EPIGENETIC MECHANISMS IN LUNG CANCER A DISSERTATION SUBMITTED TO THE FACULTY OF UNIVERSITY OF MINNESOTA BY Christopher Lee Seiler IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY ADVISER: Dr. Natalia Y. Tretyakova September 2018 © Christopher L Seiler, 2018 Acknowledgments I would like to offer my gratitude to all who have contributed both directly and indirectly to this dissertation and my education at the University of Minnesota. First and foremost, I would like to thank my adviser, Dr. Natalia Y. Tretyakova, for her continuous guidance and support during my graduate career. I would also like to thank the many other mentors who have helped shape my scientific career: Peter Villalta and Brock Matter for advancing my skills and knowledge of mass spectrometry techniques employed in this work, Linda von Weymarn and Steve Carmella for instilling a deep appreciation for chromatography, Pramod Upadhyaya and Bin Ma for exemplifying the benefits of thorough discussion, and Delshanee Kotandeniya for inspiring my research into the field of epigenetics. I would like to thank the members of my committee, Courtney Aldrich, Colin Campbell, and Robert Turesky for their guidance and suggestions during my graduate career. I am appreciative of all of the faculty and staff of the Department of Medicinal Chemistry for their influence. I am indebted to all the current and former members of the Tretyakova laboratory and the Masonic Cancer Center, as well as all of the collaborators and contributors to the work presented in this dissertation. I would like to thank Robert Carlson for his constant editorial and technical support. I would like to thank Alex Baierl and Redmond Fraser for their friendship and support through graduate school. This dissertation would not have been possible with the enthusiasm and support from my parents, family, friends, and fellow graduate students. i Most notably, I would like to thank my wife Sheila Mae Seiler for her support and motivation. ii Dedication This dissertation is dedicated to my wife Sheila Mae Seiler for her endless love and support. iii 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. iv 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 v 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. vi TABLE OF CONTENTS Abstract .............................................................................................................................. iv Table of Contents .............................................................................................................. vii List of Tables .................................................................................................................... xii List of Figures ................................................................................................................... xv List of Schemes ............................................................................................................. xxxii List of abbreviations ..................................................................................................... xxxv I. Literature Review ............................................................................................................ 1 1.1 Epigenetic regulation of gene expression ................................................................. 2 1.1.1 What is epigenetics? .......................................................................................... 2 1.1.2 DNA epigenetic marks ....................................................................................... 2 1.1.3 Histone epigenetic marks ................................................................................. 11 1.1.4 RNA epigenetic marks ..................................................................................... 16 1.2 Epigenetic deregulation and human disease ........................................................... 17 1.2.1 Role of epigenetic deregulation in disease ....................................................... 17 1.2.3 Inflammation and increased cancer risk .......................................................... 20 1.2.4 Animal models of lung cancer initiation .......................................................... 21 1.3 Methods for detection and quantification of epigenetic modifications within the genome .......................................................................................................................... 23 1.3.1 Initial detection of MeC and its oxidized forms via paper chromatography and thin layer chromatography ........................................................................................ 23 vii 1.3.2 Antibody based detection of epigenetic marks in DNA .................................. 26 1.3.4 High performance liquid chromatography and mass spectrometry