The Texas Medical Center Library DigitalCommons@TMC The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences 5-2018 INVESTIGATING THE IMPACT OF INTRAGENIC DNA METHYLATION ON GENE EXPRESSION, AND THE CLINICAL IMPLICATIONS ON TUMOR CELLS AND ASSOCIATED STROMA Michael McGuire Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations Part of the Bioinformatics Commons, Medicine and Health Sciences Commons, Molecular Biology Commons, and the Molecular Genetics Commons Recommended Citation McGuire, Michael, "INVESTIGATING THE IMPACT OF INTRAGENIC DNA METHYLATION ON GENE EXPRESSION, AND THE CLINICAL IMPLICATIONS ON TUMOR CELLS AND ASSOCIATED STROMA" (2018). 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INVESTIGATING THE IMPACT OF INTRAGENIC DNA METHYLATION ON GENE EXPRESSION, AND THE CLINICAL IMPLICATIONS ON TUMOR CELLS AND ASSOCIATED STROMA A DISSERTATION Presented to the Faculty of The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY by Michael McGuire, B.S. Houston, Texas May 19th, 2018 Dedication I dedicate this dissertation to my family: My mother, Colleen, my sisters, Jenna and Laura, and my grandmothers, Janet McGuire and Linda Hefner. Without them, I never would have been able to get into graduate school, much less carry out this dissertation. I owe them greatly for all of their love and support. My friends, of which are too numerous to name, who have always been there to share in my happiness, and lift my spirits when I was down. All of the scientists who have laid the foundation for our knowledge in biology, and who currently work toward a greater understanding of the natural world. iii Acknowledgements First and foremost, I would like to acknowledge the immense opportunity provided to me by my mentor, Dr. Anil Sood. I have learned an immense amount from Dr. Sood, both within science, and professionally as well. His encyclopedic intelligence, and tireless work ethic, both served as an inspiration to me, and a goal for me to achieve in my life. I would also like to thank my committee members greatly for their guidance during the past 5 years. Dr. Keith Baggerly, Dr. Sharon Dent, Dr. Gary Gallick, and Dr. Mien-Chie Hung for steering me down the proper path. Additionally, I would like to extend my gratitude to Dr. Menashe Bar-Eli, who graciously took Dr. Gallick’s spot on my committee after Dr. Gallick became ill. Thank you as well to Dr. KK Wong, who has supported me in multiple ways, including replacing Dr. Hung for my defense. I also would like to thank Dr. Mong-Hong Lee and Dr. Eugenie Kleinerman, who allowed me to serve in their labs during my time as a rotation student. I learned quite a lot from them during that short time, and I am very grateful for these opportunities. I also have a very special thanks for Drs. Tony Gutschner and Monika Haemmerle, who were incredibly instrumental in assisting me with my project and with how to design experiments. Tony not only gave me the idea to make a Cas9-Tet1 fusion construct, but also helped me carry out the necessary experiments. I would also like to thank all of the lab members I have met during the Sood lab and who have given iv me constructive criticism both within and outside of science. I have spent the last 5 years working in the Sood lab, and have had the opportunity to meet incredible people from diverse backgrounds. It has been extremely enriching. I would especially like to thank Dr. Sherry Wu, who is one of the best scientists I have come across, and whose support and advice were essential for finishing my Ph.D. v Abstract INVESTIGATING THE IMPACT OF INTRAGENIC DNA METHYLATION ON GENE EXPRESSION, AND THE CLINICAL IMPLICATIONS ON TUMOR CELLS AND ASSOCIATED STROMA Michael Hefner McGuire, B.S. Advisory Professor: Anil Sood, M.D. Investigations into the function of non-promoter DNA methylation have yielded new insights into epigenetic regulation of gene expression. Previous studies have highlighted the importance of distinguishing between DNA methylation in discrete functional regions; however, integrated non-promoter DNA methylation and gene expression analyses across a wide number of tumor types and corresponding normal tissues have not been performed. Through integrated analysis of gene expression and DNA methylation profiles, we uncovered an enrichment of DNA methylation sites within the gene body and 3’UTR in which DNA methylation is strongly positively correlated with gene expression. We examined 32 tumor types and identified 57 tumor suppressors and oncogenes out of 224 genes containing a correlation of > 0.5 between gene body methylation and gene expression in at least 1 tumor type. The lymphocyte-specific gene CARD11 exhibits robust association between gene body methylation and expression across 19 of 32 tumor types examined. It is significantly overexpressed in KIRC and LUAD, and has a z-score of 4 in KIRC, meaning that high expression of CARD11 in this tumor type was associated with lower patient overall survival. Contrary to its canonical function in lymphocyte NF-kB activation, CARD11 activates the mTOR pathway in KIRC and LUAD, resulting in suppressed autophagy, vi and demethylation of a CpG island within the gene body of CARD11 decreases gene expression. In addition to methylation of the open reading frame portion of a gene, other regions of site-specific DNA methylation along the gene body remain to be explored. Upon segregating the gene body into discrete functional units (5’UTR, 1st exon, 3’UTR), it was noted that the 3’UTR contained an enrichment of probes positively correlated between DNA methylation and gene expression. In 5 of 10 tumor types examined, DNA methylation of the 3’UTR is associated with patient survival in a significant number of genes. Filtering for genes in which 3’UTR DNA methylation, relative to gene body DNA methylation, is more strongly correlated with gene expression yields a list of 156 genes, enriched for functions involving T cell activation. Activating T cells ex vivo caused the immune checkpoint gene HAVCR2, but not other genes examined, to show a substantial increase in 3’UTR DNA methylation, but not adjacent exonic/intronic, or promoter DNA methylation, upon upregulation of gene expression. Furthermore, this increase in HAVCR2 gene expression can be abrogated by treatment with demethylating agents. These findings implicate the 3’UTR as a functionally relevant DNA methylation site, particularly regarding T cell activity. Additionally, they reveal a novel mechanism by which HAVCR2 is upregulated in T cells, providing a new molecular target for immune checkpoint blockade. vii Table of Contents Dedication ............................................................................................................... iii Acknowledgements ................................................................................................ iv Abstract ................................................................................................................... vi Table of Contents.................................................................................................. viii List of Figures ......................................................................................................... x List of Tables .......................................................................................................... xv Abbreviations ........................................................................................................ xvi Section 1: Introduction ........................................................................................... 1 1.1 The molecular and cellular biology of DNA Methylation................................... 1 1.1.1 Promoter Methylation ............................................................................ 8 1.1.2 Enhancer methylation ........................................................................... 9 1.1.3 Intergenic methylation ......................................................................... 12 1.1.4 Gene body methylation ....................................................................... 16 1.2 DNA Methylation and disease ...................................................................... 20 1.2.1 Methylation in cancer .......................................................................... 22 1.2.2 Clinical strategies for targeting methylation ......................................... 26 1.3 Non-coding RNA .......................................................................................... 28 1.3.1 Long non-coding RNA ........................................................................