DNA EPIGENETIC MARKS IN THE DEVELOPMENT OF INFLAMMATION ASSOCIATED CANCERS AND ALZHEIMER’S DISEASE

Abstract

Epigenetic control of gene expression via DNA methylation, histone modifications, and ncRNAs is critical for ensuring normal cellular development and homeostasis. The best studied DNA epigenetic mark is methylation of cytosine at the C5 position (5mC). This DNA epigenetic mark is introduced by de novo methyltransferases DNMT3a/b and maintained during cell division by maintenance methyltransferase DNMT1. Ten Eleven Translocation (TET) dioxygenases can iteratively oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), a process known to induce DNA demethylation and gene reactivation. A precise balance of DNA methylation and demethylation is important for establishing tissue specific gene expression patterns, maintaining cell identity, and guiding normal development. Reprogramming of DNA methylome and hydroxymethylome has been recognized as a critical event in the development of cancers. N6-methyldeoxyadenosine (N6medA) is a potential novel DNA epigenetic mark recently discovered in mammals. Although the exact biological role of N6medA in mammalian genome is still elusive, N6AMT1 and METTL4 are possible methylases that introduce N6medA, while ALKBH1 and ALKBH4 are likely demethylases that remove N6medA. N6medA has been implicated in epigenetic regulation, DNA repair, memory formation, and transposable element suppression, and it has been proposed to play a role in early embryonic development, cancer, and neurological functions. Chapter I of this Thesis provides an overview of the major mechanisms of epigenetic regulation including epigenetic marks of DNA, non-coding RNAs, and histone modifications. A further review of DNA epigenetic marks including the formation, reversibility, abundance, genomic distribution and corresponding biological effects is provided. We then discuss the experimental methodologies for quantifying global levels of epigenetic DNA modifications as well as methods for genome wide mapping of DNA epigenetic marks. Finally, Chapter I covers the role of epigenetics in serving as a molecular link between human disease and its associated risk factors, with a focus on cancer and chronic inflammation, Alzheimer’s Disease, and aging. Chapter II of this Thesis characterizes DNA methylation and hydroxymethylation changes in a mouse model of inflammatory bowel disease/colon cancer. By coupling DNA methylation and hydroxymethylation sequencing data with RNA-seq data for the same tissues, we found that inflammation mediated epigenetic changes altered gene expression levels of key tumorigenesis genes, which could contribute to colon cancer initiation. Chapter III of this thesis examines the timing and the persistence of smoking- mediated epigenetic changes in the lung using a mouse model of smoking induced lung cancer. We characterized DNA methylome, hydroxymethylome, as well as transcriptome changes in the type II alveolar cells of A/J mice exposed to cigarette smoke for 3 weeks, 10 weeks, or 10 weeks followed by a 4 week recovery period. By comparing our data with human lung adenocarcinoma dataset, we identified several cancer related genes that demonstrate early epigenetic changes upon exposure to cigarette smoking. The functional roles of these genes was explored in cell proliferation assay, leading to the identification of OSR2 as a potential protooncogene. In Chapter IV, we explored the dynamics of the novel DNA epigenetic mark N6-methyldeoxyadenosine (N6medA) in the context of human aging and AD. We characterized its global dynamics in human aging and AD via an optimized isotope dilution, high resolution nanoLC-NSI MS/MS method. Global N6medA levels positively correlated with human chronological age. In addition, N6medA was profiled across the human genome using N6medA IP-seq, revealing adenine methylation changes potentially associated with aging and AD. Finally, we identified several specific protein readers of N6medA via mass spectrometry based affinity proteomics. These protein readers have functions in DNA replication, transcription and configuration, implying a regulatory role of N6medA in DNA templated biological processes. Taken together, during the course of the studies described in this Thesis, we have characterized DNA epigenetic changes in two mouse models of inflammation associated cancer, together with gene expression changes. We have also examined the potential oncogenic roles of genes that are differentially methylated upon exposure to cigarette smoking. Finally, we investigated the potential involvement of a novel DNA epigenetic mark, N6-methyl-dA, in human aging and Alzheimer’s Disease. Overall, this work contributes to the current understanding of epigenetic deregulation in inflammation associated cancers and sheds new light on the role of a novel DNA epigenetic marks in human aging and AD.