Dissertations / Theses on the topic 'Epigenetics: histone deacetylase'

Epigenetic regulation is a mechanism by which heritable changes in gene expression are controlled by chromatin status rather than primary DNA sequence. Changes in chromatin structure affect accessibility of DNA elements to the transcriptional machinery and thus affect transcription activity of the gene. A key event in this process is reversible modification of core histones, which is catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDs, HDAs, or HDACs). In general, histone deacetylation is related to transcriptional gene silencing, whereas acetylation is associated with gene activation.To study the role of histone deacetylase in plant gene regulation and development, we generated constitutive antisense histone deacetylase 1 (CASH) transgenic plants. AtHD1 is a homolog of RPD3 protein, a global transcriptional regulator in yeast. Expression of the antisense AtHD1 caused dramatic reduction in endogenous AtHD1 transcription, resulting in accumulation of acetylated histones. Down-regulation of histone deacetylation caused a variety of growth and developmental abnormalities and ectopic expression of tissue-specific genes. However, changes in genomic DNA methylation were not detected in repetitive DNA sequences in the transgenic plants.We also identified a T-DNA insertion line in exon 2 of AtHD1 gene (athd1-t1), resulting in a null allele at the locus. The complete inhibition of the AtHD1 expression induced growth and developmental defects similar to those of CASH transgenic plants. The phenotypic abnormalities were heritable across the generations in the mutants. When the athd1-t1/athd1-t1 plants were crossed to wild-type plants, the mutant phenotype was corrected in the F1 hybrids, which correlated with the AtHD1 expression and reduction of histone H4 Lys12 acetylation. Microarray analysis was applied to determine genome-wide changes in transcriptional profiles in the athd1-t1 mutant. Approximately 6.7% (1,753) of the genes were differentially expressed in leaves between the wild-type (Ws) and the athd1-t1 mutant, whereas 4.8% (1,263) of the genes were up- or down-regulated in flower buds of the mutant. These affected genes were randomly distributed across five chromosomes of Arabidopsis and represented a wide range of biological functions. Chromatin immunoprecipitation assays indicated that the activation for a subset of genes was directly associated with changes in acetylation profiles.

2017 - 2018
The unicellular eukaryotic organism Candida albicans is one of the most important fungi in medicine, used as experimental model to study fungal pathologies and the underlying biology of dimorphic fungi. This fungus is a component of the human mucosal microbiota; it is normally found as a commensal in vaginal and oral mucosal districts, and in the gastrointestinal tract. However, at these sites it behaves as an opportunistic pathogen: following environmental changes in the host this fungus can become pathogenic, leading to invasive and lethal infections in susceptible individuals. Therefore, during the last decades, Candida has emerged as a major human fungal pathogen responsible for an extended variety of mucosal and systemic infections. The ability of this opportunistic fungus to cause and propagate successfully infections is linked to the expression of different and alternative virulence factors. Its key virulence trait is its morphological plasticity: its ability to shift from oval budding yeasts to elongated cell structures (pseudohyphal and hyphal filaments) responding to diverse and numerous environmental cues. Adaptive chromatin changes promote Candida variability and phenotypic plasticity. Therefore, epigenetic regulation of gene expression is considerably involved in the morphogenesis and virulence of this polymorphic fungus. Adaptation of C. albicans to drug pressure, yeast to hyphae transition, biofilm formation, white-opaque switch are important pathogenic mechanisms, in which posttranslational histone modifications play a prominent role. In particular, acetylation – deacetylation of histones modulates morphological switch in C. albicans and, consequently, this modification is correlated to fungal virulence. Histone H3 Lys56 acetylation (H3K56ac) is an important post-translational modification in yeast, that contributes to fungal genome stability. In C. albicans, acetylation levels of H3K56 are regulated by two enzymes with fungal-specific properties: the acetyl transferase Rtt109p and the NAD+-dependent histone deacetylase (sirtuin) Hst3p, encoded, respectively, by RTT109 and HST3 genes. HST3 is an essential gene for C. albicans: homozygous deletion mutants for this sequence are not viable. The essentiality of HST3 gene for C. albicans viability, combined with fungal-specific properties of its enzyme Hst3p, make it an attractive potential target for antifungal therapy. Focus of this study was to examine the molecular pathways regulated by Hst3p of C. albicans. Considering that deletion of this sirtuin is lethal for this fungus, it is intuitive to understand that it regulates vital process in the fungal cell. As histone deacetylase, Hst3p modulates gene expression, in particular induces the repressive state of chromatin, inhibiting transcriptional activation. Consequently, deletion of this gene or repression of its protein induces dysregulation of gene expression, leading to fungal death. Based on these considerations I focused my interest on this fungal protein, in order to characterize its role in C. albicans biology and virulence and its downstream targets, as potential new targets for the treatment of fungal infections. Substrate of Hst3p is the acetylated histone H3 Lysine 56. To analyse the effect of Hst3p inhibition on its substrate, I grew up C. albicans in the presence of nicotinamide (NAM), a non-specific sirtuin-inhibitor. Mass spectrometry analysis allowed me to evaluate, for the first time, acetylation levels of H3K56 during Candida growth and their variations upon NAM treatment. Interestingly, nicotinamide treatment induced the accumulation of H3K56 acetylation levels during C. albicans growth, demonstrating the inhibitory effect of NAM on Hst3p activity. One important attribute of Candida is its morphological variability, which is the result of the adaptive response to environmental changes which in turn this morphological plasticity triggers infection. To study the role of Hst3p in fungal virulence, I analyzed the potential involvement of this sirtuin in phenotypic switch. Morphological analyses were performed under NAM treatment to investigate the effect of Hst3p inhibition on cell duplication and filamentation. Hst3p inhibition resulted in a reduction of fungal growth rate and alteration of yeast-hyphae transition in C. albicans: NAM induced an abnormal filamentous growth, with formation of V-shaped hyphae under conditions that normally maintain the yeast shape of Candida. This phenotypic analysis was performed also on two azole-resistant strains of C. albicans to investigate the role of Hst3p in drug resistance. Hst3p inhibition had similar effect on the resistance strains compared to the control wild-type strain, inducing morphological alterations and reducing cell duplication rate. These phenotypic assays highlighted the effect of Hst3p inhibition on regulation of Candida morphology. V-shaped hyphae formation in Candida in non-inducing filamentation conditions require the structural rearrangement of the whole cell, which is a result of alteration in gene expression, induced by NAM treatment. Based on these considerations, I analysed the entire transcriptome of C. albicans strain SC5314 by RNA-sequencing to investigate whether the inhibition of Hst3p by NAM was responsible for changes in the pattern of expression of Virulence-related Genes. This analysis showed that gene categories most dysregulated upon NAM treatment are those associated with hyphal growth, adherence, white-opaque switch, drug resistance and cell wall maintenance. RNA-Sequencing analysis allowed to identify some dysregulated genes upon Hst3p inhibition; considering that no alteration in gene expression was detected for up-stream members of pathways that control these dysregulated genes, to verify if the expression of these genes is regulated epigenetically by H3K56 acetylation, future experiments of chromatin immunoprecipitation (ChIP) will be performed. To select inhibitors of Hst3p to be used as potential fungicidal compounds, I expressed and purified both the full length and a short sequence of recombinant Hst3p. These proteins did not show enzymatic activity, due probably to denaturing conditions used during purification, that were necessary considering that both the full length and the short sequence of Hst3p were complexed to the bacterial molecular chaperon GroEL. To improve protein folding in bacterial host and avoid denaturing conditions for purification, I expressed and purified recombinant Hst3p from a bacterial system over-expressing some molecular chaperons. Once determined the enzymatic activity of recombinant protein, an enzymatic assay will be set up, useful to screen and select small molecules, potential inhibitor of the fungal sirtuin Hst3p, which could be used as antifungal compounds. [edited by Author]
XXXI ciclo

Congenital heart disease is the most common congenital anomaly, affecting approximately 1% of all live births each year. Although clinical interventions are improving, many affected infants do not survive to adulthood. Congenital cardiac defects originate from disturbances during development, making the study of mammalian cardiogenesis critical to improving outcomes for infants with congenital heart disease. Development of the mammalian heart involves epigenetically-driven specification and commitment of a diverse landscape of cardiac progenitors. Recent studies determined that chromatin modifying enzymes play a previously underappreciated role in the pathogenesis of congenital heart defects. This thesis investigates the functions of Hdac1 and Hdac2, highly homologous Class I histone deacetylases, during early murine cardiac development. We establish that Hdac1 and Hdac2 cooperatively regulate cardiogenesis in distinct cardiac progenitor populations during development. Together, our findings demonstrate that Hdac1 and Hdac2 are critical mediators of the earliest stages of mammalian cardiogenesis through a variety of spatiotemporally specific, redundant, and dose-sensitive roles and indicate they may play important roles in the pathogenesis of human congenital cardiac defects.

INTRODUCTION Epigenome modifications and metabolic dysregulation have been shown to be connected to disease states like obesity and to the associated comorbidities. Epigenome modifiers such as histone deacetylases are involved in the regulation of adipose tissue pathophysiology. However, their specific role in adipocyte differentiation is still a matter of research. Moreover, in obesity excessive accumulation of fat, especially in visceral depots, triggers a low-grade inflammation that is responsible of metabolic dysfunction. The action of immune cells within adipose tissue affects normal metabolic homeostasis. PREVIOUS RESULTS Previous in vitro results from our group showed that inhibition of class I HDACs with MS-275, during early stage of adipocyte differentiation, leads to reduction of lipid droplets size. This phenomenon was accompanied by increased expression of genes regarding adipocyte functionality, lipolysis and fatty acid β-oxidation. However, these events were not observed in terminally differentiated adipocytes. Moreover, in vivo studies showed that HDAC3, a member of class I HDACs, acts as a molecular brake of metabolic rewiring that supports browning in WAT. AIM of this thesis was to further elucidate how class I HDACs are involved in adipocyte differentiation and in determining the metabolic phenotype of pre-adipocytes. Moreover, we aimed to investigate the changes of immune cell populations at different time points of high fat feeding, to fully characterize the phenotypical consequences of Hdac3 ablation in mice. In parallel, we wanted to identify key pathways and early events evoked by Hdac3 gene inactivation through transcriptomic and epigenomic analysis. RESULTS In vitro experiments on adipocytes precursors treated with MS-275 at the beginning of differentiation showed increased expression of genes belonging to mitochondrial activity and browning, which was accompanied by increased H3K27 acetylation of Pparg and Ucp1 gene enhancers. In vivo studies in H3atKO mice, exposed to HFD for different periods (4, 9, 16 weeks, respectively), showed changes in immune cell populations. The number of total macrophages significantly increased in epiWAT of H3atKO mice compared to control floxed (FL) mice at all the three periods of treatment. Moreover, H3atKO mice were able to maintain higher ratio of M2 pro-resolving vs M1 pro-inflammatory macrophages from 4 through 16 weeks of HFD feeding. In H3atKO mice fed LFD for 4 weeks we found upregulation of pathways regarding the futile cycle of simultaneous fatty acid synthesis and β-oxidation. Moreover, KO_LFD vs FL_LFD mice shown upregulation of pathways such as ferroptosis, amino acid biosynthesis and valine, leucine and isoleucine degradation pathways, while focal adhesion and extracellular receptor interaction were downregulated. One of the top downregulated gene was neuronatin (Nnat); we found a hypoacetylated region upstream the Nnat TSS. CONCLUSIONS In vitro results provided evidences regarding the role of class I HDACs in adipocyte differentiation. Their inhibition at the beginning of differentiation promotes an epigenetic imprinting towards oxidative and brown-like phenotype. In vivo experiments revealed that mice lacking Hdac3 were able to maintain a higher ratio of M2 vs M1 macrophages during an inflammatory stimulus such as HFD feeding. Such feature could support a pro-resolving inflammatory process. Omic analysis confirmed the futile cycle of fatty acid metabolism previously observed in our H3atKO model and highlighted interesting new pathways that better characterize H3atKO mice.

