Epigenetic Mechanisms in Development and Disease

Abstract

This thesis presents three ongoing research projects that each attempt to understand the earliest stages of ontogeny, the assembly of a complex multicellular organism from a single totipotent cell, and how cellular diversity arises given that these cells must interpret an identical genetic template. In chapter one, I focus on the global redistribution of cytosine methylation that occurs within the implantation-stage mouse embryo to create highly divergent embryonic and extraembryonic epigenomic landscapes. Notably, the extraembryonic landscape is characterized by globally reduced DNA methylation levels and non-canonical targeting to promoters of developmental genes, which are generally assumed to be regulated by the Polycomb Repressor Complexes (PRC) 1 and 2 within embryonic lineages but are aberrantly methylated in most cancers. By exploring the epigenetic response to certain growth factors and generating mutant embryos, I establish that this landscape can be acquired deterministically during a brief developmental window and appears to represent the downstream effects of a dedicated pathway, suggesting that this pervasively observed feature in cancer may represent a misappropriated, but developmentally-encoded, mode of genome regulation. In chapter two, I optimize a platform for evaluating mutant mouse embryos using single cell transcriptomics that can be used to investigate the roles of promiscuously utilized epigenetic regulators, which have no innate sequence specificity yet regulate a highly cell-type specific target spectrum. I then present the application of this pipeline to investigate PRC2 as it functions in early lineage commitment. Embryos lacking the essential component Eed fail to properly apportion the primitive streak, leading to a general posteriorization of the mesoderm and overproduction primordial germ cell-like cells. Notably, these defects can be detected within progenitor states before the mutant phenotype is morphologically apparent. Finally, chapter three represents ongoing efforts to develop methods for recording cellular lineage agnostically and at single cell resolution using continuous, stochastic Cas9-based genome editing in lieu of traditional methods. With this tool, my collaborators and I hope to open up new quantitative avenues for studying the robust nature of development, where differentiating cellular fields must be synchronously regulated at the organismal level to effectively reproduce a complete body plan.