No description available.

Our understanding of non-histone acetylation as a means of cellular regulation is in its infancy. Using a mass spectrometry approach we identified acetylated lysine residues and monitored acetylation changes across the proteome as a consequence of metabolic stress (hypoxia). We observed changes in acetylation status of non-histone lysines in tumor cells. Through the use of small molecule inhibitors of histone deacetylase enzymes (HDACs) and siRNA screening identified HDAC6 as a pro-survival regulator of lysine acetylation during hypoxia. The phospho-binding protein 14-3-3ζ acts as a signaling hub controlling a network of interacting partners and oncogenic pathways. We show here that lysines within the 14-3-3ζ binding pocket and protein-protein interface can be modified by acetylation. The positive charge on two of these lysines, K49 and K120, is critical for coordinating 14-3-3ζ-phosphoprotein interactions. Through screening, we identified HDAC6 as the K49/K120 deacetylase. Inhibition of HDAC6 blocks 14-3-3ζ interactions with two well-described interacting partners, Bad and AS160, which triggers their dephosphorylation at S112 and T642, respectively. Expression of an acetylation-refractory K49R/K120R mutant of 14-3-3ζ rescues both the HDAC6 inhibitor-induced loss of interaction and S112/T642 phosphorylation. Furthermore, expression of the K49R/K120R mutant of 14-3-3ζ inhibits the cytotoxicity of HDAC6 inhibition. These data demonstrate a novel role for HDAC6 in controlling 14-3-3ζ binding activity.

No description available.

Blast-induced neurotrauma (BINT) is a prevalent brain injury within both military and civilian populations due to current engagement in overseas conflict and ongoing terrorist events worldwide. In the early 2000s, 78% of injuries were attributable to an explosive mechanism during overseas conflicts, which has led to increased incidences of BINT [1a]. Clinical manifestations of BINT include long-term psychological impairments, which are driven by the underlying cellular and molecular sequelae of the injury. Development of effective treatment strategies is limited by the lack of understanding on the cellular and molecular level [2a]. The overall hypothesis of this work is that epigenetic regulatory mechanisms contribute to the progression of the BINT pathology and neurological impairments. Epigenetic mechanisms, including DNA methylation and histone acetylation, are important processes by which cells coordinate neurological and cellular response to environmental stimuli. To date, the role of epigenetics in BINT remains largely unknown. To test this hypothesis, an established rodent model of BINT was employed [3a]. Analysis of DNA methylation, which is involved in memory processes, showed decreased levels one week following injury, which was accompanied by decreased expression of the enzyme responsible for facilitating the addition of methyl groups to DNA. The one week time point also showed dramatic decreases in histone acetylation which correlated to decline in memory. This change was observed in astrocytes and may provide a mechanistic understanding for a hallmark characteristic of the injury. Treatment with a specific enzyme inhibitor was able to mitigate some of the histone acetylation changes. This corresponded with reduced astrocyte activation and an altered behavioral phenotype, which was characterized by high response to novelty. The diagnostic efficacy of epigenetic changes following blast was elucidated by the accumulation of cell-free nucleic acids in cerebrospinal fluid one month after injury. Concentrations of these molecules shows promise in discriminating between injured and non-injured individuals. To date, the diagnostic and therapeutic efforts of BINT have been limited by the lack of a mechanistic understanding of the injury. This work provides novel diagnostic and therapeutic targets. The clinical potential impact on diagnosis and therapeutic intervention has been demonstrated. / Ph. D.

Alternative splicing is an important cellular mechanism. It allows to produce multiple protein isoforms from a limited number of genes. Regulation of alternative splicing involves cis-acting elements on pre-mRNA and trans-acting splicing factors (SR and hnRNP proteins). Because splicing occurs co-transcriptionaly, chromatin structure appears to have a role in the regulation of alternative splicing. We have studied the effect of histone acetylation on alternative splicing. We have prepared splicing reporter for alternative EDB exon, which is part of the fibronectin gene. We have shown, that the inhibition of histone deacetylases affects splicing pattern of EDB exon from the reporter in the same way as the splicing of the endogenous EDB exon. Furthermore, we have shown, that the structure of the promoter affects splicing of alternative EDB exon from splicing reporter. Currently we have found out, that the structure of the promoter influences the degree of histone H4 acetylation. Inclusion of alternative EDB exon in mRNA was inversely proportional to histon acetylation on the reporter. This work might explain why various promoters have different splicing patterns of alternative exons.

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.

