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Epigenetics In Human Health and Disease Hons and PhD Program and Projects
Program 1 Understanding the MeCP2-associated regulatory complex in disease Alterations in the controls of DNA methylation and histone deacetylation play a profound role in human disease. We are interested in examining epigenetic events such as DNA methylation, histone modification and the determinants involved on chromatin to further our understanding of endogenous gene transcription. These studies are now challenging the way we view gene regulation beyond our simple understanding of "textbook" operations. Our desire to dissect the molecular details allows us to determine the roles of transcriptional regulators and provide us with a greater understanding of how they are involved in transcriptional regulation. Aims Our laboratory has a focus on endogenous gene regulation. We have generally relied on genetic and biochemical systems to study gene regulation, because, genetics excels in identifying transcription factors and regulatory complexes required for expression, whereas biochemical testing transcend what they are capable of in vitro. However, neither discipline can tell us definitely the order of events that occur naturally on endogenous genes. Understanding endogenous mechanisms of transcriptional control allows the biologist to manipulate a biological system often seen as difficult to control. Understanding epigenetic events provides an exciting platform to design transcription factors and control the Fragile X Syndrome and the FMR1 gene, which has historically eluded translational science. Relevant Reading Nature 393, 386-9 (1998) Nature Genetics 19, 187-191 (1998) Science 302, 885-889 (2003) Science 302, 890-893 (2003) J Biol Chem 279, 46490-6 (2004) Nature Genetics 37, 254-4 (2005)
PNAS 102, 17551-8 (2005) Nature Genetics 38, 962-4 (2006) Nature Genetics 38, 964-7 (2006) Aim Characterizing the MeCP2 corepressor associated SWI/SNF complex; what are the biophysical properties of MeCP2 in the brain? And what is the significance of the complex in mental health (fragile X syndrome) ? Supervisor A/Professor Sam El-Osta Keywords MeCP2; methyl-CpG binding protein 2, FPLC; fast protein liquid chromatography, transcriptional repression, chromatin, genes and disease, Fragile X Syndrome, heterochromatin, co-repressor complex
Epigenetics In Human Health and Disease Hons and PhD Program and Projects
Epigenetics In Human Health and Disease Hons and PhD Program and Projects
Epigenetics In Human Health and Disease Hons and PhD Program and Projects
Program 2 Improving molecular targeting and treatments for cancer gene therapy Given the widespread use of radiotherapy in cancer (about 50% of cancer patients are treated with x-rays), there has been a longstanding interest in the investigation of chemical compounds that can modify cellular responses to ionizing radiation. We are evaluating histone deacetylase inhibitors (HDACi) as potential radiation sensitizers. Our clonogenic survival experiments and other cell-growth assays indicate that incubation of cells with the HDAC inhibitors, Trichostatin A and valproic acid enhances radiation induced cell-death. The aim of this honours project is to further investigate the cellular and molecular basis for the radiosensitizing effect. This Hons and PhD Program will investigate the effect of HDACi on radiation-induced DSB repair in transcriptionally active or inactive euchromatin and constitutive heterochromatin analyses of γH2AX accumulation and hyperacetylation of histone H3 using chromatin immunoprecipitation (ChIP). The candidate will investigate pre-treatment with low concentration of HDACi before irradiation, results in classic hallmarks such as histone hyperacetylation and the stable accumulation of γH2AX that is more pronounced in euchromatic alleles than heterochromatic areas of the genome. At this exciting stage of gene therapy, our combined results suggest that the inhibition of HDACs can potentiate therapy by a mechanism that renders DNA more accessible to treatments by histone hyperacetylation in the absence of cytostasis, apoptosis and/or growth arrest in mammalian cells. Histone deacetylase inhibitors are emerging as a new class of targeted cancer chemotherapeutics. Several histone deacetylase inhibitors are currently in clinical trials and promising anti-cancer effects at well-tolerated doses have been observed for both hematologic and solid cancers. Histone deacetylase inhibitors have been shown to induce cell cycle and growth arrest, differentiation and in certain cases apoptosis in cell cultures and in vivo. There is growing commercial and therapeutic interest in potential clinical use of histone deacetylase inhibitors in combination with conventional cancer therapies. The focus of the Hons and PhD Program is on the different mechanisms by which histone deacetylase inhibitors significantly improve cancer therapy. Aim Improving molecular targeting and treatments for cancer gene therapy Methods This will require training in cell model immortalisations and tissue culture, protein extraction and purification. Chromatin immunoprecipitation (ChIP). Protein identification by protein blotting. Keywords Cancer, chemotherapy, radiotherapy, cancer gene therapy, DSB; double strand break, DNA repair, chromatin, HDAC; histone deacetylase, HDACi; HDAC inhibitors, �H2AX Supervisors A/Professor Sam El-Osta and Dr Tom Karagiannis
