Epigenetic Mechanisms in Lung Cancer

Abstract

Epigenetic control of gene expression involves covalent reversible modifications of DNA, RNA, and histones which lead to changes in chromatin structure and accessibility. The ability to maintain precise control over gene expression in cells and tissues is critical for ensuring normal cellular development and homeostasis. The most important epigenetic mark of DNA is methylation of cytosine at the C5 position (MeC). This stable epigenetic mark is introduced by de novo methyltransferases DNMT3a/b and maintained through cell division by maintenance methyltransferase DNMT1. Ten Eleven Translocation (TET) dioxygenases oxidize 5-methylcytosine (MeC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC), 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 development. However, inflammation and exposure to exogenous agents can lead to changes in DNA methylation patterns or “epimutations” which together with genetic mutations can lead to the development of cancer. 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. Chapter I then describes epigenetic dysregulation in cancer and other human diseases. We then go on to describe how epigenetic changes in DNA can be detected and quantified using antibodies and mass spectrometry-based approaches. After considering global quantitation of epigenetic DNA modifications, we discuss the methods available for mapping epigenetic modifications along the genome. In Chapter II of this thesis, the effects of C5-cytosine substituents with increased steric bulk on catalytic activity of maintenance DNA methyltransferase (DNMT1) were examined. This protein specifically recognizes 5-methylcytosine (MeC) bases at hemimethylated CG sites in DNA and conducts maintenance methylation. Maintenance methyltransferase activity was the highest towards DNA containing the natural DNMMT1 substrate, MeC. The enzyme was capable of performing maintenance methylation when 5-ethyl-dC was the substrate, while the more rigid and bulky C5-alkyl substituents such as 5-vinyl- dC, and 5-propyl-dC could not direct maintenance methylation. In Chapter III, we investigated the kinetics of maintenance DNA methylation towards DNA duplexes containing oxidized forms of MeC (hmC, fC, and caC). We also employed a molecular dynamics simulation of the enzyme with the DNA to understand the interactions of oxidized forms of MeC with the DNMT1 enzyme. We found that methyl transfer rates were reduced when MeC was oxidized to hmC, fC, and caC, consistent with the model that Tet mediated oxidation contributes to passive DNA demethylation. In Chapters IV and V, we investigated inflammation-mediated epigenetic changes in the lung using A/J mouse model of smoking induced lung cancer. In collaboration with NuGEN (Santa Carlos, CA), we developed a novel reduced representation bisulfite sequencing (RRBS) methodology to map both MeC and hmC genome-wide. Our studies provide evidence that inflammation of the lung induces both global and site-specific epigenetic changes in DNA methylation and hydroxymethylation, alters global histone acetylation, and deregulates gene expression. These studies also provide evidence that exposure to cigarette smoke can alter site-specific DNA methylation and hydroxymethylation of genes that are associated with the cancer phenotype. The final chapter of this dissertation (Chapter VI) employs affinity proteomics to identify protein readers of epigenetic marks of DNA in the lung. DNA duplexes functionalized with C, MeC, hmC, fC, and caC were attached to solid support and incubated with nuclear protein extracts from human bronchial epithelial cells (HBEC). Proteins specifically recognizing DNA epigenetic marks were identified using Orbitrap Velos mass spectrometer and quantified using 8-plex TMT tags. This chapter details the development of a method for carrying out the affinity proteomics experiments, including solid phase synthesis of DNA targets, peptide tagging, sample clean-up, fractionation, and nanoHPLC-ESI+-MS2 based methodology for protein identification and quantification. Overall, during the course of the studies described in this thesis, we have investigated the specificity and kinetics of human maintenance DNA methyltransferase (DNMT1), employed animal models to characterize epigenetic changes in the lung caused by inflammation and exposure to cigarette smoke, and examined novel mechanisms of epigenetic regulation at oxidized forms of MeC. Overall, this work contributes to current understanding of epigenetic regulation in normal cells and epigenetic deregulation in cancer.