Ao longo dos últimos anos observou-se um aumento no número de idosos no mundo, com isso faz-se necessário buscar terapias que amenizem os danos relacionados e também elucidar os mecanismos envolvidos neste processo. O exercício físico tem sido sugerido como uma ferramenta importante, não farmacológica, para atenuar os déficits relacionados à idade. Ainda, estudos recentes sugerem uma relação entre o processo de envelhecimento cerebral e o desequilíbrio de mecanismos epigenéticos, contudo, estes dados ainda não são conclusivos. Sabe-se que o grau de neuroplasticidade varia com a idade e que as estruturas encefálicas podem responder diferentemente à exposição ao exercício. Estudos demonstram que o córtex pré-frontal está envolvido em funções de alta ordem como atenção, tomada de decisão e memória de trabalho. Portanto, o objetivo deste trabalho foi avaliar os efeitos de diferentes protocolos de exercício físico (sessão única e exercício diário moderado) sobre a modulação de marcadores epigenéticos em córtex pré-frontal de ratos Wistar de 3 e 21 meses de idade. Os animais foram submetidos ao protocolo de sessão única (20 minutos) ou o exercício diário moderado (20 minutos durante 14 dias), 1 hora após a última sessão foram eutanasiados. O córtex pré-frontal foi dissecado e a acetilação da H4, o conteúdo da DNA metiltransferase (DNMT1 e DNMT3b), assim como a atividade da histona metiltransferase H3K27 foram analisadas. Os resultados serão apresentados na versão completa desta dissertação. / Over the past few years the number of elderly people has increased in the world, therefore it is necessary to search therapies that ameliorate age-related deficits as well as elucidate the mechanisms involved in this process. Physical exercise has been suggested as an important non-pharmacological approach to alleviate the age-related decline. Furthermore, recent studies have suggested a relationship between the process of brain aging and imbalance of epigenetic mechanisms, however, these data are not conclusive. It is well described that prefrontal cortex is involved in higher functions like attention, decision making and working memory. Then, the aim of this study was to investigate the effects of two exercise protocols (single session and daily moderate exercise) on the modulation of epigenetic markers in the prefrontal cortex from Wistar rats of 3- and 21- months-old. Animals were submitted to single session protocol (20 minutes) or the daily moderate exercise (20 minutes for 14 days), and 1hour after the last exercise session animals were euthanized. Prefrontal cortex was dissected out and acetylation of H4, the content of DNA methyl transferase (DNMT1 and DNMT3B), as well as histone methyltransferase H3K27 activity were analyzed. Results will be presented in the full version.