An understanding of the human fetal to adult hemoglobin switch offers the potential to ameliorate β-type globin gene disorders such as sickle cell anemia and β-thalassemia through activation of the fetal γ-globin gene. Chromatin modifying complexes, including MBD2-NuRD and GATA-1/FOG-1/NuRD play a role in γ-globin gene silencing, and Mi2β (CHD4) is a critical component of NuRD complexes. In the studies presented in Chapter 2, we observed that the absence of MBD2 in a sickle cell mouse model leads to a decrease in the number of sickled cells observed in the peripheral blood, and significantly increases survival in these mice. Although further studies will be necessary to fully understand the effect of MBD2 knockout in sickle cell disease mice, absence of MBD2 appears to partially ameliorate the sickle cell anemia phenotype in vivo. In the studies presented in Chapter 3, we observed that knockdown of Mi2β relieves γ-globin gene silencing in β-YAC transgenic murine CID hematopoietic cells and in CD34+ progenitor derived human primary adult erythroid cells. We show that independent of MBD2-NuRD and GATA-1/FOG-1/NuRD, Mi2β binds directly to and positively regulates both the KLF1 and BCL11A genes, which encode transcription factors critical for γ-globin gene silencing during β-type globin gene switching. Remarkably, less than 50% knockdown of Mi2β is sufficient to significantly induce γ-globin gene expression without disrupting erythroid differentiation of primary human CD34+ progenitors. These results indicate that Mi2β is a potential target for therapeutic induction of fetal hemoglobin.

Disruptions in cardiac development cause congenital heart disease, the most prevalent and deadly congenital malformation. Genetic and environmental factors are thought to contribute to these defects, however molecular mechanisms remain largely undefined. Recent work highlighted potential roles of chromatin- modifying enzymes in congenital heart disease pathogenesis. Histone deacetylases, a class of chromatin-modifying enzymes, have developmental importance and recognized roles in the mature heart. This thesis aimed to characterize functions of Hdac3 in cardiac development. We found loss of Hdac3 in the primary heart field causes precocious progenitor cell differentiation, resulting in hypoplastic ventricular walls, ventricular septal defect, and mid- gestational lethality. In primary heart field progenitors, Hdac3 interacts with, deacetylates, and functionally suppresses transcription factor Tbx5. Furthermore, a disease-associated Tbx5 mutation disrupts this interaction, rendering Tbx5 hyperacetylated and hyperactive. By contrast, deletion of Hdac3 in second heart field progenitors bypasses these defects, instead causing malformations in the outflow tract and semilunar valves, with lethality prior to birth. Affected semilunar valves and outflow tract vessels exhibit extracellular matrix and EndMT defects and activation of the Tgfβ1 signaling pathway. In normal second heart field development, Hdac3 represses Tgfβ1 transcription, independent of its deacetylase activity, by recruiting the PRC2 methyltransferase complex to methylate the Tgfβ1 promoter. Importantly, knockouts of Hdac3 in differentiated cardiac cells do not fully recapitulate the progenitor-specific knockout phenotypes. These results illustrate spatiotemporal roles of Hdac3, both deacetylase-dependent and deacetylase-independent, in cardiac development, suggesting that dysregulation of Hdac3 in cardiac progenitor cells could be a contributing factor in congenital heart disease pathogenesis.

Disruptions in cardiac development cause congenital heart disease, the most prevalent and deadly congenital malformation. Genetic and environmental factors are thought to contribute to these defects, however molecular mechanisms remain largely undefined. Recent work highlighted potential roles of chromatin- modifying enzymes in congenital heart disease pathogenesis. Histone deacetylases, a class of chromatin-modifying enzymes, have developmental importance and recognized roles in the mature heart. This thesis aimed to characterize functions of Hdac3 in cardiac development. We found loss of Hdac3 in the primary heart field causes precocious progenitor cell differentiation, resulting in hypoplastic ventricular walls, ventricular septal defect, and mid- gestational lethality. In primary heart field progenitors, Hdac3 interacts with, deacetylates, and functionally suppresses transcription factor Tbx5. Furthermore, a disease-associated Tbx5 mutation disrupts this interaction, rendering Tbx5 hyperacetylated and hyperactive. By contrast, deletion of Hdac3 in second heart field progenitors bypasses these defects, instead causing malformations in the outflow tract and semilunar valves, with lethality prior to birth. Affected semilunar valves and outflow tract vessels exhibit extracellular matrix and EndMT defects and activation of the Tgfβ1 signaling pathway. In normal second heart field development, Hdac3 represses Tgfβ1 transcription, independent of its deacetylase activity, by recruiting the PRC2 methyltransferase complex to methylate the Tgfβ1 promoter. Importantly, knockouts of Hdac3 in differentiated cardiac cells do not fully recapitulate the progenitor-specific knockout phenotypes. These results illustrate spatiotemporal roles of Hdac3, both deacetylase-dependent and deacetylase-independent, in cardiac development, suggesting that dysregulation of Hdac3 in cardiac progenitor cells could be a contributing factor in congenital heart disease pathogenesis.

Doctor of Philosophy
Psychological Sciences
Mary E. Cain
Gene-environment interactions play a significant role in drug abuse and addiction. Epigenetics (the study of how environmental stimuli alter gene expression) has gained attention in recent years as a significant contributor to many behavioral phenotypes of drug addiction. The current study sought to determine if differential rearing conditions can alter a specific epigenetic mechanism, histone deacetylase (HDAC), and how HDAC inhibition can affect drug-taking and drug-seeking behaviors differently among enriched, isolated, or standard-housed rats. Ninety male Sprague-Dawley rats were reared for 30 days in enriched (EC), isolated (IC), or standard (SC) conditions prior to amphetamine (0.03, 0.05, 0.1 mg/kg/infusion, i.v.) self-administration, extinction, or reinstatement sessions. Trichostatin A (TsA; 0.3 mg/kg, i.v.), an HDAC inhibitor, was injected 30 min prior to drug-taking or drug-seeking sessions. Results indicated that EC rats self-administered less amphetamine (0.03 mg/kg/infusion) than IC rats. No significant effects of TsA administration were found on general self-administration for any of the three amphetamine doses. While enrichment facilitated the extinction of active lever pressing, there was also a mild facilitation of extinction in IC-TsA rats compared to IC-vehicle counterparts. Lastly, TsA administration decreased cue-, but not drug-induced reinstatement, with IC-TsA rats exhibiting significantly attenuated cue-induced reinstatement compared to IC-vehicle rats. These findings suggest that differential rearing can alter HDAC mechanisms that can change drug-seeking behaviors, particularly in rats reared in isolated conditions. While TsA-induced HDAC inhibition may be less protective against general amphetamine self-administration, it may decrease drug-seeking tendencies during relapse that are induced by the reintroduction of contextual environmental cues heavily associated with drug reward.

Human adenovirus (HAdV) mainly causes minor illnesses, but can lead to severe disease and death in both immunocompromised and immunocompetent patients. In such cases, the current standards of treatment often do not improve disease outcome and no approved antiviral therapy against HAdV exists. Since HAdV relies on cellular machinery to assist in the progression of the virus lifecycle, we hypothesized that small molecules targeting certain cellular proteins/pathways, without severely affecting cell health, may serve as effective anti-HAdV compounds. Thus, we aimed to identify novel inhibitors of HAdV, and investigate the molecular mechanism to determine new therapeutic targets for intervention in HAdV infection. We first examined the antiviral properties of pan-histone deacetylase (HDAC) inhibitor SAHA and found that the drug affects multiple stages of the HAdV lifecycle, resulting in significant reductions in virus yield. SAHA was effective in decreasing gene expression from clinically relevant HAdV serotypes. Subsequent investigations on the role of HDACs in HAdV infection led us to determine that class I HDAC activity, mainly HDAC2, is necessary for optimal viral gene expression. Using a wildtype-like HAdV reporter construct that allows us to monitor virus replication by fluorescence microscopy, we then designed an efficient system for screening small molecules to identify novel HAdV inhibitors. We screened over 1300 small molecules, and the screen was sensitive enough to detect compounds with both robust and modest antiviral activity. Several positive hits were validated to reduce HAdV gene expression and yield from infected cells. Further investigation on the efficacy of these compounds and the mechanism behind their inhibition of HAdV can lead to the discovery of new pharmacological targets and the development of more effective antivirals.