Epigenetic modifications are alterations in the cell nucleus that affect gene expression and can occur in chromatin at the level of DNA methylation or histone modifications. Such ‘epigenetic marks’ can be heritable through cell division but leave the DNA sequence unchanged. Post-­translational modifications can be found on the histone proteins associated with DNA; the majority of histone modifications are found on the lysine-­rich N-‐terminal amino acid “tails”. Histone acetylation and methylation influence the chromatin structure by loosening or tightening the packaging of DNA, respectively, in association with other chromatin modifiers. Condensed chromatin is linked to transcriptional silencing and genetic imprinting and also occurs at chromosomal centromeres, where it is linked to kinetochore binding. Heart development is well studied, but the epigenetic processes involved are not yet completely understood. While active chromatin mechanisms such as histone acetylation and chromatin remodelling have been described in the heart, the role of gene repressive epigenetic mechanisms has been poorly investigated. Cardiomyocytes are post-­mitotic cells that do not divide to regenerate a damaged heart. The regeneration of cardiomyocytes after myocardial infarction is an important topic of interest in cardiovascular science. There are various approaches to heart repair after infarction, including activating cardiomyocytes so they become mitotic once again, or growing cardiomyocytes in vitro to attach to a lesion site. An important factor in these approaches is understanding the epigenetic mechanisms controlling cell division. In this thesis, we aim to advance the current knowledge of the epigenetic repressive mechanisms involved in cardiomyocyte formation and heart development to explain their lack of regenerative capacities. We studied the epigenetic changes that occur during cardiac development leading to a non-­‐regenerative state to pinpoint the moment at which these changes arise. We found that the epigenetic process is independent of whether cardiac lineage differentiation occurs during embryogenesis or during differentiation in vitro. We discovered that cardiac heterochromatin displays a singular epigenetic signature during development as compared to brain, another post-­mitotic tissue, or liver, an actively regenerative tissue. We observed an epigenetic change in the repressive histone modification histone H3 lysine 9 trimethylation that was specific to heart development. This change involved a nuclear reorganisation of heterochromatin and a reduction of the levels of this mark in E13.5 and E14.5 embryos, as compared to E10.5 embryos. This was consistent with our observations of the histone lysine methyltransferase SUV39H1, the levels of which were lower after stage E10.5 of development. However, contradictorily, in differentiated cardiomyocytes in vitro, SUV39H1 was increased but showed low levels of H3K9me3, compared to ES cells, which had low levels of SUV39H1 and high levels of H3K9me3. We detected extremely low levels of the H3K9me3 in adult heart tissue. We observed that in adult hearts, the myocardium had maintained these major changes in H3K9me3, while this effect was not observed in the epicardium. Genomic studies were carried out to determine changes at a genomic level between the two key epigenetic stages in heart development we identified at E10.5 and E13.5. Methylated DNA immunoprecipitation sequencing and chromatin immunoprecipitation sequencing for H3K9me3 analyses were carried out to find overall changes in methylation patterns. No global changes in DNA methylation were detected between these developmental stages. These results imply that the differences observed in H3K9me3 are due to remodelling of the heterochromatin during heart development and cardiomyocyte formation, rather than quantitative changes.

Cyclooxygenase (COX) is the rate limiting enzyme that catalyses the production of prostanoids, which are crucial to vascular homeostasis. Evidence suggests that endothelial dysfunction and inflammation play a role in vascular complications in aging and diabetes. Previous animal studies by our laboratory at RMIT University reported enhanced COX expression with aging in rat aortas, platelets and monocytes. Potentially, alteration in COX expression may result in an imbalanced prostanoid production favoring the synthesis of vasoconstrictors and hence increase the risk of cardiovascular events in the aging population. The regulation of altered COX expression in aging, however, is not clear. It has been suggested that histone hyperacetylation may be an important mechanism that regulates COX levels during the aging process as increased histone acetylation has been shown to occur with aging. Thus, we hypothesized that COX expression is modulated by histone hyperacetylati on. This was investigated by measuring COX expression in histone hyperacetylated cultured endothelial cells. In the case of diabetes, studies have reported that the development of diabetes and its complications is associated with persistent inflammatory activity, evident with increased inflammatory markers in the circulation. COX-mediated pathways may be involved in this inflammatory process in diabetes. Furthermore, the formation of advanced glycation end products (AGEs) is accelerated in diabetes. AGEs can bind to receptors for AGEs (RAGE), which has also been suggested to play a role in inflammation in diabetes. We hypothesized that COX- and RAGE-mediated pathways contribute to increased inflammation in diabetes and potentiate the development of diabetic vascular complications. This was investigated by measuring changes in COX-mediated pathways in both rat and human diabetic models. The current thesis reports: 1) in cultured endothelial cells, histone hyperacetylation was associated with increased COX expression; 2) an overall increase in inflammation was observed in diabetes involving COX- and RAGE-mediated pathways. This was supported by increased platelet COX-1 and monocyte COX-2 levels in Zucker rats, increased monocyte COX-2 in human Type 1 diabetes and elevated plasma TXB2 and PGE2 levels in both human Type 1 and Type 2 diabetic subjects. Up-regulation of RAGE expression was further found in platelets and monocytes in both human diabetes types. When treated with NSAIDs, plasma prostanoid levels, COX and RAGE expression were reduced significantly in both platelets and monocytes in human diabetic subjects. 3) It is unclear how COX and RAGE expression was regulated, but histone modifications may be one of the mechanisms. Data from cultured cells indicated that increased COX expression was associated with increased histone acetylation levels induced by TSA. Concurrent increases in histone acetylation and COX-2 levels were also observed in human Type 1 diabetes, but similar findings were not observed in human Type 2 diabetes. In addition, we failed to find an age-dependent increase in monocyte histone H4 acetylation in human Type 2 diabetes despite an age-dependent increase in monocyte COX-2 expression. Thus, whether histone hyperacetylation modulates COX expression and in what conditions require further investigation.

