifications compatible with increased gene expression.To elucidate the role of histone acetylation in the regulation of insulin signaling and insulin-dependent transcriptional responses in L6 myotubes, we investigated the effects of butyrate, an histone deacetylase inhibitor (HDACi), in a model of insulin resistance induced by lipotoxicity. Butyrate partly alleviated palmitate-induced insulin resistance by ameliorating insulin-induced PKB (protein kinase B) and MAPK (Mitogen-activated protein kinase) phosphorylations, downregulated with exposure to palmitate. Butyrate induced an upregulation of IRS1 gene and protein expression. The transcriptional upregulation of IRS1 was proven to be epigenetically regulated, with butyrate promoting increased histone acetylation around the TSS of the IRS1 gene.These results support the idea of the existence of a link between epigenetic modifications and insulin action. Pharmacological targeting of the epigenetic machinery might be a new approach to improve metabolism, especially in the insulin resistant condition.Key words: Muscle, insulin resistance, epigenetic, chromatin, histone acetylation, histone deacetylase inhibitor (HDACi), butyrate, palmitate

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.

In 2106, there were 171,550 new cases of blood cancers and over one million people in the United States living with one of these disorders. Bone marrow transplants have good outcomes, but these procedures require a donor who is a perfect match, and thus many patients are unable to receive treatment. It is important to find patient-derived treatments, such as molecules which stimulate hematopoietic stem cell (HSC) formation without the need for a donor. Therefore, a study was initiated to use human-induced pluripotent stem cell (hiPSC) technology to make a patient-derived, personalized HSC. Epigenetic regulators are divided into readers, writers, and erasers, and each of these classes has shown some effect on HSC formation. Writers add functional groups to deoxyribonucleic acid (DNA) and histone proteins, whereas erasers remove them. Readers are groups on transcription factors which interpret these changes and increase or decrease the recruitment of transcriptional machinery accordingly. In this study, a screen of 12 different candidate molecules with distinct epigenetic targets in casper zebrafish was conducted at 36 hours postfertilization (hpf) to reveal increases or decreases in definitive HSC development. The two writer molecules, C646 (histone acetyltransferase, or HAT, inhibitor) and OICR9249 (WDR5 inhibitor), and the two eraser molecules, Ex-527 (Sirt1 inhibitor) and JIB-04 (bromodomain inhibitor), showed varying degrees of increasing HSC formation. Of these molecules, C646 created the most significant increase and was further tested in the zebrafish at 48 and 72 hpf and in a murine model using ex vivo technique and a colony-forming unit (CFU) assay. In contrast to these results, the two eraser molecules, entinostat (class I histone deacetylase, or HDAC, inhibitor) and vorinostat (general HDAC inhibitor), were found to decrease HSC formation in zebrafish. The overall findings of this study provide insight into specific epigenetic regulators in HSC development and identify particular epigenetic markers that could regulate HSC formation from endothelial cells. This discovery will be a stepping stone in utilizing patient-derived hemogenic endothelial cells as a novel source of bone marrow-independent HSCs to treat patients with leukemia, lymphoma, and bone marrow cancers. / 2019-07-11T00:00:00Z