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A doença de Alzheimer (DA) é uma desordem neurodegenerativa crônica caracterizada clinicamente pela perda progressiva de função cognitiva, distúrbios neuropsiquiátricos e comportamentais. Patologicamente esta doença caracteriza-se pelo acúmulo anormal do peptídeo β-amilóide (Aβ) no córtex e no hipocampo, emaranhados neurofibrilares intracelulares formados por tau hiperfosforilada, disfunção progressiva sináptica e, posteriormente perda neuronal. As opções terapêuticas disponíveis melhoram os sintomas, mas não impedem a progressão da doença, portanto, ainda está faltando uma estratégia terapêutica efetiva para DA. Há estudos relacionados à utilização de terapia epigenética para o tratamento da DA, a terapêutica mais desenvolvida é a que envolve a classe dos inibidores das deacetilases (HDACs). Assim, este trabalho tem por objetivo investigar o efeito protetor do inibidor da HDAC ácido hidroxâmico de suberoilanilida (SAHA) em um modelo de DA em camundongos. Para isso, foram utilizados 50 camundongos Swiss adultos, pesando entre 30-35 g, divididos em dois experimentos. No primeiro, os camundongos foram divididos em 6 grupos que receberam uma injeção de Aβ1-42 via intracerebroventricular (i.c.v.) no início da experiência (exceto o grupo Sham que foi utilizado como controle) para investigar a atividade das histonas   acetiltransferase (HATs) e HDAC, determinação dos níveis do fator neurotrófico derivado do cérebro (BDNF), expressão do mRNA de BDNF e modulação da via (cAMP/PKA/CREB) em uma curva de tempo (6 horas, 1, 3, 7 e 21 dias). Ao final de cada tempo, os animais foram submetidos ao teste cognitivo e foram eutanasiados. O córtex pré-frontal e o hipocampo foram removidos para posteriores análises. No segundo experimento, os camundongos foram dividos em 4 grupos: Grupo Controle (sham+veículo); Grupo Aβ1-42 (Aβ1-42 + veículo); Grupo SAHA (25 mg/kg, via intraperitoneal) (sham + SAHA); Grupo Interação (Aβ1-42 + SAHA). O peptídeo Aβ1-42 ou o veículo foram infundidos por injeção i.c.v. e, um dia depois, iniciou-se o tratamento, por via i.p., durante 21 dias. Ao final do experimento os animais foram submetidos ao teste cognitivo, eutanásiados para retirada das estruturas cerebrais. As amostras foram utilizadas para a determinação dos níveis de BDNF, expressão do mRNA de BDNF, atividade enzimática das histonas (HDAC e HATs) e regulação da via cAMP/PKA/CREB. O presente estudo observou deficiências significativas causadas pela Aβ1-42 na memória (Labirinto Aquático de Morris), bem como causou desequilíbrio das enzimas HAT/HDAC, redução de cAMP, PKA e CREB e BDNF no córtex pré-frontal e hipocampo de camundongos. A inibição de HDAC, com SAHA demostrou neuroproteção nas alterações comportamentais e neuroquímicas induzidas por Aβ1-42. Estes dados mostram que a acetilação através da inibição do HDAC, desempenha um papel fundamental na mediação da memória e demonstra que SAHA poderá ser uma ferramenta médica promissora na abordagem terapêutica para o tratamento da DA.
Alzheimer's disease (AD) is a chronic neurodegenerative disorder characterized clinically by the progressive loss of cognitive function, neuropsychiatric and behavioral disorders. Pathologically this disease is characterized by the abnormal accumulation of β-amyloid peptide (Aβ) in the cortex and hippocampus, intracellular neurofibrillary tangles formed by hyperphosphorylated tau, progressive synaptic dysfunction and, later, neuronal loss. The available therapeutic options improve the symptoms, but they do not prevent the progression of the disease, therefore, an effective therapeutic strategy for AD is still lacking. There are studies related to the use of epigenetic therapy for the treatment of AD, the most developed therapy is that involving the class of deacetylase inhibitors (HDACs). Thus, this work aims to investigate the protective effect of the HDAC inhibitor hydroxamic acid suberoilanilide (SAHA) in an AD model in mice. For this, 50 Swiss adult mice weighing between 30-35 g were used, divided in two experiments. In the first, the mice were divided into 6 groups that received an injection of Aβ1-42 via the intracerebroventricular (i.c.v.) at the beginning of the experiment (except the Sham group that was used as control) to investigate histone activity acetyltransferase (HATs) and HDAC, determination of brain derived neurotrophic factor (BDNF) levels, expression of BDNF mRNA and modulation of the pathway (cAMP / PKA / CREB) in a time curve (6 hours, 1, 3, 7 and 21 days). At the end of each time, the animals were submitted to the cognitive test and were euthanized. The prefrontal cortex and hippocampus were removed for further analysis. In the second experiment, the mice were divided into 4 groups: Control Group (sham + vehicle); Group Aβ1-42 (Aβ1-42 + vehicle); SAHA group (25 mg / kg, intraperitoneal route) (sham + SAHA); Interaction Group (Aβ1-42 + SAHA). The Aβ1-42 peptide or vehicle was infused by i.c.v. and one day later the treatment was started i.p. for 21 days. At the end of the experiment the animals were submitted to the cognitive test, euthanasia for removal of the cerebral structures. The samples were used for the determination of BDNF levels, expression of BDNF mRNA, histone enzymatic activity (HDAC and HATs) and regulation of the cAMP / PKA / CREB pathway. The present study observed significant deficiencies caused by Aβ1-42 in memory (Morris Aquatic Labyrinth), as well as caused imbalance of HAT / HDAC enzymes, cAMP, PKA and CREB and BDNF reduction in the prefrontal cortex and hippocampus of mice. Inhibition of HDAC with SAHA demonstrated neuroprotection in behavioral and neurochemical changes induced by Aβ1-42. These data show that acetylation through inhibition of HDAC plays a key role in memory mediation and demonstrates that SAHA may be a promising medical tool in the therapeutic approach to AD.

Cancer is the second leading cause of death in the United States (more than half a million people each year), and even with billions of dollars in medical effort patients are rarely cured. This dissertation research is devoted to meeting this medical need by providing new cancer therapeutics that are more potent and safer than current chemotherapies. This is achieved by using two state of the art anticancer “warheads”: 1) gold nanoparticle (AuNP) technology and 2) a new class of epigenetic anticancer small molecules, histone deacetylase inhibitors (HDACi). These warheads are then selectively delivered to cancer cells via “homing devices” targeted to receptors that are overexpressed in the cancers. This work primarily focuses on the androgen receptor (AR) to target prostate cancer. The 1st chapter sets the stage, providing scientific rationale and background for the central hypothesis: small molecules that engage the AR can, upon conjugation to a therapeutic agent, enable selective delivery of that agent to prostate cancer cells. Chapter 2 delves into the structural molecular biology of the androgen receptor. There is a survey of the crystallographic data for all nuclear receptors, providing structural information which is used to build AR homology models for antagonist and inverse agonist modes of ligand binding. These models are used to design AR targeting ligands (Chapters 3, 5, 6 and 7). The application of the targeting technology is illustrated by attaching them to AuNPs for selective delivery to prostate cancer cells (Chapter 3). Next, in order to appreciate the importance of using targeting agents in HDACi cancer therapeutics, we reviewed this recently emerged field in Chapter 4. In this chapter we argue that the failure of HDACi in solid tumors, despite more than 500 clinical trials in the last decade, is primarily due to an inability of these small molecules to accumulate at effective concentrations in the cancer. We provide an analysis of the paradigms being pursued to overcome this barrier, including HDAC isoform selectivity, localized administration, and targeting cap groups to achieve selective tissue and cell type distribution. In Chapter 5, this last approach (targeting cap groups, or a “homing device”) is illustrated with HDACi targeted to prostate cancer via antiandrogens that bind the AR. The second generation of improved “homing devices” is disclosed in Chapter 6 (for both AuNPs and HDACi), in addition to preliminary ADMET data and safety studies in mice. Excitingly, our three dimensional understanding of binding to the AR allowed design and structure-activity-relationship studies that lead to the first reported examples of AR inverse agonists (Chapter 7) Several points of significance: • AuNP targeted to AR ∙ have the strongest binding affinity ever reported (IC50 ~14 picomolar) ∙ are actively recruited to prostate cancer cells ∙ overcome treatment resistance in advanced prostate cancer cells ∙ exhibit nanomolar anticancer potency ∙ resolved the identity of the “membrane AR” as the GPRC6A • HDACi targeted to AR ∙ have HDACi activity and AR binding affinity superior to their clinical precursors ∙ exhibit potent AR antagonist activity ∙ induce AR translocation to the nucleus in a HDACi dependent fashion ∙ selectively and potently kill prostate cancer cells that express AR ∙ are safer than Tylenol®, as tested in small animals • Pure AR binding ligand studies ∙ resulted in the discovery of the first examples of AR inverse agonists, which are vastly more potent that clinically available antiandrogens for prostate cancer ∙ work via a never-before-seen mechanism of action, by localizing to the nucleus and recruiting corepressors to actively shut off AR genes

L’épigénétique représente les modifications de l’expression génique, sans altérer la séquence nucléique de l'ADN. L'un des mécanismes de régulation est le remodelage de la chromatine qui s’effectue via les histones acétyltransférases et les histones désacétylases (HDAC) permettant ou non la transcription de gènes. Une expression anormale des HDAC est corrélée à de nombreuses maladies (dépendance à l'alcool, inflammation ainsi que les maladies cardiovasculaires et neurodégénératives, cancers…). Il est primordial de cibler la sélectivité d’une isoforme parmi les 11 connues des HDAC zinc dépendantes pour éviter les effets secondaires. Le but de la recherche était de concevoir et de synthétiser de nouveaux composés, de vérifier leur activité inhibitrice vis-à-vis des HDAC de classe I ou II et leur cytotoxicité sur quatre lignées cellulaires: HaCaT, V79-4, SH-SY5Y et PC12. Ainsi, nous nous sommes concentrés sur les pharmacomodulations du ZBG, de l’espaceur et de la tête de molécules connues tels que le MS-275 (sélectif de la classe I des HDAC), les SAHA et TSA (espaceur en C5 ou C6) avec une forte activité inhibitrice vis-à-vis des HDAC, mais non sélectifs. Nous nous sommes concentrés sur les pharmacomodulations de l'HDACI connu modifiant le domaine de liaison au zinc ZBG (sulfonylhydrazide, catéchol), la nature de l’espaceur (alkyl, aryl) et le groupe de reconnaissance de surface (bis-aryl, adamantyl, indolopyridazinone). Une bibliothèque de 57 nouveaux composés a été créée en trois séries. Aucun d'entre eux n'a montré d'activité inhibitrice satisfaisante. Les composés sélectionnés n'ont pas montré d'activité cytotoxique sur les lignées de cellules neuronales. Sur la base de cette recherche, il est possible de créer de nouveaux composés dans la série indolopyridazinone afin de les tester
Epigenetics represents changes in gene expression without altering the nucleic sequence of DNA. One of the main mechanisms of regulation of gene expression is chromatin remodeling via histone acetyltransferases and histone deacetylases (HDAC), which may or may not allow gene transcription. An abnormal expression of HDACs is correlated with many diseases (alcohol dependence, inflammation as well as cardiovascular and neurodegenerative diseases, cancers...). It is essential to target the selectivity of one isoform among the 11 known zinc-dependent HDACs to avoid side effects. The aim of the research was to design and synthesize new compounds, verify their inhibitory activity against class I or II HDACs and their cytotoxicity on four cell lines: HaCaT, V79-4, SH-SY5Y and PC12. We focused on the pharmacomodulations of ZBG, the linker and the cap of known molecules such as MS-275 (selective for class I of HDACs), SAHA and TSA (spacer in C5 or C6) with a strong inhibitory activity towards HDACs, but not selective. We concentrated on the pharmacomodulations of known HDACI modifying the zinc binding domain (sulfonylhydrazide, catechol), the nature of the spacer (alkyl, aryl) and the surface recognition group (bis-aryl, adamantyl, indolopyridazinone). A library of 57 new compounds was designed in three series. None of them showed satisfactory inhibitory activity. The selected compounds did not show cytotoxic activity on neuronal cell lines. Based on this research, it is possible to create new compounds in the indolopyridazinone series in order to test them

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.

The MBD2-NuRD co-repressor complex is an epigenetic regulator of the developmental silencing of embryonic and fetal β-type globin genes in adult erythroid cells as well as aberrant methylation-dependent silencing of tumor suppressor genes in neoplastic diseases. Biochemical characterization of the MBD2-NuRD complex in chicken erythroid cells identified RbAp46/48, HDAC1/2, MTA1/2/3, p66α/β, Mi2α/β and MBD2 to comprise this multi-protein complex. In the work presented in Chapter 2, we have pursued biophysical and molecular studies to describe a previously uncharacterized domain of human MBD2 (MBD2IDR). Biophysical analyses show that MBD2IDR is an intrinsically disordered region (IDR). Despite this inherent disorder, MBD2IDR increases the overall binding affinity of MBD2 for methylated DNA. MBD2IDR also recruits the histone deacetylase core components (RbAp48, HDAC2 and MTA2) of NuRD through a critical area of contact requiring two contiguous amino acid residues, Arg286 and Leu287. Mutation of these critical residues abrogates interaction of MBD2 with the histone deacetylase core and impairs the ability of MBD2 to repress the methylated tumor suppressor gene Prostasin in MDA-MB-435 breast cancer cells. These findings expand our knowledge of the multi-dimensional interactions of the MBD2-NuRD complex that govern its function. In Chapter 3, we have discussed a novel mechanism for MBD2-mediated silencing of the fetal γ-globin gene. Through microarray expression analyses in adult erythroid cells of MBD2-/- mice, we identified ZBTB32 and miR-210 as downstream targets of MBD2. Over-expression of ZBTB32 and miR-210 in adult erythroid cells causes increased expression of the silenced fetal γ-globin gene. Thus, our results indicate that MBD2 may regulate γ-globin gene expression indirectly though ZBTB32 and miR-210 in adult erythroid cells.

