Skeletal muscle specification and differentiation programs are regulated by the myogenic regulatory factors which include Myf5, MyoD, myogenin and Mrf4. Upstream of the MRFs, the transcription co-activators and other intracellular and extracellular signals play crucial roles in regulating skeletal myogenesis. Histone acetyltransferase activity of p300 is required for Myf5 and MyoD expression. Furthermore, the MyoD core enhancer region is indispensable for MyoD expression. However, the mechanism by which p300 activates MyoD gene expression is to be determined. The histone acetyltransferase activity of p300 can be inhibited by small molecule inhibitors such as curcumin. Thus, using the inhibitor approach on stem cells is useful to investigate the role of p300 in activating MyoD expression during myogenesis. We here show that curcumin was able to inhibit stem cell determination and differentiation into skeletal myocytes. We also show that p300 is present, and histone acetylation is high at the core enhancer region. Therefore, we provide evidence that p300 is directly involved in MyoD gene expression during skeletal myogenesis.

Skeletal muscle specification and differentiation programs are regulated by the myogenic regulatory factors which include Myf5, MyoD, myogenin and Mrf4. Upstream of the MRFs, the transcription co-activators and other intracellular and extracellular signals play crucial roles in regulating skeletal myogenesis. Histone acetyltransferase activity of p300 is required for Myf5 and MyoD expression. Furthermore, the MyoD core enhancer region is indispensable for MyoD expression. However, the mechanism by which p300 activates MyoD gene expression is to be determined. The histone acetyltransferase activity of p300 can be inhibited by small molecule inhibitors such as curcumin. Thus, using the inhibitor approach on stem cells is useful to investigate the role of p300 in activating MyoD expression during myogenesis. We here show that curcumin was able to inhibit stem cell determination and differentiation into skeletal myocytes. We also show that p300 is present, and histone acetylation is high at the core enhancer region. Therefore, we provide evidence that p300 is directly involved in MyoD gene expression during skeletal myogenesis.

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Epigenetic mechanisms play tremendous roles in the development and management of neural processing. The important mechanisms include inactivation of transcription via methylation, histone modification via acetylation/deacetylation, and miRNA regulation. These modifications allow for expression or silencing of genes, without manipulation of nucleotide sequence. An individual's internal and external environments provide input for quotidian epigenetic regulation. Aberrations in the form of regulation have been increasingly linked to neurological disorders, in addition to the established correlation to tumorigenesis. In recent years, deviations from normal epigenetic patterns have been observed in cases of Alzheimer's disease (AD). The brains of patients with AD have been shown to display significantly less methylation overall, as compared to age-matched controls. Of particular concern, the methylation, which normally keeps the promoter of the APP gene silenced, occur far less frequently in AD patient allowing for the progression of amyloid deposition and subsequent tau pathology. In addition to the hypomethylation present in AD, many AD cases present with a concurrent hypoacetylation on histones in the hippocampus. There is strong evidence suggesting that the reduced levels of acetylation are due to over-activation of histone deacetylases. Post-mortem examinations of the brains of AD patients have shown that the brain-derived neurotrophic gene, which is crucial for neural processing associated with maturation and memory, has low levels of acetylation halting its transcription. While low levels of methylation and acetylation seem to contribute to the pathogenesis of AD, regulatory miRNA levels can have adverse effects whether they are aberrantly reduced or increased. Patients with AD tend to show abnormally augmented expression of miRNA-125b, miRNA-128, and miRNA-9 in the hippocampus, while a reduced expression of miRNA-107. Deregulation of these miRNAs have been linked to the progression of AD and include amyloid deposition, tau pathology, and oxidative stress through inflammatory processes. The latter quandary of oxidative stress has been shown to be crucial for the early progression of AD. Reactive oxygen species disallow the methylation of genes due to steric hindrance at the CpG islands of DNA where DNA methyltransferases act. Research shows that increases in oxidative stress are correlated to decreases in methylation, which allows for APP expression. While these alterations to normal epigenetic patterns occur internally, there is a breadth of changes that the external environment imposes to exacerbated AD pathogenesis. Most heavily studied of these external environmental factors is lead exposure. There is a strong correlation between lead exposure in individuals who carry the ApoE4 gene and increased mRNA transcription of the APP gene. Lead is thought to demethylate the promoter of the APP gene and allow for amyloid processes to occur. Inadequate nutrition, specifically deficits in choline and folate, has been linked to hypomethylated states due to an inefficient "methylation/remethylation cycle" leading to an accumulation of homocysteine characteristic of AD. With the emphasis epigenetic deregulation has in the progression of AD, epigenetic treatments need to be seriously considered as therapeutic avenues. Current drugs treat the symptoms and acute conditions of AD, but through epigenetic modifications, the pathology of the diseases can be directly addressed. Potential therapeutic avenues include the use of methyl donors, highly specific histone deacetylase inhibitors, and miRNA biomarkers. Methyl donors can help alleviate the hypomethylated state and prevent further APP expression and amyloid deposition. Currently, the histone deacetylase inhibitors are being used as global inhibitors, but have adverse effects including non-specific and premature cell death. By further researching these inhibitors and finding a mechanism to attack specific histone deacetylases (such as HDAC6 in AD), the efficacy of this aspect of treatment will be greatly increased. The current use of miRNAs as epigenetic regulators to turn off unwanted genetic expression is ineffective due to a major problem of effective delivery to target zones. By using the gene sequences of miRNAs as biomarkers, an AD patient's genomic sequence can be mapped, marking which areas require regulation. This process is necessary because of the inter-individuality of miRNA regulation between each case of AD. Also, the problem of some anti-miRNA molecules not being able to cross the blood brain barrier needs to be addressed using a novel transport mechanism, as direct brain injections are not feasible. The simplest, and highly effective, therapeutic avenue is a healthy lifestyle. Daily exercise and proper nutrition hinder inflammatory process and oxidative stress and can prevent progression of AD through allowing higher brain perfusion for cognitive functioning.

