EASTBIO -The Requirement for Epigenetic Mechanisms in establishing and maintaining Cell Identity

Cellular memory is regulated by epigenetic mechanisms that ensure the heritable characteristics of cells and functional differences between cell types, without changes to the DNA sequence. These epigenetic changes tie in with the influence of the signalling environment of the embryo on gene regulation. DNA methylation is a repressive epigenetic modification involving the covalent addition of a methyl group to cytosine by DNA methyltransferases (DNMTs). The relationship between DNA methylation and gene silencing is indirect for many genes, as global methylation levels affect silencing via the function of histone modifying complexes and the formation of higher order 3D chromatin structures. However, maintenance (Dnmt1) and de novo DNA methyltransferases (Dnmt3 and Dnmt3b) are essential for mouse development. Their inactivation leads to developmental failure during gastrulation stage that is associated with gene misexpression, implying that DNA methylation is required for later stage embryos when differentiation occurs, and methylation patterns are propagated through cell lineages. In vitro cultured mouse embryonic stem cells (mESCs) can self-renew without DNA methylation but are impaired for differentiation to most cell fates.
It is still not well understood why repressive epigenetic pathways are necessary for development and when they become operational in the transition from stem cells to differentiated cells. We have reported that dominant transcription factor networks operate independently from repressive epigenetic processes in mESCs and hypothesise that they engage with these epigenetic mechanisms during a transition process upon differentiation.
To address these research questions, we will use mESC model systems, in which the global DNA methylation levels can be controlled. Colour markers in the cells will report on DNA methylation and differentiation status, or alternatively, cell stress status. The first aim is to determine the lower threshold of DNA hypomethylation necessary for differentiation. We will assess how this affects the generation of cell types of the three germ layers in embryoid body differentiation models and other embryo-like assemblies. We will identify factors critical for these transitions at threshold conditions based on gene expression changes. Conversely, we aim to determine the effects of controlled DNA methylation loss on somatic cells derived from these mESCs, to determine the DNA methylation threshold for hypomethylated somatic cells that is necessary to retain their identity. We will identify the factors involved in this process and compare the requirements of different cell types, including neuronal, heart and liver cells, of which our labs have expertise. Candidate gene expression will be individually validated in cultured embryos, and in vitro models for embryonic blastocyst and gastrulation assemblies and somatic cell organoids.
This project will contribute novel insights into ‘the rules of life’ by asking a fundamental question of how our cells would function in the absence of epigenetic mechanisms, to determine how cells rely on these pathways. Previous work suggests that the integrity of epigenetic mechanisms is linked with healthy ageing and understanding its precise role may identify possible new targets for regenerative therapy of chronic and degenerative conditions.