Enviromental and nutritional stimuli can affect chromatin modifications. Several evidences highlighted the role of epigenetics in the regulation of energy homeostasis. Among the epigenetic modifications, the acetylation/deacetylation of histone tails it has been associated to metabolic disorders such as obesity and diabetes that are often linked with defects in oxidative metabolism. In this doctorate thesis it has been investigated the effect of the selective inhibition of class I histone deacetylases (HDACs) in metabolic profile and functionality of key organs participating in energy metabolism. Cultured myotubes and primary brown adipocytes treated with a class I-specific HDAC inhibitor showed higher expression of Pgc-1α, increased mitochondrial biogenesis, and augmented oxygen consumption. In vitro and in vivo ChIP experiments suggested that these beneficial effects are mediated by inhibition of HDAC3. Treatment of diet induced obese mice with a class I-selective HDAC inhibitor MS275 reduced body weight and improved glucose tolerance. Moreover inhibition of class I HDACs increased thermogenic capacity and brown adipose tissue (BAT) functionality, reduced abdominal fat and decreased size of subcutaneous and visceral adipocytes. Visceral and subcutaneous white adipose tissues from these mice showed higher expression of functionality markers, along with enhanced oxidative metabolism. Interestingly in this mouse model MS275 was able to induce browning of visceral white adipose tissue. In vitro experiments indicated that early inhibition of class I HDACs in adipose precursors reprograms cell fate of adipose precursors toward an oxidative and more functional adipose phenotype.

Histone deacetylases (HDACs) are epigenetic enzymes that modulate chromatin structure through the deacetylation of lysine residues in histones, playing a crucial role in cell viability, cell cycle progression and tumorigenesis. Yet the role of individual HDACs in these biological processes remains enigmatic. Inappropriate recruitment of HDACs is involved in the pathogenesis of several forms of leukemia and several lines of evidence point to a role for HDACs in tumor progression, consistent with the anti-proliferative and apoptotic effects of HDAC inhibitors (HDACi). In this regard, HDACs are considered promising targets for development of new molecules for cancer therapy. To date, some HDACi which have a broad antitumor activity and low toxicity towards normal cells, such as Romidepsin (Depsipeptide or FK228) and SAHA have been approved by U.S. food and drug administration (FDA) for the treatment of cutaneous T-cell lymphoma (CTCL). Moreover, HDACi are undergoing clinical trials for the treatment of hematological malignancies as well as for solid tumors. Most of the HDACi available at the moment are not isoform specific, being active on more than one HDAC. Thus, to design more selective HDACi for cancer therapy it is important to elucidate the role of individual HDACs. Acute Promyelocytic Leukemia (APL) is the first model disease in which the involvement of HDACs has been documented. APL is a subtype of Acute Myeloid Leukemia (AML), a cancer of blood and bone marrow, which is characterized by hyperproliferation of immature granulocytes blocked at the promyelocytic stage. It is genetically associated with a chromosomal translocation t(15;17)(q22;q21), which encodes the oncogenic fusion protein PML-RARα found in more than 90% of APL patients. In murine models of APL that recapitulate the human disease, PML-RARα induces a “pre-leukemic” stage with long latency and without an overtly dramatic phenotype before full leukemic transformation. In fact, for this reason it is assumed that in addition to this oncogenic fusion protein, other genetic hits are required for clonal expansion of leukemic blasts. This fusion protein recruits a number of chromatin modifier enzymes such as HDACs and DNA methyltransferases (DNMTs) to the promoter of retinoic acid (RA) target genes and transcriptionally silence them, leading to the myeloid differentiation block. Furthermore, PML-RARα causes the impairment of p53 pathway by deacetylation and degradation of p53 through the recruitment of HDAC- containing complexes. HDACs from class I (HDACs 1, 2 and 3) have been found associated with PML-RARα paving the way for the use of HDACi for APL treatment. Recently, a study on APL, which has been conducted by Santoro et al., showed that among class I HDACs, HDAC1 and to a lesser extent HDAC2 have a dual role in APL development and maintenance. In fact, while they behave as oncosuppressors at the early stages, they function as oncogenes in established tumor cells. Since inhibition of HDAC1 and HDAC2 in pre-leukemic stage leads to the acceleration of the disease in murine models of leukemia, it suggests caution in the clinical utility of epidrugs that target any of these two HDAC isoforms. Moreover, it has been shown that the expression of HDAC3, which associates with nuclear hormone corepressor and silencing mediator of retinoid hormone (NCoR/SMRT) complex, is frequently increased in tumors, while Hdac3 downregulation results in reduced proliferation and survival of tumor cells. In view of these observations, in this study we functionally assessed the role of HDAC3 in the development and maintenance of APL. To achieve our goal, we have dissected the role of HDAC3 in two different phases of the disease: pre-leukemic phase and full-established leukemia. The murine model of APL, which we used, is the mCGPR/PR mouse model in which PML-RARα is expressed under the control of the cathepsin G promoter. The mice show a very long latency (the pre-leukemic phase) associated with high penetrance (more than 90% of the mice develop APL). We characterized the role of HDAC3 through a functional knock-down approach, assessing its impact on cellular differentiation, proliferation and the ability to influence the transplantation of HSCs and APL cells. Indeed, Hdac3-KD in vitro reduced the proliferative potential of both pre-leukemic and full leukemic cells and boosted their differentiation, suggesting that HDAC3 plays the role of an oncogene in APL initiation and progression. These results were not restricted to APL, because lymphoma driven by c-myc overexpression and leukemia driven by MLL-AF9, were both impaired in cell growth upon Hdac3-KD. In vivo, inoculation of Hdac3-KD pre-leukemic cells into lethally irradiated recipient mice or inoculation of Hdac3-KD APL cells into the recipient mice did not result in leukemia development or progression, respectively. These results suggest that HDAC3 can be considered as a target for epidrugs in the treatment of hematological malignancies. Thus, we assessed this hypothesis with the treatment of pre-leukemic and leukemic cells with the HDAC3 selective inhibitor, RGFP966. Indeed, inhibition of HDAC3 enzymatic activity with RGFP966, phenocopied Hdac3-KD phenotypes in pre-leukemic and leukemic cells confirming the putative oncogenic role of HDAC3. In conclusion, my PhD project has expanded our comprehension about the role of HDAC3 in hematological malignancies and is beginning to unravel alternative views on the targets of epidrugs for the treatment of leukemic patients.

In this study, we investigated the role of individual class I histone deacetylases (HDACs) namely Hdac1, -2 and -3 using retroviral RNA interference to define their specific contribution to control an inducible gene expression program, namely inflammatory gene expression in 3T3 fibroblasts and primary macrophages. In addition to genes showing the expected transcriptional de-repression, we observed 8/20 genes in a test set being down-regulated following individual HDAC depletion. The requirement for HDACs function in gene induction as opposed to the more commonly observed role as transcriptional repressors may either underlie an indirect consequence of impaired HDAC mediated repression or a direct involvement of Hdac1 and Hdac3 in inducible gene activity. Therefore, we extended both the in vitro and in vivo analyses using conditional knockout (KO) mice. Genetic deletion of Hdac3 indicates that Hdac3 is required for the activation of 45% of the lipopolysaccharides (LPS)-induced genes. Global analysis of histone H4 acetylation showed that transcriptional down-regulation in Hdac3-/- cells did not correlate with increased histone acetylation, suggesting the possible involvement of indirect or secondary effects. We found that the LPS-inducible, Hdac3-dependent genes include a large group of interferon-β (IFNβ)-inducible genes (eg. IP10, Irf1) and another group (eg. Il-6) that may be regulated by AP-1 family proteins. In addition, gene expression analyses identified interferon-signalling pathway as being impaired in Hdac3-/- cells. Basal and inducible Ifnβ transcriptions require cJun/AP-1 and the decreased amount of AP-1 family proteins in Hdac3-/- cells may explain the lack of Ifnβ activation and the increased acetylation in genomic regions.

DNA methylation-dependent gene silencing, catalyzed by DNA methyltransferases (DNMTs) and mediated by methyl binding domain proteins (MBDs) and histone deacetylases (HDACs), is essential for mammalian development, with the nervous system demonstrating particular sensitivity to perturbations. Little is known, however, about the role of DNA methylation in the stage-specific differentiation of neurons. In the olfactory epithelium (OE), where neurogenesis is continuous and the cells demonstrate a laminar organization with a developmental hierarchy, we identified sequential, transitional stages of differentiation likely mediated by different DNMT, MBD and HDAC family members. Biochemically, HDAC1 and HDAC2 associate with repressor complexes recruited by both MBD2 and MeCP2. HDAC1 and HDAC2, however, are divergently expressed in the OE, a pattern that is recapitulated in the brain. Rather than simultaneous inclusion in a complex, therefore, the individual association of HDAC1 or HDAC2 may provide specificity to a repressor complex in different cell types. Furthermore, distinct transitional stages of differentiation are perturbed in the absence of MBD2 or MeCP2. MeCP2 is expressed in the most apical immature olfactory receptor neurons (ORNs), and is up-regulated with neuronal maturation. In the MeCP2 null OE there is a transient delay in ORN maturation and an increase in neurons of an intermediate developmental stage. Two protein variants of MBD2 are expressed in the OE, with MBD2b expressed in cycling progenitor cells and MBD2a in the maturing ORNs. MBD2 null ORNs undergo increased apoptotic cell death. There is also a significant increase in proliferating progenitors in the MBD2 null OE, likely due, at least in part, to feedback from the dying ORNs, acting to up-regulate neurogenesis. Increased cell cycling in the MBD2 null is also observed post-lesion, however, in the absence of feedback back from the ORNs, a phenotype that is recapitulated by an acute inhibition of HDACs with valproic acid. Therefore, disruptions at both transitional stages of ORN differentiation are likely in the MBD2 null mouse. Together, these results provide the first evidence for a sequential recruitment of different MBD proteins and repressor complexes at distinct transitional stages of neuronal differentiation.