Social stress is the most common stressor experienced by humans and exposure to social stress is thought to cause or exacerbate neuropsychiatric illness. Social stress also leads to behavioral and physiological responses in many animal models that closely mirror the symptoms of fear and anxiety in humans. Our laboratory uses Syrian hamsters to study behavioral responses to social stress. Hamsters are highly territorial, but after losing an agonistic encounter, hamsters exhibit a striking behavioral change, abandoning all territorial aggression and instead becoming highly submissive. This behavioral shift is termed conditioned defeat. Epigenetic modifications, such as changes in histone acetylation, are a possible molecular mechanism underlying such behavioral shifts. Histone deacetylase (HDAC) inhibitors have been shown to enhance fear learning and conditioned place preference for drugs of abuse, while suppressing histone acetylation with histone acetyltransferase (HAT) inhibitors impairs long-term memory formation. The first goal of this study was to test the hypothesis that histone acetylation is a molecular mechanism underlying conditioned defeat. We found that animals given an HDAC inhibitor systemically before social defeat later exhibited increased conditioned defeat. This treatment also suppressed defeat-induced immediate-early gene activity in the infralimbic cortex but not the basolateral amygdala. Next, we demonstrated that administration of an HDAC inhibitor in the infralimbic cortex before defeat enhanced stress-induced behavioral responses while HAT inhibition blocked these behavioral changes. Although both males and females exhibit conditioned defeat, the behavioral expression is more pronounced in males. We next used transcriptomic analysis to investigate potential genetic mechanisms leading to this sexually dimorphic expression and to further delineate the role of acetylation in stress-induced behavioral changes. We sequenced the whole brain transcriptome of male and female hamsters as well as the transcriptome of basolateral amygdala, a nucleus necessary for the acquisition and expression of conditioned defeat, of dominant, subordinate, and control animals. Our analysis revealed that numerous genes relating to histone acetylation, including several HDACs, were differentially expressed in animals of different social status and between sexes. Together, these data support the hypotheses that histone modifications underlie behavioral responses to social stress and that some of these modifications are sexually dimorphic.

Mechanical ventilation, a lifesaving intervention for patients with acute respiratory distress syndrome (ARDS), also unfortunately contributes to excessive mechanical stress and impaired lung physiological and structural integrity. We have elsewhere established the pivotal role of increased nicotinamide phosphoribosyltransferase (NAMPT) transcription and secretion as well as its direct binding to the toll-like receptor 4 (TLR4) in the progression of this devastating syndrome; however, regulation of this critical gene in ventilator-induced lung injury (VILI) is not well characterized. On the basis of an emerging role for epigenetics in enrichment of VILI and CpG sites within the NAMPT promoter and 5'UTR, we hypothesized that NAMPT expression and downstream transcriptional events are influenced by epigenetic mechanisms. Concomitantly, excessive mechanical stress of human pulmonary artery endothelial cells or lipopolysaccharide (LPS) treatment led to both reduced DNA methylation levels in the NAMPT promoter and increased gene transcription. Histone deacetylase inhibition by trichostatin A or Sirt-1-silencing RNA attenuates LPS-induced NAMPT expression. Furthermore, recombinant NAMPT administration induced TLR4-dependent global H3K9 hypoacetylation. These studies suggest a complex epigenetic regulatory network of NAMPT in VILI and ARDS and open novel strategies for combating VILI and ARDS.

