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Date of Award
Doctor of Philosophy (PhD)
Epigenetics is the study of heritable changes in gene activity that are not caused by changes in the DNA sequence. It involves the addition of various chemical groups to the DNA or histone tails. By controlling the chromatin accessibility, epigenetics can regulate gene expression, and as a result, influence biological processes at various different levels. DNA methylation is a heritable epigenetic mechanism with important roles in diverse biological processes ranging from gene-environment interactions underlying complex diseases in humans to the evolution of epigenomes across multiple species. The investigation of DNA methylation and its relationship with genetics is the focus of this dissertation project. My research encompasses several domains in the fields of epigenome evolution, and in epigenetics of complex diseases, particularly addiction and metabolic diseases.
At the epigenome level, I took a comparative epigenomics approach and investigated principles of epigenome evolution using next-generation sequencing data in several species. Advanced sequencing technologies can characterize the DNA methylomes at high resolution in multiple samples. In this project, I utilized MeDIP-seq and MRE-seq technologies to generate DNA methylomes in multiple tissue types in rats. I have also collected and studied publically available methylation data, including MeDIP-seq, MRE-seq and genome wide bisulfite sequencing (WGBS) data, to comprehensively analyze DNA methylomes of different tissues in multiple species. To our knowledge, this study is the first comprehensive characterization of three tissue types (blood, brain, sperm) in rats; the work is also the first to examine epigenetic conservation using multiple tissues in multiple species. I showed that tissue-specifically differentially methylated regions (tsDMRs) strongly associate with tissue-specific regulatory elements. Comparisons between species revealed that 10-40% of this tissue-specific DNA methylation pattern is conserved, a phenomenon that we defined as epigenetic conservation. Conserved DNA methylation is also accompanied by conservation of other epigenetic marks including histone modification. Examination of the genetic underpinning of epigenetic conservation suggests that primary sequence conservation is a driving force behind epigenetic conservation. In contrast, analyses of evolutionarily dynamic regions harboring tissue-specific DNA methylations implicated the maintaining or turning over of binding sites of important transcription factors. Our study thus provides a framework into investigation of principles of epigenetic evolution.
At the complex diseases level, I conducted two projects that applied sequencing and microarray technologies to study methylation patterns in morphine addiction in model rats and in adiposity traits in humans, respectively. The purpose of the studies is to improve our understanding of DNA methylation in studying complex diseases, particularly in the context of gene-environment interactions that lead to the manifestation of disease phenotypes. In the morphine addiction study, I applied the DNA methylome sequencing technologies and the M&M analysis method to compare methylation patterns in morphine treated and placebo treated rats. I generated 36 DNA methylomes and investigated the effects of morphine on differential DNA methylation in three tissue types: blood, brain and sperm, across three generations including the morphine/placebo treated rats (F1 generation), the male offspring of the F1 generations (which were not exposed to the treatments - F2), and the precise fathers of the F2 generation). While a number of minor alterations were detected, I did not observe dramatic difference in DNA methylations between the morphine-treated samples and placebo-treated samples. The results seemed to suggest that the acute environmental assault of a short-period (3-day) of morphine treatment in the rat model was not enough to induce dramatic DNA methylation change in blood, brain and sperm. We venture that an increased or sustained exposure to morphine is needed to induce more substantial change in the epigenome and a refined sample type (use specific region in the brain such as nucleus accumbens and ventral tegmental area) is necessary to detect the difference. In the adiposity traits study, I applied methylation microarray technology to survey global methylation marks in peripheral blood cells (PBC) in 16 Caucasians selected from a linkage study of body mass index (BMI) and 8 unrelated controls. Association analysis of BMI with methylation levels at 27K CpG sites was followed by family-based association test of methylation levels at the significant sites with GWAS single-nucleotide polymorphisms (SNPs). I found 140 CpG sites significantly associated with BMI (p<5x10-3), many with regulatory potentials, including one in a CpG island in the promoter region of HDAC11 that encodes for a histone deacetylase important to transcriptional regulation. Hierarchical clustering applied to methylation levels at the significant sites showed clear familial aggregation of methylation patterns. The family-based GWAS of methylation levels at the significant sites identified 1,710 unique "methylation SNPs" (mSNPs) associated (p<10-3) with methylation at >= 1 of 112 sites, 243 of which were associated with more than 6 sites; one on Chr 2 with 7 sites (including FSTL1 and KCNQ1, previously implicated in obesity and CVD). While many of the significant PBC methylation marks possibly do not have direct functional role in adiposity, the findings provide evidence of familial epigenetic signatures for adiposity by DNA methylation in PBC that may provide new revenues for studying epigenetic-mediated GxE interactions.
In summary, in the present research I applied latest technologies for DNA methylation analysis to study principles of epigenome evolution and epigenetics in complex diseases. The investigation at the evolution level conducted the first study of comparative epigenomics based on genome-wide DNA methylation in 3 major tissue types across 3 species and made some exciting discoveries about epigenome conservation and the underlying factors driving epigenome conservation. The real studies of global methylation patterns in morphine addiction and in adiposity traits provided some insights about the effect of acute environmental assault (morphine treatment) and sustained long-term environmental exposure on the methylomes in model animals and in humans.
Due to the modest sample sizes and sampling limitations of the real studies, further confirmation or replication of the findings are in order. Nonetheless, our work illustrates some important aspects of principles of epigenome evolution and epigenetics of complex diseases. The findings provide a basis for further study of epigenome evolution, and for continual exploration of methylomic variations as epigenetic markers and risk factors to study epigenetic-mediated gene-environment interactions in complex diseases in humans.
Chair and Committee
Charles Gu, Theodore Cicero, Justin Fay, Nancy Saccone, Gary Stormo
Zhou, Jia, "Principles of Epigenome Evolution" (2014). Arts & Sciences Electronic Theses and Dissertations. 343.
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