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

Cholesterol Homeostasis: involvement of histone deacetylases in the molecular regulatory pathway of Cholesterol 7α-hydroxylase Cholesterol 7α-Hydroxylase (CYP7A1) is the major check-point of bile acid (BA) biosynthesis, quantitatively the most important route of cholesterol disposal in mammals. BA returning to the liver via enterohepatic circulation repress CYP7A1 expression. BA induce histone deacetylase 7 (HDAC7) translocation to nucleus and the sequential recruitment of HDAC7, 3, 1 and of the corepressor SMRTα creates a repressor complex on the nuclear receptor HNF-4 bound to CYP7A1 promoter in a human hepatoma cell line. Previous results showed that non-selective HDAC inhibitors increase CYP7A1 expression in vitro and in vivo by preventing the negative feedback exerted by BA and reduce serum cholesterol in mice. Based on these seminal findings, our aim was to define the role of specific HDACs and corepressors in the regulation of CYP7A1. To this end, we tested class selective HDAC inhibitors in vitro and in vivo. By using a reporter cell line containing CYP7A1 promoter upstream of luciferase gene, we demonstrated that the class I selective HDAC inhibitor MS275 totally prevented the repressive effect of BA on CYP7A1; class II selective inhibitor MC1568 only elicited a partial recovery of CYP7A1 promoter driven transcription. In addition, MS275 significantly increased liver Cyp7a1 expression in C57Bl/6J mice. A mild induction, albeit not statistically significant, was observed even after SAHA and MS275 treatment. HDAC1, 3 and 7 silencing in the same reporter cell line pointed out the importance of HDAC1 and 7 in the regulation of CYP7A1 transcription To unravel the role of specific HDACs and corepressors we cloned shRNA against Hdac1, 3, 4, 5, 7 and Smrt in adenovectors and we tested them in primary hepatocytes. Hdac1, 7 and Smrt silencing significantly increased Cyp7a1 transcription, highlighting their involvement in the regulation of this gene. Adenoviral infection of C57BL/6J mice confirmed Hdac7 and Smrt as crucial players in the regulation of BA metabolism in vivo. Generation of HDAC7 liver-specific KO mice fed western diet allowed to investigate the role of HDAC7 in vivo: we observed body weight and LDL-cholesterol reduction, increased liver BA consistent with increased Cyp7A1 expression, lower liver lipid accumulation and a major fraction of lipid poor pre-beta HDL profile compared to wild type mice. The results obtained with the investigations performed during my doctorate training show that specific HDACs affect CYP7A1 transcription highlighting their role in the regulation of cholesterol and lipid homeostasis and represent a step forward contributing to shed light on the mechanisms of CYP7A1 regulation and their impact in lipid homeostasis.

Despite recent improvements in the clinical outcomes of vital pulp treatment, existing approaches remain non-specific and unpredictable. Developing biologically-based therapies that promote pulp regeneration is critical. Epigenetic modifications of DNA and histones control cellular processes, including proliferation, mineralisation and stem cell fate, and therefore offer exciting therapeutic opportunities. Chromatin acetylation can be altered pharmacologically using histone-deacetylase-inhibitors (HDACis), which relax its structure and modulate transcription. This project investigated regenerative-associated HDACi effects in vitro on a cell-line and primary dental-pulp-cells (DPCs), using proliferation, viability, mineralisation, cell-migration, enzyme activity, high-throughput gene/protein expression and pathway analyses. HDACis increased DPC differentiation and mineralisation-associated gene/protein expression at concentrations, which did not reduce viability. Primary DPC mineralisation was promoted without altering cell viability/apoptosis, indicating a resistance to HDACi-mediated toxicity compared with cell-lines. HDACi-induced DPC reparative processes were mediated by matrix metalloproteinase (MMP) expression and activity. MMP-13 inhibition further increased mineralisation-associated events, but decreased cell-migration indicating a novel role for MMP-13 in pulpal repair. HDACi solutions released a range of previously characterised and unreported bioactive dentine matrix components, which may further supplement regenerative capability in vivo. Results demonstrate that HDACi directly stimulate DPC repair-associated events, highlighting their potential for augmenting dental materials or pulp-engineering scaffolds for regenerative endodontics.

La mucoviscidose (CF) est causée par des mutations sur le gène CFTR codant pour une protéine indispensable au maintien de l'homéostasie des transports hydro-électrolytiques au sein de l'épithélium des organes cibles de la pathologie, dérivés de l'endoderme (poumon, pancréas, appareil reproducteur). Entre ces différents tissus et au cours du développement fœtal, l'expression du gène varie, particulièrement dans le tissu pulmonaire où une répression est observée à l'âge adulte.Ce travail propose dans une première partie la caractérisation des modifications épigénétiques associées à la régulation spatio-temporelle physiologique de l'expression du gène CFTR dans les tissus humains sains adultes et fœtaux. Les résultats obtenus soulignent le rôle important des modifications post-traductionnelles des histones dans la régulation in vivo. Nous avons notamment observé i) un équilibre fin entre marques d'ouverture (acétylation) et de fermeture (H3K27Me3) de la chromatine sur la région promotrice du gène CFTR et ii) l'acétylation significative de régions cis-régulatrices intragéniques.La deuxième partie de ce travail consiste en l'évaluation des effets du SAHA, un inhibiteur d'histones déacétylases (HDACi) dans un modèle ex vivo d'épithélium nasal de patients atteints de mucoviscidose. Les résultats montrent que le SAHA ne restaure pas l'adressage membranaire de la protéine CFTR en contexte pathologique mucoviscidose (mutation p.(Phe508del)) dans des cellules CF différenciées en épithélium ex vivo. De plus, le SAHA induit une modification du profil inflammatoire des épithélia et une dé-différenciation épithéliale dans le modèle ex vivo montrant que les mécanismes d'action de cette molécule sont multiples et réversibles.Ce travail souligne la nécessité d'analyser in vivo les mécanismes physiopathologiques impliqués dans la mucoviscidose et d'évaluer l'impact des molécules thérapeutiques sur les protéines endogènes dans un modèle d'épithélium différencié
Cystic fibrosis (CF) is caused by mutations in the CFTR gene encoding for a protein essential to maintain the homeostasis of fluid and electrolyte transport in the epithelium of endoderm-derived organs (lung, pancreas, reproductive tract) that are affected in CF patients. CFTR expression greatly varies between these tissues and during fetal development, particularly in the lung where repression is observed in adulthood .In the first part of this work, we characterized epigenetic modifications associated with the spatio-temporal regulation of CFTR gene expression in healthy human adult and fetal tissues. Our results emphasize the important role of histone post-translational modifications in this regulation in vivo. Specifically, we observed i) a fine balance between active (acetylation) and repressive (H3K27Me3) marks in the promoter region and ii) significant acetylation in intragenic cis-regulatory regions.In the second part of this work, we evaluated the effects of SAHA, a histone deacetylase inhibitor (HDACi) in an ex vivo model of nasal epithelium of CF patients. Our results show that SAHA can not restore CFTR protein to the apical membrane in a p.(Phe508del)-CFTR context in ex vivo CF differentiated epithelial cells. In addition, SAHA induces a change in the inflammatory profile of epithelia and epithelial dedifferentiation in the ex vivo model showing that the mechanisms of action of this molecule are multiple and reversible.This work highlights the need to analyze in vivo the pathophysiological mechanisms involved in Cystic Fibrosis and to evaluate the impact of therapeutic molecules on the endogenous proteins in a differentiated epithelium model

Hohe Telomeraseaktivität bedingt durch genomische TERT-Rearrangements definiert eine Gruppe an Hochrisiko-Neuroblastompatienten mit ungünstiger Prognose. Das Abzielen auf Telomerase ist ein hochpriorisierter Ansatzpunkt in der Therapie, für die es bislang keine klinisch erfolgreichen Inhibitoren gibt. Der Einsatz von epigenetisch wirksamen Histondeacetylase Inhibitoren (HDACi) stellt dabei eine interessante Therapieoption dar. In TERT-rearrangierten Neuroblastomzellen erzielte die Behandlung mit verschiedenen pan-, Klasse I oder spezifischen HDAC1/2 Inhibitoren eine Supprimierung der TERT mRNA Expression und der Telomeraseaktivität. RNA-Interferenz Studien bestätigten, dass HDAC1 und HDAC2 die TERT Expression positiv regulieren. Die transiente Überexpression von TERT zeigte einen partiellen Rescue des HDACi-bedingten anti-proliferativen Effekts. Der präventive und therapeutische Einsatz von HDACi Panobinostat verlangsamte das Xenografttumorwachstum, die TERT-Expression und Telomeraseaktivität in subkutanen NMRI-Foxn1nu/nu Mausmodellen des TERT-rearrangierten Neuroblastoms bei klinisch relevanten Dosen. Dies zeigt das translationale Potential und die klinische Durchführbarkeit der Panobinostat-Behandlung. ChIP Sequenzierung und Methylierungsanalyse zeigten keine bedeutenden Unterschiede der Histonmodifikationen und der Methylierung von CpG Dinukleotiden am TERT Lokus nach Panobinostatbehandlung. Die Inhibierung der de novo RNA Synthese zeigte, dass die Stabilität des TERT mRNA Transkripts nach Panobinostatbehandlung verringert war. Dies deutet darauf hin, dass die reduzierte Transkriptstabilität der zugrundeliegende molekulare Mechanismus ist. Zusammenfassend konnte gezeigt werden, dass die hohe Telomeraseaktivität in TERT-rearrangierten Neuroblastommodellen durch den Einsatz zugelassener HDACi supprimiert werden kann.
Telomerase activation by genomic TERT-rearrangements defines a subgroup of high-risk neuroblastomas with adverse outcome. Accordingly, telomerase activity presents a high-priority drug target with no currently available clinical inhibitors. It was assessed whether telomerase activity could be inhibited through histone deacetylase (HDAC) inhibition in models of TERT-rearranged neuroblastoma. Treatment with a panel of seven pan-, class I- or specific HDAC1/2 inhibitors suppressed TERT mRNA expression and telomerase activity in TERT-rearranged neuroblastoma cells at clinically achievable concentrations. RNA interference-based studies confirmed that HDAC1 and HDAC2 positively regulate TERT transcript levels. Enforced TERT expression partly rescued the anti-proliferative effect of HDAC inhibition indicating a causal role of TERT suppression in the HDAC inhibitormediated tumor-suppressive phenotype. Panobinostat treatment, in preventive and therapeutic settings, considerably attenuated tumor growth in subcutaneous TERT-rearranged neuroblastoma xenograft models in NMRI-Foxn1nu/nu mice and suppressed TERT transcript levels and telomerase activity at clinically relevant doses, thus demonstrating translational potential and clinical feasibility. ChIP sequencing detected no major differences in the chromatin context of the TERT locus between HDAC inhibitor-treated and control cells. Likewise, HDAC inhibition did not substantially alter the methylation profile in the TERT region. Blocking de novo RNA synthesis, however, reduced TERT mRNA transcript levels in HDAC inhibitor-treated cells, suggesting reduced TERT transcript stability as the underlying molecular mechanism. In summary, high-level telomerase activity caused by genomic rearrangements in neuroblastoma models is suppressed by treatment with clinically approved HDAC inhibitors, suggesting indirect druggability and a potential molecular rationale for therapeutic intervention.