Epigenetics In Human Health and Disease Hons and PhD Program and Projects
Epigenetics In Human Health and Disease Hons and PhD Program and Projects
Epigenetics In Human Health and Disease Hons and PhD Program and Projects
Epigenetics In Human Health and Disease Hons and PhD Program and Projects
Program 3 Identification of new regulatory mechanism of transcriptional repression in response to cancer chemotherapy The general focus of this project is to investigate the mechanisms by which specific MBD1 regulatory complexes serve to integrate and repress gene in models of cancer. Specifically, the project will define the functional roles of specific components of the co-repressor complexes in gene repression events and characterise the link to cancer-associated loci such as the multidrug resistance gene (MDR1) using genetic and genomic approaches. The central focus of this aim takes the Teams latest findings on transcriptional repression to the next logical step and further extends the mechanism, as currently understand and published recently in Nature Genetics (2005) 37:254-264 and Oncogene (2005) 24:8061-7075. The project is aimed to examine the controversial point of MBD1 repression independent of direct DNA methylation. Human methylation dependent proteins are well characterized and the best characterized is MeCP2 which is strongly associated with transcriptional silencing. However, little is known of the role of other MBD co-repressor complexes such as MBD1 in gene silencing. Repression of transcription exerts an equally fundamental role in gene regulation as activation. We have functionally defined components of the corepressor and coactivator complexes that are required for regulatory activities, proving that a regulated exchange of coactivator and corepressors dictates the level of expression of specific genes in different disease models. We hypothesize that one of the critical functions of these complexes is to integrate MBD1 recruitment in response to input from multiple signaling pathways. The presence of MBD1 associated components in multiple complexes, coupled with their biological roles in development, presents an ideal model for elucidation of basic principles of regulatory gene expression and the generality of this regulation. In cancer tumor suppressor genes arte often silenced and characterized by profound epigenetic changes. However, it is not known whether an epigenetic program and in particular MBD1 corepressor complex is implicated in gene regulation. The team has exciting experimental evidence that MBD1 corepressor complex is critical in regulating gene activity associated with cancer. Aim Identification of new regulatory mechanism of transcriptional repression in response to cancer chemotherapy Methods Chromatin immunoprecipitation (ChIP), High-resolution separation of proteins according to size using FPLC. This will require training in cell model immortalisations and tissue culture, protein extraction and purification. Protein identification by protein blotting and protein-protein immunoprecipitation. Keywords Cancer, chemotherapy, drug resistance, MBD1; methyl-CpG binding protein 1, FPLC; fast protein liquid chromatography, transcriptional repression, chromatin, heterochromatin, euchromatin Supervisors A/Professor Sam El-Osta and Dr Emma Baker
Epigenetics In Human Health and Disease Hons and PhD Program and Projects
Epigenetics In Human Health and Disease Hons and PhD Program and Projects
Program 4 Epigenetic memory in diabetes and metabolic syndrome Diabetes and its consequence of accelerated vascular complications is a major global clinical problem. Diabetic complications occur as a result of pathological processes activated by chronic hyperglycaemia. Of particular interest is the phenomenon of “metabolic memory” where diabetic patients, despite improved glycaemic control, develop complications as a result of prior poor glycaemic control. In recent clinical evaluates from the large Diabetes Control and Complications Trial, and its follow-up, the prospective Epidemiology of Diabetes Interventions and Complications trials, diabetic subjects have continued to develop ongoing end-organ injury, as a result of previous periods of poor glycaemic control. This has been interpreted as indicating that there is a memory of past glycaemia known as “metabolic memory” which is a major determinant of subsequent development of vascular complications. Several hypotheses have been raised to explain this phenomenon, including the possibility of “epigenetic memory”. However, such epigenetic pathways relevant to diabetic complications have not been previously determined. Such a possibility is further suggested from twin studies where it was shown in identical twins that there is a lack of concordance in complications. Although this could be related to environmental differences, epigenetic mechanisms have been postulated to explain this discordancy between identical twins. It remains unknown as to the mechanism whereby hyperglycaemic