<p>The human tumour suppressor protein p53 is an effective inhibitor of cell growth, by inducing cell cycle arrest and apoptosis. However, p53-induced cell growth inhibition can be detrimental for virus multiplication. Therefore, viruses encode for proteins, which can interfere with the functions of the p53 protein. Human adenoviruses encode for a transcription repressor protein named E1B-55K, which inhibits the activity of the p53 protein during a lytic adenovirus infection.</p><p>In this thesis, we have studied the biochemical characteristics of the E1B-55K protein and how the E1B-55K protein interferes with the function of p53 as a transcription factor.</p><p>Our data show that the E1B-55K protein interacts with the Sin3 co-repressor complex in adenovirus transformed and in adenovirus infected cells. Furthermore, the E1B-55K protein recruites a histone deacetylase activity, indicating that the E1B-55K protein is associated with a functional chromatin modifying complex. We also show that in addition to repressing p53-activated transcription, E1B-55K could also relieve p53-mediated repression of the survivin and Map4 promoters.</p><p>Previous results have shown that E1B-55K inhibits p53 as a transcriptional activator of the p21/CDKN1A promoter. Here we show that the E1B-55K protein prevents p53 from inducing histone H3 and H4 acetylation on p21/CDKN1A promoter, which coincided with the inhibition of p21/CDKN1A protein expression. Notably, the Sin3 complex was detected in the vicinity of the p53 binding site on the p21/CDKN1A promoter, suggesting that the E1B-55K protein blocked p53-mediated histone acetylation by recruitment of a histone deacetylase activity. Inhibition of p21/CDKN1A protein expression might be the reason, why the E1B-55K protein alleviates p53-dependent transcriptional repression of the survivin promoter. </p><p>Finally, we show that oligomerisation of the E1B-55K protein is important for the defined subcellular localization of the protein and for its function as a repressor of p53-activated transcription.</p>

The human tumour suppressor protein p53 is an effective inhibitor of cell growth, by inducing cell cycle arrest and apoptosis. However, p53-induced cell growth inhibition can be detrimental for virus multiplication. Therefore, viruses encode for proteins, which can interfere with the functions of the p53 protein. Human adenoviruses encode for a transcription repressor protein named E1B-55K, which inhibits the activity of the p53 protein during a lytic adenovirus infection. In this thesis, we have studied the biochemical characteristics of the E1B-55K protein and how the E1B-55K protein interferes with the function of p53 as a transcription factor. Our data show that the E1B-55K protein interacts with the Sin3 co-repressor complex in adenovirus transformed and in adenovirus infected cells. Furthermore, the E1B-55K protein recruites a histone deacetylase activity, indicating that the E1B-55K protein is associated with a functional chromatin modifying complex. We also show that in addition to repressing p53-activated transcription, E1B-55K could also relieve p53-mediated repression of the survivin and Map4 promoters. Previous results have shown that E1B-55K inhibits p53 as a transcriptional activator of the p21/CDKN1A promoter. Here we show that the E1B-55K protein prevents p53 from inducing histone H3 and H4 acetylation on p21/CDKN1A promoter, which coincided with the inhibition of p21/CDKN1A protein expression. Notably, the Sin3 complex was detected in the vicinity of the p53 binding site on the p21/CDKN1A promoter, suggesting that the E1B-55K protein blocked p53-mediated histone acetylation by recruitment of a histone deacetylase activity. Inhibition of p21/CDKN1A protein expression might be the reason, why the E1B-55K protein alleviates p53-dependent transcriptional repression of the survivin promoter. Finally, we show that oligomerisation of the E1B-55K protein is important for the defined subcellular localization of the protein and for its function as a repressor of p53-activated transcription.