Ovarian cancer is the leading cause of death among gynecologic malignancies. The risk of developing ovarian cancer in a woman’s lifetime is 1 to 2 in 100. This high rate of development and death from the tumorigenesis is a result of its asymptomatic manifestation. Ovarian cancer is usually found in its advanced stage; therefore the survival rate is lower than for other types of cancers. The most common type of ovarian cancer, serous epithelial ovarian cancer, arises from the surface epithelium of the ovary and less frequent from the fallopian tubes or uterus. The treatment of surgery is limited by the fact that most ovarian cancers are detected after they have metastasized. Chemotherapy is often difficult because of the lack of sufficient target specific drugs. Typically, platinum in combination with other drugs is provided as the standard treatment. These combinations exhibit higher toxicity, are often not target specific, and frequently despite treatment, the tumor relapses. Current studies suggest epigenetics plays a significant role in carcinogenesis by the silencing of tumor suppressor genes (TSG). Histone modifications and the methylation of specific cytosine phosphate guanosine (CpG) residues in the upstream region of genes silence the TSG. Many clinical trials are in progress to develop combination therapies utilizing histone deacetylase inhibitors (HDACi), and DNA methyl transferase I (DNMTI) inhibitors, in combination with other cytotoxic agents. HDACi are known to be effective against different types of leukemia’s, such as Cutaneous T-cell Lymphoma; however, they are not as effective against solid tumors when used as a single agent. Our laboratory was one of the first to demonstrate that HDACi, in addition to its known property to increase histone acetylation, additionally decrease CpG island methylation in the upstream region of TSG. This demethylation causes re-expression of TSG. Our laboratory hypothesizes that re-expression of TSG sensitize cancer cells to other cytotoxic drugs. In an effort to develop improved therapy for ovarian cancer, we employed a combination therapy, which includes epigenetic drugs, HDACi, in combination with calpain protease inhibitor, calpeptin. Calpain is a ubiquitous protease usually activated in cardiovascular diseases and cancer cells. The present study discerns that combination of HDACi and calpeptin produce more than additive growth inhibition of diverse ovarian cancer cells. HDACi re-expressed TSG. Additionally, the observed growth inhibition of ovarian cancer cells was caused by cell-cycle arrest, induction of apoptosis, followed by autophagy. The phosphorylation of growth promoting signaling protein, Mitogen Activated Protein Kinase 1 (ERK), was inhibited. In addition, the inhibitors also partially inhibited phosphorylation of anti-apoptotic protein V-ask Murine Thyomoma Viral Oncogene Homolog 1 (Akt). Collectively, the outcome of this study suggests that epigenetic drugs (HDACi) sensitize the diverse ovarian cancer cell lines by re-expression of TSG, followed by cell death, when treated in combination with calpain inhibitor, calpeptin.

Periodontitis (PD) is one of the most common bone loss pathologies in adults and currently affects more than 60% of the population in its destructive form. Ineffective or surgically invasive treatment options can result in patient noncompliance, gingival recession, alveolar bone destruction and eventual loss of teeth. Aside from the psychosocial effects of poor dental health, PD has been associated with a variety of systemic conditions, such as rheumatoid arthritis and cardiovascular disease, exacerbating their onset and severity. Histone deacetylase (HDAC) enzymes are molecules that control cellular activity through modifications to gene expression and protein function at an epigenetic level. Alterations to HDAC expression or function can cause diminished physiological cell regulation, thought to be an essential factor in the pathogenesis of disease. The use of selective HDAC inhibitors (HDACi) targeting candidate HDACs may be an effective, non-invasive therapeutic tool to treat both inflammation and bone loss in PD. The aim of this research was to investigate the effects of novel HDACi designed to target individual HDAC isoforms as a PD treatment. In vitro investigations identified therapeutic potential for targeting HDAC 1 and HDAC 2 during the inflammatory response and catabolic actions of human monocytes and osteoclasts. Whereas HDAC 5 inhibition holds regenerative potential, as osteoblastogenesis and matrix mineralisation was induced by its suppression. With the additional identification and characterisation of candidate HDAC isoforms in an in vivo mouse model of PD, the results of this thesis will provide a strong foundation for future investigations and clinical translation of HDACi.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2018

Indiana University-Purdue University Indianapolis (IUPUI)
Epigenetic alterations have been shown to control cell type specification and differentiation leading to the changes in chromatin structure and organization of many genes. HDACs have been well documented to play an important role in both neurogenesis and gliogenesis in ganglionic eminence and cortex-derived cultures. However, the role of HDACs in glial cell type specification and differentiation in the optic nerve has not been well described. As a first step towards understanding their role in glial cell type specification, we have examined histone acetylation and methylation levels as well as the expression levels and patterns of the classical HDACs in both murine and chick optic nerve. Analysis of mRNA and protein levels in the developing optic nerve indicated that all 11 members of the classical HDAC family were expressed, with a majority declining in expression as development proceeded. Based on the localization pattern in both chick and murine optic nerve glial cells, we were able to group the classical HDACs: predominantly nuclear, nuclear and cytoplasmic, predominantly cytoplasmic. Nuclear expression of HDACs during different stages of development studied in this project in both murine and chick optic nerve glial cells suggests that HDACs play a role in stage-dependent changes in gene expression that accompany differentiation of astrocytes and oligodendrocytes. Examination of localization pattern of the HDACs is the first step towards identifying the specific HDACs involved directly in specification and differentiation of glia in optic nerve.

Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutations in the Dystrophin gene. DMD is characterized by continuous cycles of muscle contraction/degeneration, leading to muscular tissue replacement by fibrotic and fat infiltrations deposed by the fibro-adipogenic progenitors (FAPs). Although there is currently no cure for DMD, the Histone Deacetylase Inhibitors (HDACi) represent the first generation of epigenetic drugs used to counteract DMD progression. Our lab has identified a regulatory network targeted by HDACi to repress FAPs differentiation into fibro-adipogenic cells, while activating a latent pro-myogenic phenotype (Saccone et al. 2014). However, HDACi pharmacological efficacy is restricted at early stage of DMD (Mozzetta et al. 2013). The mechanism during dystrophic FAPs aging that confers resistance to HDACi treatment is still unknown. To address this issue, we performed ChIP-seq for H3K9/14ac and H3K27ac in FAPs from early and late stage dystrophic mice, treated or not with HDACi. Coupling ChIP-seq with RNA-seq we identified differentially acetylated regions responsible of gene expression changes and unveiled the stage-specific epi-signature of both HDACi efficacy and resistance. This work paves the way to future diagnostic and therapeutic perspective for extending the efficacy of HDACi for the treatment of DMD.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Zuzana Faustová Supervisor: Prof. Doutor Jorge Miguel de Ascenção Oliveira PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: The impact of SIRT1 inhibition on zebrafish morphology and behavior After discovery of connection between yeast Silent Information Regulator 2 (Sir2) and its ability to alter lifespan, Sir2 and its seven mammalian orthologs became very attractive therapeutic target. These so called sirtuins are members of a histone deacetylase family. They possess unique catalytic activity having nicotinamide adenine dinucleotide as a cofactor and their function can be influenced by environmental factors. The aim of this diploma thesis was to extend knowledge of Sirtuin 1 (SIRT1), which is from all mammalian sirtuins considered to have the closest relation to yeast Sir2. At first we tested the impact of SIRT1 inhibition on early developmental stages of zebrafish (Danio rerio) embryos and larvae, finding out that SIRT1 is important for normal development and SIRT1 inhibition or malfunction result in cardiovascular defects, delayed development, and death. Additionally, we tried to learn more about SIRT1 and its connection with Parkinson's disease by combining nontoxic doses...

Indiana University-Purdue University Indianapolis (IUPUI)
The epigenetic state of any cell is, in part, regulated by the interaction of DNA with nuclear histones. Histone tails can be modified in a number of ways that impact on the availability of DNA to interact with transcriptional complexes, including methylation, acetylation, phosphorylation, ubiquituination, and sumoylation. Histones are acetylated by a large family of enzymes, histone acetyl transferases (HATs), and deacetylated by the histone deacetylases (HDACs). Acetylated histones are generally considered markers of genomic regions that are actively being transcribed, whereas deacetylated and methylated histones are generally markers of regions that are inactive. The goal of the present study was to 1) study the epigenetic state with regard to the presence of euchromatin and heterochromatin in the developing chick and murine retina, 2) study and compare the localization patterns of the classical HDACs in the developing chick and murine retina with respect retinal progenitors and early differentiated cell types 3) to test the hypothesis that overall HDAC activity is required for dividing retinal progenitors to leave the cell cycle and differentiate. Our results showed that the classical HDACs were ubiquitously expressed in the developing chick and murine retinas. Species specific differences as well as stage dependent variations were observed in the localization of the HDACs in the cell types that were studied in the chick and murine retina. Our preliminary results also showed that HDAC inhibition may lead to the inability of the cell types to leave the cell cycle and a subsequent increase in the number of progenitor cells present in the developing chick retina.

Pubertal timing displays wide, normally distributed variation in a healthy population of sexually maturing adolescents. However, like many complex traits, factors contributing to the variation are not well understood. Epigenetic regulation may contribute to some of the population variation. The role that epigenetics, specifically DNA methylation and histone acetylation, may play in regulating pubertal timing was investigated in C57BL/6 female mice: investigating whether population variation in pubertal timing among inbred mice could be explained by environmental factors; whether perturbing the epigenome using a histone deacetylase inhibitor or methyl-donor would alter pubertal timing; and examining genome-wide methylation patterns in hypothalami of early versus late maturing mice. Results demonstrate that measurable micro-environmental factors have only negligible effects on pubertal timing; pubertal timing was significantly altered by administration of epigenetic modifying agents; differences in methylation patterns are subtle. This initial evidence supports the involvement of epigenetic mechanisms in regulating pubertal timing.