During cell activation the tightly compacted DNA is made available to DNA-binding proteins allowing the induction of gene transcription. In the resting cell, DNA is packaged into chromatin whose fundamental subunit is the nucleosome, composed of an octamer of four core histones (H) 3, 4, 2A and 2B. During the induction of gene transcription, modification of histones, by acetylation, methylation etc., results in unwinding of the DNA, permitting access of large DNAbinding proteins, such as RNA polymerase II, and subsequent induction of gene transcription. This investigation initially examined the effects of pro-inflammatory stimuli LPS and TNF-a on the production of IL-8 in a macrophage cell line (U937 cells) and in two T-cell lines (Jurkat and HUT-78 cells) as a marker of NF-KB-directed inflammatory gene expression. The ability of dexamethasone (Dex) and triamcinolone acetonide (TA) (synthetic glucocorticoid agonists) to suppress expression of the inflammatory cytokine IL-8 and to regulate histone acetylation was also investigated in these cells. LPS and TNF-a caused an increase in IL-8 expression, which was further enhanced by the histone deacetylases inhibitor trichostatin A (TSA), suggesting a role for histone acetylation in IL-8 production in these cells. Dex and TA, repressed LPS- and TNF-a -induced IL-8 expression in all three cell lines. This effect of both Dex and TA was attenuated by TSA in all cell lines studied, where the effect of TSA was greater in TA stimulated cells. Stimulation of all cell lines with LPS and TNF-a induced acetylation of H4 lysine residues (K5, 8, 12 and 16), the highest elevation of which was for K8 and K12. Also demonstrate is a K5 and K16 specificity of acetylation by glucocorticoids, apparent in all cell lines studied. Dex and, to a greater extent, TA suppressed LPS- and TNFa-induced K8 and K12 acetylation. TSA attenuated the inhibitory effect of the glucocorticoids for all three cell lines. An inCrease in HDAC activity with GCs was observed and ChiP assay showed these events occur on the native IL-8 promoter via histone acetylation. Further studies investigated whether there were any links between histone acetylation and the regulation of apoptosis. It was showed that TSA induced apoptosis in cells previously stimulated with the inducer of oxidative stress hydrogen peroxide (H20 2). Studies into the activation of caspase 3 in LPS- and TNF-a stimulated cells revealed that the combinatory effect of Dex or TA with TSA Significantly enhanced expression of the marker in all three cell lines. In resting cells, Dex, and TA, in the presence of TSA downregulated caspase 3 expression. These findings support the notion that glucocorticoid actions on apoptosis is mediated, at least in part, through an action on histone acetylation. Finally, histone acetylation was investigated in vivo in two rat models of inflammation and in human subjects with inflammatory bowel disease (IBD). The results showed an increase in histone H4 acetylation lysine specificity of acetylation on K8 and K12 in inflamed tissue and Peyer's patches in animal models and in IBD patients. Whereas H3 acetylation was not elevated to the same extent in tissue and was restricted to the mantle zone of Peyer's patches. In general, the present studies on histone acetylation and inflammation (in animal models and IBD patients) underlined the possibility of a general mechanism linking activation of the transcription factor NFKB with histone acetylation. The ultimate objective of this work is to aid in the understanding of the mechanisms of how deregulation of chromosome structure leads to progression of the disease state. This knowledge may aid in the development of new therapeutic approaches or improved glucocorticoids.

