Uncovering signals regulating epigenetic establishment and maintenance in mammals

The central dogma of biology is that genes code for proteins, which in turn are responsible for building cells and bodies. Our bodies are composed of diverse cell types, all specialized for specific tasks, which ensure our correct development and survival. For example, liver cells express the genes necessary to detoxify the body whilst neurons in our brain express genes necessary to transmit electrical and chemical signals. A fascinating phenomenon is that this diversity in cellular function and behavior arises and persists despite the fact that all these different cell types contain identical sets of genes (genomes) encoding a human being. To understand how different cells form and behave, we need to understand how the expression of all these different genes is controlled and regulated. Access to different genes is partly controlled by the expression of cell type-specific proteins that control gene expression (transcription factors). However, there is another layer of control that helps determine gene expression and represents the field of my interest. This so-called 'epigenetic' regulation consists of modifications to the genome and the way it is packaged which affect gene expression states without changing the underlying DNA sequence. The DNA that constitutes a cell's genome is tightly packaged into a highly organized structure within the nucleus of each cell called a nucleosome. Nucleosomes can be thought of as 'beads on a string', with each nucleosome consisting of DNA (the 'string') wrapped around a core of proteins called histones (the 'beads'). Chemical modifications can be added or removed from histones, which can have an affect on the ability of the associated DNA to attract other proteins. In addition, the DNA itself can also be subjected to modification. One of the bases of the genetic code, cytosine (C), can undergo a modification called methylation (m). Although this does not change the sequence (the mC is still read as a C), methylation contributes to gene silencing. As well as being crucial to our normal development and well being, disturbances in epigenetic regulation have the capacity to alter which genes are expressed which can have dire consequences on cell function and identity. Epigenetic alterations have been documented during aging and are a common feature of many noninfectious, age-related human diseases, such as auto-immunity, complex psychiatric disorders and cancer. In order to understand how disease-related changes arise, we need to gain a better understanding of the way epigenetic modifications are both established and accurately transmitted. This forms the basis of my proposed project. Using mice as an animal model and mouse embryonic stem (ES) cells, I aim to identify proteins involved in the establishment and maintenance of DNA methylation. I will also investigate the role of a specific modification on a histone protein in DNA methylation establishment in mouse embryos. Understanding how these modifications are normally regulated will provide insight into how things can go wrong, which could be exploited for disease diagnosis and treatment. ES cells offer the potential to generate healthy organs and tissues for use in regenerative medicine and ES cells are known to be highly active in epigenetic processes. Their possession of a unique 'epigenetic signature' underlies their ability to differentiate into many different cell types and a greater understanding of how epigenetic modifications are established and maintained will assist in their manipulation.

Epigenetic processes regulate the transmission of gene transcription states during cellular differentiation, involved in the maintenance of pluripotency and differentiation. Aberrant gene expression is responsible for most noninfectious diseases and disease-associated epigenetic changes strongly correlate with a number of age-related human disorders, such as cancer, autoimmune disease and complex psychiatric disorders. How epigenetic states are established and maintained remains poorly defined and elucidating the mechanisms will help in biomarker development for screening purposes, as well as the identification of drug targets for disease treatment. The objectives outlined in this proposal will further define mechanisms involved in both the establishment and maintenance of DNA methylation. To identify regulators of maintenance methylation, I will generate an ES cell line carrying a GFP reporter targeting the Avy agouti locus for use in an RNAi screen. A second, independent RNAi screen will identify candidate germ cell-specific factors involved in DNA methylation establishment, using retroviral infection of ES cells. Objectives 3 and 4 of this proposal will follow up on my previous findings, showing that a regulator of DNA methylation establishment, Dnmt3L, binds the N terminus of the nucleosomal protein histone H3. This binding is inhibited by methylation at lysine 4 (H3K4), suggesting that factors affecting H3K4 methylation are involved in DNA methylation establishment. To test this, I will generate mice carrying a point mutation in the Dnmt3L gene, preventing the protein from binding histone H3. These mice will provide insight into the general importance of histone recognition by Dnmt3L. I also will examine H3K4 methylation status at DNA methylation target loci in mouse germ cells using a combination of mini-ChIP and IF-FISH. Analysis of factors known to affect H3K4 methylation will highlight functionally important factors for future study by mouse genetics.

The aetiology of most noninfectious diseases involves disturbances in gene expression. Epigenetic modifications affect transcription and are altered in age-related diseases such as cancer and neurodegenerative disorders. Potential outcomes of my research could improve disease prevention, diagnosis and treatment, improving human health. Cancer and neurodegenerative disorders pose a serious threat to the health of the developed world. One in three individuals are predicted to suffer from cancer at some point in their lives and three-quarters of cases occur in people over the age of 60. Dementia, an umbrella condition that includes Alzheimer's, is not only a health issue, but is of social significance affecting the family unit and friends of sufferers. The proportion of people over the age of 60 is increasing, with the fastest growth in individuals older than 85, who are the group most at risk of developing the diseases mentioned. Immediate beneficiaries from my proposed research are the fields of epigenetics and gene regulation. Because of the role of DNA methylation in mammalian biology, my proposed work will also impact studies on mammalian development, gametogenesis and fertility. DNA methylation is an important modification regulating the expression of imprinted genes. My work will impact on organisations seeking to understand the aetiology of imprinting syndromes, such as Angelman Syndrome Support Education and Research Trust (ASSERT, UK) and Foundation for Prader-Willis Research (FPWR, USA). The development of therapeutics with the potential of impacting DNA methylation will benefit Assisted Reproductive Technology (ART), as improved understanding of how DNA methylation is regulated could make this procedure safer, reducing the incidence of imprinting syndromes. Impact is also expected in diseases where alterations in DNA methylation have been observed to affect transcription. Aberrant acquisition of DNA methylation contributes to gene silencing. This is observed in diseases such as cancer, which would benefit from agents capable of focused removal of DNA methylation as well as the restoration of methylation at sequences of functional consequence. Studies into neurodegenrative disorders may also benefit as methylation abnormalities have been reported in Alzheimer's. Regenerative medicine will also benefit from my proposed research. Reprogramming differentiated cells into multi- and pluripotent states involves loss of methylation at silent pluripotency genes. Finally, gene therapy may also benefit. Commonly used gene-delivery vectors are based around stem cell viruses and methylation-associated silencing is a problem. Understanding how DNA methylation is targeted could be exploited to either prevent or reactivate silent viral vectors, thereby prolonging the duration of therapy. Discoveries from my proposed research will be communicated to the scientific community through publication in peer-reviewed journals and presentations at international meetings and conferences. Researchers requesting resources resulting from my work will be provided with them. Collaboration has been a key aspect of my studies to date and I anticipate this to continue. Throughout my career, I have demonstrated the ability to publish my results in high-impact journals. The main publication resulting from my postdoctoral studies has garnered over 100 citations in just over 2 years since publication and I anticipate my proposed future research to have a similar impact. UCI houses a number of clinical researchers working on experimental therapeutics and has strong links to The University College Hospital. This environment will maximize collaborative efforts in translating my research e.g. analysing disease samples for lesions in factors identified from my research. Disseminating my results to the general public is encouraged by UCL, which organizes The Livery Science Day Event, aimed at introducing scientific methodology to school children.