memory leads to a program of diabetic vascular complications. We postulate that epigenetic pathways, specifically histone code changes, participate in this phenomenon. This Hons and PhD Program will investigate the epigenetic pathways that act as a bridge linking hyperglycaemia to the central molecular and cellular events, which lead to vascular injury in diabetes. We have identified that in the context of a hyperglycaemic milieu, transcriptional competence is directly linked with epigenetic changes. These findings present a new paradigm for histone methyltransferase function and epigenetic modification that is relevant to our understanding of the transcriptional response to glucose. Finally, these findings will provide new targets for generating end-organ protective agents for the common and devastating clinical problems of diabetic vascular complications. Aim The role of the histone code hypothesis on hyperglycemic memory Methods Endothelial cell models, chromatin immunoprecipitation (ChIP), High-resolution separation of proteins according to size using FPLC. This will require training in cell model immortalisations and tissue culture, protein extraction and purification. Training is provided in the laboratory. Keywords Diabetes, hyperglycaemia, histone methyltransferase, histone methylation, histone code hypothesis, transcriptional activation, chromatin Supervisor A/Professor Sam El-Osta
Epigenetics In Human Health and Disease Hons and PhD Program and Projects
Epigenetics In Human Health and Disease Hons and PhD Program and Projects
Program 5 Heart disease, Stem cells and Epigenetics An enlarged heart, or cardiac hypertrophy, results from an increase in the thickness of the heart muscle in response to stress on the heart and blood system and is the heart’s mechanism of coping with the increased effort of pumping blood around the body. The stressor is often high blood pressure and prolonged hypertrophy is associated with an increased risk of heart disease. Whereas it has been known for years that epigenetic factors, specifically genomic methylation, function to modulate gene transcription, their role in the gene expression profile of compromised hearts associated with aging, hypertrophy and heart failure, remains unknown. The key issue that this project is designed to address relates to how pathological stimuli are finally converted into an altered gene expression profile that eventually leads to hypertrophy and heart failure. A better understanding of the epigenetic factors that control gene transcription in the compromised heart will advance the current knowledge of the mechanism responsible for molecular remodeling in the diseased organ. The planned studies will generate valuable data to answer important questions including whether modulating genomic methylation can alter hypertrophic growth and whether different gene expression profiles in aging or hypertrophic hearts could be partly attributable to epigenetic mechanisms. Furthermore, these studies could inform on the potential of novel therapeutic targets aimed at modulating epigenetic factors to alter or reverse unwanted gene transcription. We are offering honours and PhD students the opportunity to join the Human Epigenetics Laboratory at the Baker IDI Heart and Diabetes Research Institute and work towards reducing death and disability arising from heart disease in the community. Stem cells have the unique capacity for self-renewal and differentiation into specialised cell types. We are offering projects using stem cell cultures to understand how the healthy heart develops and hence how heart problems can arise when development goes awry. We will look at important developmental genes and determine how they are controlled as the heart forms from embryonic, to foetal, to neonatal-like stages. Aim 1 The role of epigenetic factors on transcriptional regulation in the failing heart Aim 2 Defining the impact of the foetal gene-expression program during the differentiation of stem cell-derived heart cells. Methods Cardiac cell models, chromatin immunoprecipitation (ChIP), High-resolution separation of proteins according to size using FPLC, Stem cell culture, Generation of transgenic stem cells. This will require training in tissue culture, protein extraction and purification. Training is provided in the laboratory. Keywords Heart disease, hypertrophy, stem cells, differentiation, development and epigenetic changes Supervisor A/Professor Sam El-Osta
Epigenetics In Human Health and Disease Hons and PhD Program and Projects
