Epigenetic modifications are genomic alterations which regulate the expression and activity of genes by changing the structure of chromatin. These mechanisms of regulation expand the proportion of the genome that is functional well beyond the comparably rare instances of protein coding genes, which, in humans, only correspond to ~2% of the genome. The aim of this dissertation is to leverage advances in the genomic identification and annotation of epigenetic modifications to explore questions regarding the (1) role of DNA methylation in X chromosome regulation through comparative genomic analyses, (2) the organization and (3) evolution of enhancers identified from histone modifications.
In the second chapter of this thesis, we consider the role of DNA methylation in an iconic example of epigenetic regulation, namely the X chromosome inactivation (XCI). XCI is the process by which one of the two female X chromosomes is silenced to balance the expression of X-linked genes in male and female genomes and is functionally conserved in two branches of mammals (eutherians and marsupials). In eutherians, it is well established that DNA methylation plays a role in establishing XCI through the silencing of the lncRNA Xist on the active X chromosome as well as in the long-term maintenance of inactive X-linked genes. However, the role of DNA methylation in marsupials remains controversial. We utilize novel multi-tissue, sex-inclusive Whole Genome Bisulfite Sequencing (WGBS) coupled with improved genomic annotations to elucidate the role of DNA methylation in X chromosome regulation in a representative marsupial, the modern koala (Phascolarctos cinereus). Consequently, we clarify conserved and divergent roles of DNA methylation on the regulation of XCI in marsupials and eutherians.
In the following two chapters, we integrate multi “-omics” datasets including whole genome chromatin state maps and gene expression data from a diverse set of tissues to elucidate the organization and evolution of human enhancers, a hallmark of the (epi)genomic regulatory landscape. Enhancers are short, mostly non-coding DNA sequences that orchestrate the context- and developmental time-specific expression of associated genes. Enhancers are often studied as highly tissue-specific regulatory elements in what has been deemed a “paradigm of modularity.” However, contrary evidence, indicating that a subset of enhancers may be repurposed in multiple tissue and/or developmental contexts, is mounting. In this study, we characterize the previously unknown frequency and genomic characteristics of these highly “pleiotropic” enhancers. We further evaluate the organization of the larger gene-enhancer interaction network considering (1) the distribution of enhancer pleiotropy, (2) the variations in the number of enhancer-target gene links, and (3) the expression breadth of target genes.
Furthermore, we explore the evolution of human enhancer through genomic duplication events. Duplications are a canonical reservoir of the raw material needed for the evolution of novel functional elements in the genome and have been studied extensively with respect to genes. The selective processes governing the maintenance of duplicate genes are well characterized, and similar evolutionary mechanisms have been proposed for non-coding regulatory elements. However, whether duplication events affect enhancer evolution and maintenance is currently unknown. Through sequence homology analyses, we identify likely candidate duplicate enhancers in our large dataset to determine the frequency of duplicate enhancer retention in the human genome. Additionally, we determine the characteristics of duplicate enhancers contributing to their evolutionary maintenance. We demonstrate that duplication of enhancers has significant footprint on pleiotropic enhancers and that recently duplicated human enhancers exhibit signatures of accelerated evolution and specialized for immune related functions.
Together, these studies reveal previously unknown patterns of conservation and divergence of epigenetic regulatory mechanisms along two deep branches of mammals, as well as elucidate the molecular architecture and the impact of duplication on the genomic landscape of enhancer-gene regulation.Ph.D