Introdução: O Adenocarcinoma ductal pancreático é um tumor bastante agressivo que apresenta uma alta de letalidade e, para o qual, poucas opções terapêuticas estão disponíveis. Isto pode ser parcialmente explicado pela complexidade derivada de múltiplas aberrações genéticas e da população celular mista presente em um tumor pancreático, o que também pode explicar o curso clínico heterogêneo observado na prática diária. Ultimamente, pesquisas científicas têm contribuído para ampliar o conhecimento sobre o impacto das alterações epigenéticas no desenvolvimento de múltiplos tipos de câncer, porém no pâncreas essas alterações ainda são incertas e, por isso, foco de investigação. A desregulação epigenética parece estar envolvida no ciclo celular da célula tumoral, incluindo o crescimento celular, diferenciação, progressão tumoral e morte celular, e a acetilação das histonas é um importante mecanismo que regula a transcrição de genes envolvidos nesses processos. Padrões globais de modificações das histonas foram recentemente apontados como preditores de desfecho em pacientes com câncer, mas poucos estudos têm sido realizados nesta área, inclusive em Adenocarcinoma ductal pancreático (ADP). Objetivos: O presente estudo foi desenvolvido a fim de investigar o padrão de modificação de acetilação das histonas em adenocarcinoma pancreático, através da análise imunohistoquímica. Materiais e métodos: Uma análise clinicopatológica retrospectiva foi realizada em 119 pacientes diagnosticados com câncer de pâncreas entre os anos de 2005 e 2011, e realizado estudo imunohistoquímico com os anticorpos contra H4K12ac, H3K9ac e H3K18ac. Marcação nuclear positiva para cada histona foi medida quanto à intensidade e expressão, sendo classificadas em grupos de baixa ou de alta intensidade/expressão. Os resultados foram analisados em relação aos parâmetros clinicopatológicos de cada paciente. Resultados: Houve uma relação positiva entre diferenciação tumoral e alta expressão de H4K12ac (P <0,05), bem como a intensidade forte dos três marcadores correlacionou-se positivamente com o estágio do tumor (P <0,01). Análise univariada mostrou pior sobrevida em pacientes com níveis elevados de expressão de H4K12ac (p = 0,038) e H3K18Ac (P = 0,033). Modelo de risco proporcional de Cox revelou o efeito prognóstico independente de níveis elevados de H4K12ac H3K18ac (taxas de risco de 1,6 e 1,7, respectivamente, p <0,05), especialmente para pacientes em estágios iniciais. Sugerimos como hipótese que as modificações na acetilação das histonas H4K12 e H3K18 podem ser consideradas fatores prognósticos importantes para o câncer de pâncreas, embora o mecanismo envolvido necessite de mais investigação. Aumentando a compreensão e o conhecimento sobre o padrão de acetilação das histonas, poder-se-ão finalmente gerar novas idéias para um diagnóstico molecular racional e novas abordagens terapêuticas. / Introduction: Ductal pancreatic adenocarcinoma (DPAs) is a highly aggressive tumor, with a high letality rate, for which few therapeutic options are available. This may be partially explained by the notorious complexity derived from the multiple genetic aberrations and mixed cellular population present in a pancreatic tumor, which can also explain the heterogeneous clinical course observed in daily practice. Lately, there is an increase in the literature about the impact of epigenetic changes on the development of several cancer, however in the pancreas these changes are still uncertain. Epigenetic deregulation may be involved in tumor cell biology, including cell growth, differentiation, tumor progression and cell death, and histone acetylation is a major mechanism that regulates gene transcription. Patterns of global histone modifications have been recently suggested as outcome predictors in cancer patients, but few studies have been conducted on pancreatic ductal adenocarcinomas. Objectives: This study was designed to investigate the predictive value of histone acetylation modifications on pancreatic cancer. Material and methods: A retrospective clinicopathologic analysis was undertaken in 119 patients diagnosed with PDAC between 2005 and 2011, and immunohistochemistry performed with antibodies against H4K12ac, H3K9ac and H3K18ac. Positive nuclear staining for each histone was measured as the intensity and expression, being classified into low or high-staining groups. Results were analyzed in relation to patients’ clinicopathologic parameters. Results: There was a positive relationship between tumor differentiation and H4K12ac high scores (P<0.05) and staining of the three markers correlated positively with tumor stage (P<0.01). Univariate analysis showed worse survival in patients with high detection levels of H4K12ac (p=0.038) and H3K18Ac (P=0.033). A backwards Cox proportional hazards model revealed the independent prognostic effect of high H4K12ac and H3K18ac levels (hazard ratios of 1.6 and 1.7 respectively, p<0.05), especially for patients at early stages. We hypothesize that acetylation of H4K12 and H3K18 may be considered valuable prognostic factors for pancreatic cancer, although the mechanism involved needs further investigation. Increasing insights into histone acetylation modifications can ultimately generate new ideas for rationally and molecularly based diagnostic and therapeutic approaches.