Program 6 Mechanisms of genetic changes in imprinting disorders Genomic imprinting is a particularly attractive example of epigenetic regulation, since in the same cell one of the two parental alleles is stably repressed by epigenetic modifications, whereas the other allele is maintained in an active state. This allele-specific regulation is entirely dependant on whether the gene is inherited from the mother or from the father. Imprinted genes are organised in clusters that are regulated by imprinting control regions (ICRs). To achieve allele-specific expression, ICRs are regulated by epigenetic modifications (DNA methylation and modifications of the histone proteins) that differentially mark the parental alleles as either active or repressed. These epigenetic modifications are controlled by DNA methyltransferases, histone modifying proteins, methyl CpG binding domain proteins, insulator proteins and chromatin-modifying complexes. Genomic imprinting is a multistep process involving the active erasure of existing epigenetic marks in primordial germ cells and the establishment of novel imprints later during gametogenesis (maternal marks in oocytes and paternal marks in male germ cells). Imprinted genes play key functions in development, and more particularly in foetal growth and their epigenetic deregulation results in different human diseases, including cancer. Two imprinted foetal growth disorders [the Beckwith-Wiedemann (BWS) and the Silver-Russell (SRS) syndromes] display opposite phenotypes: overgrowth in BWS and growth retardation in SRS. These two disorders are caused by disruption of imprinting of the 11p15 region by epigenetic mechanisms that are opposite: gain of methylation in BWS and loss of methylation in SRS of the same 11p15 ICR. Although abnormal methylation of ICRs have been demonstrated in various imprinting disorders, the exact molecular mechanisms resulting in abnormal methylation of ICRs remains unknown. Several lines of evidence suggest that the imprint error occurs after fertilization and concerns factors involved in the maintenance of imprints after fertilization. Moreover, whether other epigenetic defects than abnormal DNA methylation may result in imprinting disorders has not been investigated. Aim 1 - Elucidating the mechanism(s) resulting in loss of imprinting in patients with epigenetic defects by analysing key trans-acting regulatory factors involved in the maintenance of imprinting during early foetal development. Aim 2 - Evaluating if biochemical modification of core histones may represent an alternative and/or complementary mechanism to methylation defects in patients with imprinting disorders. This project will give new insight into the mechanisms of imprinting disorders including cancer. Furthermore, recent studies indicate that the use of assisted reproductive technology can affect the epigenetic cycle of imprinting and result in BWS and SRS by a mechanism that is still unidentified. The outcome of the research will therefore highlight our understanding of the epigenetic risk of assisted reproductive technology. Recommended reading (Nat Genet 2005; 37: 1003) (Bioessays 2006; 28: 453) (Best Pract Res Clin Endocrinol Metab 2008; 22: 1) Keywords Foetal growth, imprinting disorders, reproduction, development and epigenetic changes Supervisors Dr Christine Gicquel and A/Professor Sam El-Osta
Epigenetics In Human Health and Disease Hons and PhD Program and Projects
Program 7 Genome wide approaches to the study of gene-environment interactions and chromatin modifications The two metre human genome is a dynamic physical structure comprised of protein-DNA associations, all of which participate in nuclear functions such as transcriptional regulation, replication and repair and epigenetic inheritance. Therefore it is imperative we look beyond mRNA expression profiling as an endpoint measure. Understanding when DNA binding proteins are bound or released from gene sequences is just as important as where this occurs on the genome. The availability of large-scale high throughput epigenomic strategies and more specifically, next generation sequencing coupled with chromatin immunopurification (ChIP) approaches will allow us to explore these questions of spatial and temporal landscapes with exquisite detail. Examples of the application the El-Osta team are currently interested include but not limited to genome wide mapping of DNA methylation using bisulfite sequencing protocols, determination of histone modifications and nucleosome positioning, chromatin accessibility for the determination and monitoring of changes in chromatin structures and remodeling, chromasome capture conformation and long range interaction and chromatin communication (ChIP-loop, 4C) and mapping of transcription factor binding sites. Aim 1 - Development of computational tools that integrate genome-wide data sets for proximal and distal transcription binding sites and histone modification mapping. Aim 2 - Development of ChIP approaches and strategies to the broad application of determining epigenomic signatures in models of human health and disease. Aim 3 - Development of epigenomic protocols and strategies for low cell numbers from clinical isolates. Recommended reading (Nature Review Genetics 2008) (Mol Biotechnol 2008) (Front Biosci 2008) Keywords Chromatin immunoprecipitation, bisulfite sequencing, chromatin conformation, next generation sequencing, bioinformatics Supervisor A/Professor Sam El-Osta
Epigenetics In Human Health and Disease Hons and PhD Program and Projects
Contact Details for Further Information about all Human Epigentics projects for students and postdoctoral scientists Associate Professor Sam El-Osta Head, Epigenetics in Human Health and Disease Laboratory Director, Epigenomics Profiling Facility Baker IDI Heart and Diabetes Institute 75 Commercial Road, Melbourne, Victoria 3004 Phone: 613-8532-1389 Fax: 613-8532-1100 mailto:[email protected]