Cancer is a multifactorial disease caused by multiple unrepaired DNA mutations and/or epigenetic changes, occurred during cell life cycle that the organism cannot detect and reverse. Acute lymphocytic leukaemia (ALL) is a disease characterized by a rapidly proliferation of early lymphoid precursors (namely lymphoblast), arrested in an early stage of its development, that replace normal hematopoietic cells in the bone marrow and cause decline in the production of normal marrow cells. Chronic lymphocytic leukaemia (CLL) is a disorder characterized by a progressive accumulation of lymphocytes that are apoptosis resistant, being the most common type of chronic leukaemia found in adults and among men over 55 years, hardly affecting children. Each type of cancer not only has its own recurrent genomic aberrations but also has its characteristic epigenetic changes which demonstrate its importance to cancer pathogenesis. Epigenetic modifications participate in the silencing and/or activation of genes that are the main key to cell and tissue differentiation. The most studied epigenetic changes in cancer are DNA methylation of CpG islands in regulatory regions of gene and post- translational modifications of histones. In ALL and CLL, aberrant DNA methylation, such as hypermethylation of CpG islands of gene promoters and abnormal histone deacetylation have been associated with cancer. Thus, DNA hypomethylating agents and histone deacetylase inhibitors are two categories of epigenetic therapies that are being studied as possible new therapies for ALL and CLL. The present study has two main goals. First, evaluate the therapeutic potential of two DNA methyltransferase inhibitors (Azacytidine and Decitabine) and two histone deacetylase inhibitors (Panobinostat and Vorinostat), in monotherapy and in combination, in two B-ALL cell lines (697 and KOPN8 cells) and in peripheral blood mononuclear cells (PBMC) obtained from CLL patients. Second, evaluate the methylation profile of the B-ALL cell lines and CLL samples. To this end, two B-ALL cell lines (697 and KOPN8 cells) and 31 individuals participated in this study, 21 patients diagnosed with CLL and 10 non¬neoplastic controls. ALL cell lines (KOPN8 and 697 cells) and PBMCs from CLL patients were incubated with 5-AC, DAC, LBH589, and SAHA, in monotherapy (single dose and daily administration) and in combination, during 72h and 48h, respectively. The cytotoxic effect of drugs was evaluated by fluorometric microculture cytotoxicity assay (FMCA). Cell death and cell cycle were analysed by flow cytometry through annexin V/Propidium iodide and PI/RNAse assays, respectively. Methylation patterns were determined by MS-MLPA and levels of 5-mC were determined by intracellular labelling with anti-5-mC antibodies by flow cytometry. CD5 and CD19 antibodies were used to identify normal B cells (CD5-/CD19+), neoplastic B cells (CD5+/CD19+), normal T cells (CD5+/CD19-) and other mononuclear cells (CD5"/CD19-). Methylation study was determined by MS-MLPA. For the methylation study, DNA was extracted from samples and controls by salting out protocol. The evaluation of the differences between monotherapy doses, combination schemes, cell cycle and cell death data were determined by applying the nonparametric ANOVA Kruskal-Wallis test (Dunn's multiple comparisons test). MS-MLPA obtained data were analysed using nonparametric ANOVA Kruskal- Wallis test (Dunn's multiple comparisons test) for cell lines and chi-squared (X2) test for patients. A probability value of p<0.05 was considered statistically significant for both cell lines and patient studies. The in vitro studies showed that epigenetic modulators have a cytotoxic effect, being able to reduce cell viability in B-ALL cell lines, 697 and KOPN8. All four therapies demonstrated to have effect on cell viability that is concentration, incubation time and cell type dependent. 697 cells seem to be more sensitive to all four therapies, since the IC50 dose was reached with lower concentrations compared with KOPN8 cell line, where the IC50 was not reached with tested doses. In 697 cells, DAC demonstrated to have more effect than 5-AC, when comparing same doses. For KOPN8 cells, the combinations studied do not show beneficial results compared to those obtained in monotherapy. On the other hand, for 697 cells, the combination of LBH589 and SAHA with DAC leads to a higher reduction in cell viability compared to those observed in cells treated with drugs in monotherapy, independent of the administration scheme. The best administration scheme was the administration of LBH589 and SAHA, 3 hours after the administration of DAC. All four tested drugs induced cell death by apoptosis, confirmed by changes in morphologic aspects. It was also observed that those drugs, in a global way, they showed antiproliferative effect, inducing cell cycle arrest at G0/G1 phase. Moreover, 5-AC and DAC in monotherapy and in therapeutic combination, induced a decrease in 5-mC levels. Methylation data showed that none condition tested caused an alteration on gene promoter methylation levels, compared with control. CLL studies also demonstrated that DNA hypomethylating agents and histone deacetylase inhibitors induced similar effects and all four therapies reduced cell viability in a dose-dependent manner. For the combination of therapies, none demonstrated to be statistically significant in comparison to monotherapy. 5-AC in combination with LBH589 obtained better results on cell viability than the ones obtained for monotherapy, causing an increased reduction on cell viability. However, this reduction was not significant. The results obtained for cells treated with the epidrugs, in a daily dose administration scheme, were very similar to the combination therapy, where none demonstrated to be statistically significant in comparison to monotherapy. DNA hypomethylating agent therapies and histone deacetylase inhibitors arrested cell cycle in S phase. Neoplastic B lymphocytes demonstrated to be more affected than normal B and T cells, which was expected. Only LBH589 and SAHA IC50 doses were considered statistically significant, compared with control, with p<0.01 for both conditions. However, all four therapies demonstrated to induce much more apoptosis on neoplastic B lymphocytes than on normal lymphocytes and other mononuclear cells. Methylation data showed that CLL patients had a significant higher methylation frequency of MSH6 (86%, 18/21), KLLN (67%, 14/21), WT1 (86%, 18/21) and GATA5 (71%, 15/21) gene promoters, when compared with controls, suggesting the involvement of DNA methylation on CLL development. Results also suggest that the methylation of tumour suppressor genes is a common event in CLL patients and that epigenetic modulators induce a cytotoxic effect in cells, reducing cell viability in a time- and dose-dependent manner. Therefore, these results are promising and encourage further studies
O cancro é uma doença multifatorial, provocada por múltiplas mutações do DNA e/ou alterações nos processos epigenéticos, que ocorrem durante o ciclo celular e que o organismo não consegue reverter. A leucemia linfocítica aguda (LLA) é uma doença caracterizada por uma rápida proliferação de células percursoras da linhagem linfoide, imaturas, designadas de linfoblastos, que se acumulam na medula óssea e provocam declínio na produção de células normais. A leucemia linfocítica crónica (LLC) é uma desordem caracterizada pela acumulação progressiva de linfócitos maduros resistentes à apoptose. É o tipo de leucemia crónica mais comum em adultos do sexo masculino com idade superior a 55 anos, sendo um evento raro em crianças. Cada tipo de cancro é caracterizado não só por alterações genómicas recorrentes, mas também aberrações epigenéticas, demonstrando a sua importância no desenvolvimento destas patologias. As modificações epigenéticas são importantes para o silenciamento e/ou ativação de genes, fundamental para a diferenciação celular e de tecidos. As modificações epigenéticas mais estudadas no cancro são a metilação das ilhas CpG dos promotores de genes e as modificações pós-traducionais das histonas. Na LLA e na LLC, as duas modificações epigenéticas mais estudadas e associadas à sua patogénese são as alterações na metilação de DNA, como o caso da hipermetilação, e a desacetilação de histonas. Desta forma, os agentes hipometilantes e os inibidores das desacetilases das histonas são duas categorias de modeladores epigenéticos que estão em fase de estudo para possível utilização na terapêutica da LLA e LLC. O presente estudo apresenta dois objetivos: avaliar o potencial terapêutico de dois agentes hipometilantes (Azacitidina e Decitabina) e de dois inibidores das desacetilases de histonas (Panobinostat e Vorinostat), em monoterapia e combinação, em duas linhas celulares de LLA-B (células 697 e KOPN8) e em células mononucleares de sangue periférico de doentes com LLC; avaliar o perfil de metilação das linhas celulares de LLA-B e das células mononucleares de LLC. Desta forma, este estudo envolveu duas linhas celulares de LLA da linhagem B (células 697 e KOPN8) e 31 indivíduos (21 diagnosticados com LLC e 10 controlos não-neoplásicos). As duas linhas celulares de LLA-B (células 697 e KOPN8) e as células mononucleares de sangue periférico de doentes com LLC foram incubadas com Azacitidina, Decitabina, Panobinostat e Vorinostat, em monoterapia (doses únicas e administração fracionada) e em combinação, durante 72h e 48h, respetivamente. O efeito citotóxico dos fármacos em estudo foi avaliado pelo ensaio de citotoxicidade de microcultura fluorométrica (FMCA). O estudo da morte e ciclo celular foram obtidos por Citometria de fluxo, com recorrência a ensaios de Anexina V/Iodeto de Propídio e Iodeto de Propídio/RNAse, respetivamente. Os padrões de metilação foram obtidos pela técnica MS-MLPA e os níveis de 5-mC foram determinados por Citometria, com recurso a marcação intracelular com anticorpos anti-5mC. Os anticorpos para deteção das proteínas CD5 e CD19 permitiram a distinção de linfócitos B normais (CD5-/CD19+), linfócitos B neoplásicos (CD5+/CD19+), linfócitos T normais (CD5+/CD19-) e outras células mononucleares (CD5-/CD19-), nos estudos de morte celular em amostras de LLC. O DNA utilizado nos estudos de metilação foi extraído das amostras de LLC e dos controlos pelo protocolo de extração por salting out. A avaliação das diferenças entre as doses de monoterapia, os esquemas de combinação, o ciclo celular e os dados de morte celular foram determinados pela aplicação do teste estatístico não paramétrico Kruskal-Wallis (teste de comparações múltiplas de Dunn). Os dados obtidos pela técnica MS-MLPA foram analisados utilizando também o teste não paramétrico Kruskal-Wallis (teste de comparações múltiplas de Dunn) apenas nas linhas celulares. Nos doentes foi aplicado o teste de qui-quadrado (X2). Um valor de probabilidade de p <0,05 foi considerado estatisticamente significativo, tanto para as linhas celulares de LLA quanto para os estudos com doentes de LLC. Os estudos in vitro demonstraram que os moduladores epigenéticos apresentam efeito citotóxico, reduzindo a viabilidade celular nas linhas de LLA-B, células 697 e KOPN8, sendo esse efeito dependente da concentração, tempo de incubação e tipo celular. As células 697 demonstraram ser mais sensíveis a todos os fármacos, uma vez que o IC50 foi alcançado com doses inferiores comparando com as células KOPN8, nas quais o IC50 não foi atingido com as doses testadas. As mesmas células demonstraram sofrer mais efeito quando tratadas com 5-AC do que com DAC. Os resultados obtidos das células KOPN8 demonstraram que as combinações não apresentam beneficio comparativamente à monoterapia. Por outro lado, nas células 697, as combinações de Panobinostat e Vorinostat com a Decitabina provocaram uma maior redução na viabilidade celular, comparando com os resultados obtidos na monoterapia, sendo independente dos esquemas de administração. O melhor esquema de administração demonstrou ser a administração de LBH589 e SAHA, 3 horas após DAC. Os quatro fármacos em estudo induziram morte celular por apoptose, tendo sido confirmada por alterações no aspeto morfológico das células. Também se observou um efeito anti-proliferativo, induzindo a paragem do ciclo celular na fase G0/G1. Adicionalmente, os fármacos 5-AC e DAC, em monoterapia e em combinação, induziram uma diminuição nos níveis de 5-mC. Os estudos do perfil de metilação demonstraram que nenhum dos fármacos, testados em monoterapia e combinação, provocaram a alteração no perfil de metilação de nenhum gene em ambas as linhas celulares. Os estudos em LLC também demonstraram que os agentes hipometilantes e os inibidores das desacetilases de histonas reduziram a viabilidade celular dependente da dose. As combinações celulares não demonstraram benefícios em comparação com a monoterapia. 5-AC em combinação com LHB589 demonstraram melhores resultados que a monoterapia, provocando maior redução na viabilidade celular, contudo não foi estatisticamente significativo. Os esquemas de administração fracionada também não demonstraram benefícios, comparativamente à monoterapia. Ambos os agentes hipometilantes e os inibidores das desacetilases de histonas provocaram uma paragem na fase S do ciclo celular. Os quatro fármacos também demonstraram ter influencia apenas nos linfócitos B neoplásicos. Apenas as concentrações de IC50 dos dois inibidores das desacetilases de histonas (LBH589 e SAHA) apresentaram diferenças significativas na morte celular (p<0,01), induzindo mais apoptose que o controlo. Contudo, os dois agentes hipometilantes também provocaram um aumento da morte celular por apoptose nos linfócitos B neoplásicos. Os estudos de metilação demonstraram que os doentes com LLC apresentam níveis de metilação elevados dos promotores dos genes MSH6 (86%, 18/21), KLLN (67%, 14/21), WT1 (86%, 18/21) e GATA5 (71%, 15/21), quando comparados com o controlo, sugerindo o envolvimento da metilação no desenvolvimento da LLC. Os resultados obtidos neste estudo sugerem que a metilação de genes supressores tumorais é um evento comum em doentes com LLC e que os modeladores epigenéticos induzem um efeito citotóxico dependente do tempo e da dose, reduzindo a viabilidade celular de células de LLA e LLC. Desta forma, os resultados são bastante promissores, encorajando estudos futuros
Mestrado em Bioquímica

Human genetic data suggests DISC1 (Disrupted-in-schizophrenia 1) is a susceptibility gene for schizophrenia and depression. Disc1 Q31L-/- mutants show depression-like behaviour and Disc1 L100P-/- mutants schizophrenia-like behaviour. Heterozygous mutants show an intermediate phenotype. In a gene-environment interaction study, we exposed heterozygotes to chronic social defeat (CSD) stress and phenotyped behaviour. Disc1, Bdnf(III) and Pde4b mRNA levels were also measured. Moreover, as epigenetic mechanisms may mediate some effects of CSD, we also exposed wildtype mice to CSD concurrently with the histone deacetylase inhibitor valproate. We found that CSD increased anxiety in L100P-/+ mutants, and that levels of Disc1, Bdnf(III) and Pde4b mRNA were higher in this mutant. Valproate treatment did not correct CSD-induced behavioural changes. In conclusion, we have demonstrated an interaction between a strong susceptibility gene for psychiatric disease and an environmental manipulation similar to stressors known to affect mental illness.