Introdução: O Adenocarcinoma ductal pancreático é um tumor bastante agressivo que apresenta uma alta de letalidade e, para o qual, poucas opções terapêuticas estão disponíveis. Isto pode ser parcialmente explicado pela complexidade derivada de múltiplas aberrações genéticas e da população celular mista presente em um tumor pancreático, o que também pode explicar o curso clínico heterogêneo observado na prática diária. Ultimamente, pesquisas científicas têm contribuído para ampliar o conhecimento sobre o impacto das alterações epigenéticas no desenvolvimento de múltiplos tipos de câncer, porém no pâncreas essas alterações ainda são incertas e, por isso, foco de investigação. A desregulação epigenética parece estar envolvida no ciclo celular da célula tumoral, incluindo o crescimento celular, diferenciação, progressão tumoral e morte celular, e a acetilação das histonas é um importante mecanismo que regula a transcrição de genes envolvidos nesses processos. Padrões globais de modificações das histonas foram recentemente apontados como preditores de desfecho em pacientes com câncer, mas poucos estudos têm sido realizados nesta área, inclusive em Adenocarcinoma ductal pancreático (ADP). Objetivos: O presente estudo foi desenvolvido a fim de investigar o padrão de modificação de acetilação das histonas em adenocarcinoma pancreático, através da análise imunohistoquímica. Materiais e métodos: Uma análise clinicopatológica retrospectiva foi realizada em 119 pacientes diagnosticados com câncer de pâncreas entre os anos de 2005 e 2011, e realizado estudo imunohistoquímico com os anticorpos contra H4K12ac, H3K9ac e H3K18ac. Marcação nuclear positiva para cada histona foi medida quanto à intensidade e expressão, sendo classificadas em grupos de baixa ou de alta intensidade/expressão. Os resultados foram analisados em relação aos parâmetros clinicopatológicos de cada paciente. Resultados: Houve uma relação positiva entre diferenciação tumoral e alta expressão de H4K12ac (P <0,05), bem como a intensidade forte dos três marcadores correlacionou-se positivamente com o estágio do tumor (P <0,01). Análise univariada mostrou pior sobrevida em pacientes com níveis elevados de expressão de H4K12ac (p = 0,038) e H3K18Ac (P = 0,033). Modelo de risco proporcional de Cox revelou o efeito prognóstico independente de níveis elevados de H4K12ac H3K18ac (taxas de risco de 1,6 e 1,7, respectivamente, p <0,05), especialmente para pacientes em estágios iniciais. Sugerimos como hipótese que as modificações na acetilação das histonas H4K12 e H3K18 podem ser consideradas fatores prognósticos importantes para o câncer de pâncreas, embora o mecanismo envolvido necessite de mais investigação. Aumentando a compreensão e o conhecimento sobre o padrão de acetilação das histonas, poder-se-ão finalmente gerar novas idéias para um diagnóstico molecular racional e novas abordagens terapêuticas. / Introduction: Ductal pancreatic adenocarcinoma (DPAs) is a highly aggressive tumor, with a high letality rate, for which few therapeutic options are available. This may be partially explained by the notorious complexity derived from the multiple genetic aberrations and mixed cellular population present in a pancreatic tumor, which can also explain the heterogeneous clinical course observed in daily practice. Lately, there is an increase in the literature about the impact of epigenetic changes on the development of several cancer, however in the pancreas these changes are still uncertain. Epigenetic deregulation may be involved in tumor cell biology, including cell growth, differentiation, tumor progression and cell death, and histone acetylation is a major mechanism that regulates gene transcription. Patterns of global histone modifications have been recently suggested as outcome predictors in cancer patients, but few studies have been conducted on pancreatic ductal adenocarcinomas. Objectives: This study was designed to investigate the predictive value of histone acetylation modifications on pancreatic cancer. Material and methods: A retrospective clinicopathologic analysis was undertaken in 119 patients diagnosed with PDAC between 2005 and 2011, and immunohistochemistry performed with antibodies against H4K12ac, H3K9ac and H3K18ac. Positive nuclear staining for each histone was measured as the intensity and expression, being classified into low or high-staining groups. Results were analyzed in relation to patients’ clinicopathologic parameters. Results: There was a positive relationship between tumor differentiation and H4K12ac high scores (P<0.05) and staining of the three markers correlated positively with tumor stage (P<0.01). Univariate analysis showed worse survival in patients with high detection levels of H4K12ac (p=0.038) and H3K18Ac (P=0.033). A backwards Cox proportional hazards model revealed the independent prognostic effect of high H4K12ac and H3K18ac levels (hazard ratios of 1.6 and 1.7 respectively, p<0.05), especially for patients at early stages. We hypothesize that acetylation of H4K12 and H3K18 may be considered valuable prognostic factors for pancreatic cancer, although the mechanism involved needs further investigation. Increasing insights into histone acetylation modifications can ultimately generate new ideas for rationally and molecularly based diagnostic and therapeutic approaches.