Listeria monocytogenes est l'un des meilleurs organismes modèles pour l'étude des interactions bactérie-hôte. Ce pathogène intracellulaire facultatif peut infecter, survivre et se répliquer dans le cytoplasme des cellules eucaryotes, démontrant la co-évolution étroite de Listeria avec son hôte. Le style de vie intracellulaire de ce pathogène implique la manipulation de divers composants de la cellule hôte, dont l'un est la chromatine. En induisant des modifications de la chromatine au niveau des histones, Listeria peut influencer le programme transcriptionnel de l'hôte. Ce projet de thèse porte sur une modification spécifique des histones, la désacétylation de la lysine 18 de l'histone H3, induite par la désacétylase de l'hôte Sirtuin 2 (SIRT2) lors de sa relocalisation du cytoplasme vers le noyau pendant l'infection. Le détournement de SIRT2 par Listeria fournit un système idéal pour étudier les mécanismes de la localisation subcellulaire de SIRT2, qui est mal comprise, et c'est le but de cette thèse. En utilisant la spectrométrie de masse, nous avons identifié une nouvelle modification posttraductionnelle de SIRT2, la phosphorylation de la sérine 25 (S25), ciblée spécifiquement par l'infection, et essentielle pour l'association de SIRT2 à la chromatine. Nous avons caractérisé le complexe moléculaire impliqué dans la déphosphorylation de SIRT2-S25 et nous montrons que cette modification est essentielle pour contrôler la fonction de SIRT2 en tant que répresseur transcriptionnel, et est nécessaire pour une infection efficace. Notre approche protéomique a aussi permis la caractérisation d'un interactome de SIRT2. De nombreuses protéines ont été identifiées et quelques-unes ont été confirmées et étudiées pour leur rôle dans le transport nucléo-cytoplasmique de SIRT2. De plus, une collaboration au laboratoire a mis au jour un mécanisme de subversion de la réponse aux dommages de l'ADN de l'hôte par Listeria. Dans son ensemble, ce travail a contribué à la compréhension de mécanismes originaux de l’interaction entre les bactéries et la chromatine et a révélé un processus cellulaire contrôlant la localisation subcellulaire et la fonction de la protéine de l’hôte SIRT2. / One of the best model organisms for the study of bacterial-host interactions is Listeria monocytogenes. This facultative intracellular pathogen can infect, survive, and replicate in the cytoplasm of eukaryotic cells, demonstrating the close co-evolution of Listeria with itshost. The intracellular life style of this pathogen involves manipulation of various host cellcomponents, one of which is chromatin. By inducing chromatin modifications at the level of histones, Listeria can influence the transcriptional program of the host. This thesis focuses on one specific histone modification, deacetylation of histone H3 of lysine 18, which is induced by the host deacetylase Sirtuin 2 (SIRT2) upon its relocalization from the cytoplasmto the nucleus during infection. Hijacking of SIRT2 by Listeria provides an ideal system tostudy the mechanisms of SIRT2 subcellular localization, which is poorly understood, and is the purpose of this thesis. By using mass spectrometry we have identified a novel posttranslational modification of SIRT2, Serine 25 (S25) phosphorylation, specifically targeted byinfection, and essential for SIRT2 chromatin association. We have characterized themolecular complex involved in dephosphorylating SIRT2-S25 and we show that this modification is essential for controlling SIRT2 function as a transcriptional repressor andnecessary for productive infection. Our proteomic approach further allowed the characterization of a SIRT2 interactome. Many proteins were identified and a few wereconfirmed and studied for their role in nucleo-cytoplasmic shuttling of SIRT2. In addition, a laboratory collaboration uncovered a mechanism for subversion of the host DNA DamageResponse by Listeria. As a whole, this work has contributed to the understanding of original mechanisms of chromatin-bacteria cross talk, and has revealed a cellular process controlling subcellular localization and function of the host protein SIRT2.

Humans are exposed to arsenicals through many routes with the most common being drinking water. Exposure to arsenic has been associated with an increased incidence of skin, lung, liver, prostate, and bladder cancer. Although the relationship between arsenic exposure and carcinogenesis is well documented, the mechanisms by which arsenic participates in tumorigenesis are not fully elucidated. We evaluated the potential epigenetic component of arsenical action by assessing the histone acetylation and DNA methylation state of 13,000 human gene promoters in a cell line model of arsenical-mediated malignant transformation. We show changes in histone H3 acetylation and DNA methylation occur during arsenical-induced malignant transformation, each of which is linked to the expression state of the associated gene. These epigenetic changes occurred non-randomly and targeted common promoters whether the selection was performed with arsenite [As(III)] or with the As(III) metabolite monomethylarsonous acid [MMA(III)]. The epigenetic alterations of these promoters and associated malignant phenotypes were stable after the removal of the transforming arsenical. One of the affected regions was the promoter of WNT5A. This gene is transcriptionally activated during arsenical induced malignant transformation and its promoter region exhibited alterations in each of the four histone modifications examined which were linked to its transcriptional activation. Experimental reduction of WNT5A transcript levels resulted in abrogated anchorage independent growth, suggesting a participative role for the epigenetic remodeling of this promoter region in arsenical-induced malignant transformation. Taken together, these data suggest that arsenicals may participate in tumorigenesis by stably altering the DNA methylation and histone modifications associated with targeted genes, uncovering a likely set of participative genes and representing a mechanism to potentially explain the latency associated with arsenic-induced malignancy.

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.