In our two previous studies, we reported the discovery and the optimization of novel 1,4-dihydropyridine-based sirtuin ligands. Starting from SIRT1-activating 1,4-DHPs, bearing benzyl group at N1, we identified carbonyl group at N1 to be responsible for an increased SIRT3 activation (MC2791 (1a) and MC2789 (1u)). However, the moderate potencies of 1a and 1u prompted us to screen for more potent derivatives. We generated new series of compounds by varying their “top” or “bottom” with other substituents at C1 or C4 position of the DHP scaffold, respectively. Hence, we reported the discovery and characterization of potent and specific activators for Sirt3 and/or Sirt5. The 1,4-DHP-based activators bind to the sirtuin catalytic core independent of bound substrates and increase the enzyme’s turnover. The compounds are selective for Sirt3 or Sirt5 and show cellular activity. Overall, our results provided a scaffold for potent and specific sirtuins activation and an activation model for Sirt3 and Sirt5 as a basis for functional studies and further drug development. Additionally, HBV-infected cells treated with our potent and selective Sirt3 activator 1a, demonstrated that 1a regulates the antiviral activity of cccDNA in HepAD38 cells. The treatment of HBV-infected cells with 1a through SIRT3 stimulation led to histone H3 and/or H4 hypoacetylation and reduction in the transcription from a viral cccDNA template, accompanied by a reduction in HBV replication. Together these results indicate that the Sirt3 activator 1a can modulate the acetylation status of cccDNA-bound H3/H4 histones, thus providing a novel therapeutic approach for the treatment of chronic HBV infection.
Very recently, the trend to shift towards epi-polypharmacology drugs has been taken into account in order to acquire a superior therapeutic effect and eventually reduce drug-related doses and toxicity, as well. Based on these evidence, we design and synthesize novel dual HDAC/EZH2 inhibitors to achieve higher anticancer effect by regulating sinergically the expression of a huge number of tumor suppressor genes. For our purpose, we chose the HDAC pharmacophoric model due to its wide structural variety and feasibility to accommodate on the surface binding cap a high degree of different chemical entities. In our first investigation, we combined the well-known vorinostat HDACi moiety to the already optimised pyrazole and pyrrole EZH2i scaffolds MC3629 and MC3707, respectively. The first preliminary screening of our two hybrid compounds MC4134 and MC4128 showed that only the pyrrole derivative MC4128 was able to simultaneously inhibit EZH2 and HDAC, also exhibiting an interesting isoform selectivity for HDAC6. Prompted by these findings, we decided to develop a series of dual inhibitors of EZH2 and HDAC (8a-g and 6a-g), combining the well-known HDACi moieties to the already optimised EZH2i scaffold MC3707. Therefore, according to the HDACi pharmacophoric model, we have chosen different types of spacer: the aliphatic one (Vorinostat), the benzoic one (Entinostat) and the cinnamic one (Panobinostat and Belinostat). As zinc binding group we used, in turn, a hydroxamic acid or an ortho-amino anilide. Overall, compounds 8a-g and 6a-g have been confirmed to be dual inhibitors of EZH2 and HDACs in vitro, showing an interesting selectivity profile towards HDAC isoforms. In preliminary assays in U-937 AML cells, 8c (MC4128) decreased cell viability and induced apoptosis, with increased levels of acetyl-histone H3 and acetyl-α–tubulin. Importantly, we expect that our novel HDAC/EZH2 dual inhibitors can display a potent and synergic anti-cancer activity in vivo, thus becoming an attractive therapeutic approach to fight cancer.
Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative mitochondrial disorder caused by an unstable GAA trinucleotide (TTC) repeat expansion in the first intron of the frataxin gene (FXN). In FRDA patients, the expanded GAA·TTC repeats lead to partial transcriptional silencing resulting in expression of structurally and functionally normal frataxin, but at lower levels compared to the normal. FRDA can be considered as an epigenetic disease due to the identification of several associated epigenetic marks, including 1) increased levels of methylated histones (H3K9me2, H3K9me3, H4K20me3) in regions flanking the GAA repeats, 2) increased DNA methylation at specific CpG sites upstream of the GAA repeats and, 3) reduced acetylation of several H3 and H4 lysine residues. Due to the importance of H4K20me to genomic integrity, very recently A-196 has been discovered as the first-in-class chemical probe of Suv4-20H1 and Suv4-20H2, with an IC50= 25 nM and IC50=144 nM, respectively. Despite the in vitro potency of A-196, confirmed by biochemical and cellular assays, preliminary in vitro metabolic studies in human liver microsomes (HLM) have shown some metabolic liability and potentially low solubility, with a Clint (µL/min/mg protein) =191 and a t1/2 (min)=7.28. Prompted by these findings, a lead chemical optimisation has been carried out with the aim to ameliorate chemical and physical properties of A-196. Preliminary biological results of our final compounds (1a-m and 2c) in HEK293 cell model of FRDA showed that only compound 1b (RM02) demonstrated similar effects (or slight better) than the reference compound A-196, with a luciferase fold reactivation of 1.20. The other compounds, unfortunately, showed no increase in frataxin expression beyond that one induced by the vehicle DMSO. Preliminary data for the evaluation of the cytotoxicity (adenylate kinase scores) showed that our compounds are probable non-toxic to the cells.

The enhanced expression of histone deacetylases (HDACs) in a variety of malignancies drew attention to investigate a new category of anti-cancer drugs that are based on the inhibition of those enzymes. Valproic acid (VPA) is a well-known antiepileptic drug that exhibits antitumor activities through inhibition of HDACs class I and IIa. Cancer stem cells (CSCs) have been recognized to drive the tumor growth and progression hence; attention has been given to target this small subpopulation of CSCs rather than the whole bulk tumor cells. CD133 is considered to be a CSC marker in several tumors and its transcription is strongly influenced by epigenetic changes that will be altered upon administration of histone deacetylase inhibitors (HDACi) in cancer treatment. Therefore, we evaluated the epigenetic and cytotoxic effects of treatment with 1 mM VPA in combination with other chemotherapeutics and its influence on the expression of CD133 in human neuroblastoma (NB) cell lines. Our results revealed that addition of VPA to DNA-damaging chemotherapeutics induced a synergistic anti-tumor effect that was associated with caspase-3 dependent induction of apoptosis in UKF-NB-4 cells. This synergism was related to the increase of the acetylation status of histones H3 and H4 and was only produced either by...

The major evolutionary transition from single-celled to multicellular life is believed to have occurred independently of the main metazoan lineages in the cellular slime moulds, of which Dictyostelium discoideum is the best-studied species. Unusually, in this case multicellular development is a facultative trait and part of an asexual life cycle. It is triggered by starvation and involves aggregation of hitherto independent and possibly unrelated free-living cells. The consequences of multicellularity in D.discoideum are strongly influenced by the environment and meaningful external perturbations are easily carried out. This makes the organism ideally suited to a study of epigenetic factors that regulate development. In an attempt to understand how conserved epigenetic pathways are integrated within the developmental framework, two likely players were chosen for investigation - heat shock protein 90 (Hsp90) and histone deacetylases (HDACs). Hsp90 has been implicated in diverse biological processes such as protein folding, cell cycle control, signal transduction, and morphological evolution. The role of Hsp90 in D.discoideum life cycle was studied using a specific inhibitor, geldanamycin. Inhibition of Hsp90 function in D.discoideum caused a delay in aggregation and an arrest of development at the ‘mound’ stage. A reduction in Hsp90activity in starving cells of D.discoideum resulted in the generation of a range of phenotypes. The study suggests that Hsp90 is required for a specific developmental transition of the social amoeba and is important in generating a reliable outcome of the developmental process. Histone acetylation regulates gene expression and leads to the establishment and maintenance of cellular phenotypes during development of plants and animals. To study the roles of HDACs in D.discoideum, biochemical, pharmacological and genetic approaches were employed. The inhibition of HDAC activity by trichostatin A resulted in histone hyperacetylation and a delay in cell aggregation and differentiation. Cyclic AMP oscillations were normal in starved amoebae treated with trichostatin A but the expression of a subset of cAMP-regulated genes was delayed. Bioinformatic analysis indicated that there are four genes encoding putative HDACs in D.discoideum. One of these four genes, hdaB, was found to be dispensable for growth and development under laboratory conditions; but formed spores with lower efficiency than the wild type in chimeras. The work shows that HDAC activity is important for regulating two aspects of multicellular development: (a) heterochrony, namely the relative timing of developmental events, and (b) modulating the behaviour of single cells in a manner that is sensitive to their social environment.

abstract: Alzheimer’s disease (AD) is characterized by the degeneration of cholinergic basal forebrain (CBF) neurons in the nucleus basalis of Meynert (nbM), which provides the majority of cholinergic input to the cortical mantle and together form the basocortical cholinergic system. Histone deacetylase (HDAC) dysregulation in the temporal lobe has been associated with neuronal degeneration during AD progression. However, whether HDAC alterations play a role in cortical and cortically-projecting cholinergic nbM neuronal degeneration during AD onset is unknown. In an effort to characterize alterations in the basocortical epigenome semi-quantitative western blotting and immunohistochemistry were utilized to evaluate HDAC and sirtuin (SIRT) levels in individuals that died with a premortem clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment (MCI), mild/moderate AD (mAD), or severe AD (sAD). In the frontal cortex, immunoblots revealed significant increases in HDAC1 and HDAC3 in MCI and mAD, followed by a decrease in sAD. Cortical HDAC2 levels remained stable across clinical groups. HDAC4 was significantly increased in prodromal and mild AD compared to aged cognitively normal controls. HDAC6 significantly increased during disease progression, while SIRT1 decreased in MCI, mAD, and sAD compared to controls. Basal forebrain levels of HDAC1, 3, 4, 6 and SIRT1 were stable across disease progression, while HDAC2 levels were significantly decreased in sAD. Quantitative immunohistochemistry was used to identify HDAC2 protein levels in individual cholinergic nbM nuclei immunoreactive for the early phosphorylated tau marker AT8, the late-stage apoptotic tau marker TauC3, and Thioflavin-S, a marker of mature neurofibrillary tangles (NFTs). HDAC2 nuclear immunoreactivity was reduced in individual cholinergic nbM neurons across disease stages, and was exacerbated in tangle-bearing cholinergic nbM neurons. HDAC2 nuclear reactivity correlated with multiple cognitive domains and with NFT formation. These findings identify global HDAC and SIRT alterations in the cortex while HDAC2 dysregulation contributes to cholinergic nbM neuronal dysfunction and NFT pathology during the progression of AD.
Dissertation/Thesis
Doctoral Dissertation Neuroscience 2018