La CREB Binding Protein (CBP) a une activité lysine acétyltransférase intrinsèque et fonctionne aussi comme un co-activateur transcriptionnel. L'activité acétyltransférase et la fonction de coactivateur transcriptionel sont toutes deux essentielles pour la formation de mémoire à long terme. De plus, la dérégulation de CBP a été observée dans des maladies neurodégénératives comme la maladie d'Alzheimer et la maladie de Huntington. L'objectif de ma thèse était d'étudier le rôle de la CBP et de son activation pharmacologique dans le cadre de la formation de la mémoire spatiale, une forme de mémoire qui est démantelé très tôt dans la MA. Les données obtenues à partir de ma thèse montrent que l'activation de la fonction acétyltransférase CBP par l’activateur CSP-TTK21 améliore les processus mnésiques chez des souris adultes normales et aussi dans un modèle murin de MA (THY-Tau22). Ainsi, la stratégie d’activation pharmacologique de l'activité acétyltransférase de CBP a un énorme potentiel pour une utilisation en tant qu'agent thérapeutique pour le traitement des maladies liées à l'altération de la mémoire tel que la maladie d'Alzheimer. / CREB Binding Protein (CBP) has an intrinsic lysine acetyltransferase activity and alsofunctions as a transcriptional co-activator. Both the acetyltransferase activity and the transcriptional co-activator function are critical for long-term memory formation. Importantly, CBP dysregulation has been observed in neurodegenerative conditions like in Alzheimer’s disease and Huntington’s disease. The focus of my thesis was to study the role of CBP and its activation by a new pharmacological tool, in the context of spatial memory formation, a form of memory that is very early dismantled in AD. Data obtained from my thesis clearly suggests that activation of CBP acetyltransferase function by small molecule activator CSP-TTK21 can improve memory related processes in healthy adult mice and also in a mouse model of AD, (THY-Tau22). Therefore, the strategy of pharmacological activation of CBP acetyltransferase activity has tremendous potential for use as therapeutics for the treatment of diseases related to memory impairment such as Alzheimer’s disease.

Splicing is the process when introns gets removed and exons are spliced together. This is an important step to form a clean mRNA with no unnecessary sequences that could interrupt protein synthesis. There are different types of splicing and some of them need a complex called spliceosome. The spliceosome requires ATP, small nuclear RNAs and splicing factors. The spliceosome and the process splicing can be regulated by epigenetics, and one epigenetic mechanism is histone modification. There are four types of histone modifications; methylation, phosphorylation, ubiquitination and acetylation. They regulate splicing to different extents by altering the chromatin structure, affect the assembly of the spliceosome and regulate the attraction of splicing factors. This review will investigate if histone modifications affect splicing and to what extent. Suggestions for further research regarding the relationship between splicing and histone modifications will also be provided. The review is based on 30 articles and two books and the search was conducted between 30th of March 2020 and 13th of April 2020. Ubiquitination and phosphorylation have a minor effect on splicing meanwhile methylation and acetylation affect splicing in great extent.

京都大学 / 0048 / 新制・論文博士 / 博士(理学) / 乙第13369号 / 論理博第1573号 / 新制||理||1666(附属図書館) / (主査)教授 杉山 弘, 教授 深井 周也, 教授 秋山 芳展 / 学位規則第4条第2項該当 / Doctor of Science / Kyoto University / DGAM

No description available.

DNA methylation and histone modifications have emerged as key mechanisms in transcriptional regulation. The target of methylation-induced silencing 1 (TMS1) is a bipartite protein. Recent studies have indicated that methylation-associated silencing of TMS1 occurs in many cancers. However, whether and how TMS1 is regulated by epigenetic mechanisms in cancers remains unknown. In this study we showed that methylation of the TMS1 promoter occurred in five of six hepatocellular carcinoma (HCC) cell lines. TMS1 expression was reduced in four HCC cell lines and correlated with methylation status. Furthermore, the TMS1 promoter was completely methylated and mRNA expression was undetectable. TMS1 expression could be restored by 5-aza-2′-deoxycitidine (5-Aza-dC) (a DNA methyltransferase inhibitor) or trichostatin A (TSA) (a histone deacetylase inhibitor) alone and the promoter methylation. was partially reversible. TSA was more efficient than 5-Aza-dC in inducing TMS1 expression, and the combination of 5-Aza-dC and TSA resulted in markedly synergistic reactivation of the gene and completely reversed promoter methylation. Interestingly, TMS1 promoter methylation-associated gene silencing was accompanied by histone H3 Lysine 9 (H3K9) hypoacetylation and trimethylation. 5-Aza-dC and/or TSA also had some effect on conversion of methylated to acetylated H3K9 in restoring TMS1. This conversion was dynamic at the TMS1 promoter and a decrease in H3K9 trimethylation preceded an increase in H3K9 acetylation after 5-Aza-dC and/or TSA treatment. Our results thus suggest that epigenetic inactivation of TMS1 expression is regulated by promoter hypermethylation and H3K9 modifications in a coordinated way.

Covalent modifications of histones play a critical role in many important biological processes such as transcription, DNA repair and recombination. Among the major modifications known so far, histone H3 acetylation at lysines 9 and 14 (H3K9/14), monoubiquitination of histone H2B at lysine123 (H2BK123) and H3 lysine 4 methylation (H3K4) are among the more studied ones. The importances of these modifications have been further stressed by its connection to various human diseases including cancers. Previous biochemical studies have shown that H2BK123 ubiquitination is mandatory for methylation at histone H3K4. However, little is known about the regulatory mechanisms of H3K4 methylation by H2B ubiquitination in vivo. Thus, the prime focus of this study is to understand the factors involved in the regulation of H2B ubiquitination, the regulatory mechanisms of the cross-talk between H2BK123 ubiquitination and H3K4 methylation and the role of these covalent modifications in transcriptional regulation under physiological conditions. Here in this study, I have shown that Ubp8p, a histone deubiquitinase, is a bona fide subunit of SAGA (Spt3-Ada-Gcn5 acetyltransferase) co-activator complex and selectively regulates both di and trimethylation of histone H3K4 at the core promoter of a SAGA-dependent gene in vivo. However, over the open reading frames for a subset of constitutive genes H2B ubiquitination selectively upregulates only H3K4 trimethylation but not dimethylation. Moreover, such an upregulation of H3K4 trimethylation has no impact on the RNA Polymerase II (RNAPII) recruitment and hence transcription of the respective genes. Interestingly, at an inducible gene, histone H2B ubiquitination promotes transcription elongation independently of H3K4 methylation. Furthermore, this study also demonstrates for the first time, the molecular mechanism for the cross-talk between H2B ubiquitination and H3K4 methylation in vivo. Evidently a COMPASS subunit, Cps35p, is necessary for the trans-tail cross talk between histones H2B and H3. Finally, this study also shows that Sgf73p, a SAGA subunit, is required for SAGA recruitment at the promoters of several SAGA dependent genes and facilitates transcription in both HAT-dependent and HAT-independent manner. Collectively, the results from this study not only provide deep insights into the regulatory mechanisms of H2B ubiquitination and H3K4 methylation (and their role in transcription) but also give a new functional dimension to SAGA subunit, Sgf73p, under physiological conditions. Given the role of histone acetylation, ubiquitination and methylation in many human diseases, the results from this study is of tremendous clinical value unveiling new therapeutical targets.

Melatonin is an indoleamine hormone with neuromodulatory and neuroprotective properties. It mediates many of its effects by its two G protein-coupled receptors, MT1 and MT2. We have shown that valproic acid (VPA) induces melatonin receptor expression in cultured rat C6 glioma cells, and in the rat hippocampus. VPA is known to affect gene expression through several mechanisms, including the modulation of intracellular kinase pathways and/or transcription factors, as well as the inhibition of histone deacetylase (HDAC) activity. In this study, we show that HDAC inhibitors of distinct chemical classifications, including suberanilohydroxamic acid (SAHA) and 4-(dimethylamino)-n-[7-(hydroxyamino)-7-oxoheptyl] benzamide (M344), parallel the effects of VPA on MT1 induction in vitro. However valpromide, a VPA analogue that lacks the ability to inhibit HDAC activity, does not. The observed increase in MT1 expression by VPA is matched by an increase in global histone H3 acetylation. More importantly, an enrichment of histone H3 acetylation occurs along the rat MT1 promoter following treatment with VPA, indicating that histone acetylation and chromatin remodelling are a primary mechanism underlying this induction. Independent of VPA, the rat MT1 gene may be regulated by a number of intracellular kinase pathways and transcription factors, which are also targeted by VPA. KG501-mediated CREB inhibition did not block MT1 upregulation by VPA. Blockade experiments targeting the PKC, PI3K/AKT, or GSK3β signaling pathways suggest that VPA induces melatonin receptor expression independent of these intracellular signaling cascades as well. The relevance of melatonin receptor upregulation was assessed using in vivo VPA and melatonin combination treatments on neuroprotective gene expression. The results of this study provide evidence that expression of the melatonin receptor is epigenetically induced by VPA by means of promoter histone acetylation. Melatonin receptor upregulation by VPA, or other HDAC inhibitors, may represent a therapeutic strategy for the management of several nervous system disorders. / Dissertation / Doctor of Philosophy (PhD)