Washington University in St. Louis Washington University Open Scholarship All Theses and Dissertations (ETDs) 5-31-2012 Determining the Roles that DICER1 and Noncoding RNAs Play in Endometrial Tumorigenesis Katherine Chiappinelli Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/etd Recommended Citation Chiappinelli, Katherine, "Determining the Roles that DICER1 and Noncoding RNAs Play in Endometrial Tumorigenesis" (2012). All Theses and Dissertations (ETDs). 946. https://openscholarship.wustl.edu/etd/946 This Dissertation is brought to you for free and open access by Washington University Open Scholarship. It has been accepted for inclusion in All Theses and Dissertations (ETDs) by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Division of Biology and Biomedical Sciences Developmental, Regenerative, and Stem Cell Biology Dissertation Examination Committee: Paul J. Goodfellow, Chair Douglas Chalker Sarah C. R. Elgin Susana Gonzalo Sheila A. Stewart Ting Wang Determining the Roles that DICER1 and Noncoding RNAs Play in Endometrial Tumorigenesis by Katherine Bakshian Chiappinelli A dissertation presented to the Graduate School of Arts and Sciences of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy August 2012 Saint Louis, Missouri copyright by Katherine Bakshian Chiappinelli 2012 ABSTRACT OF THE DISSERTATION Cancer is both a genetic and epigenetic disease. Changes in DNA methylation, histone modifications, and microRNA processing promote tumorigenesis, just as mutations in coding sequences of specific genes contribute to cancer development. In my thesis work I sought to determine the role that noncoding RNAs play in endometrial tumorigenesis. Aberrant methylation of the promoter region of the MLH1 DNA mismatch repair gene in endometrial cancer is associated with loss of MLH1 expression and a "mutator phenotype" in endometrial and other cancers. The molecular and cellular processes leading to aberrant methylation of the MLH1 promoter region are largely unknown. I tested the hypothesis that the EPM2AIP1 antisense transcript at the MLH1 locus could be involved in MLH1 transcriptional silencing. I characterized the MLH1/EPM2AIP1 bidirectional promoter region in endometrial cancer and normal cell lines and found an abundance of forward and reverse transcripts initiating from a large region of nucleosome-free DNA in expressing cells. The DICER1 protein, which is necessary for processing small RNAs involved in post-transcriptional silencing, is downregulated in many cancers, including endometrial cancer. I used genomic methods (RNA-Seq and MeDIP/MRE) to characterize the transcriptome and methylome of endometrial cancer cells depleted of DICER1. Using a combination of computational and wet lab methods I showed that reduced DICER1 triggers an interferon response in cancer cells because of accumulation of pre-microRNAs that activate immune sensors of viral dsRNA. The methylome of DICER1 knockdown cells revealed subtle changes in methylation, including decreased methylation at the Alu family of repetitive elements. ii Small RNAs processed by DICER1 may thus be involved in silencing repetitive regions. Non-coding RNA has effects on endometrial cancer cells that may contribute to tumorigenesis, such as influencing the active state of the MLH1 gene and modulating the immune response. iii ACKNOWLEDGEMENTS I thank Paul J. Goodfellow who has truly been the best Ph. D. mentor I could have asked for. His patience, constructive criticism, and quiet encouragement have made graduate school an excellent experience for me. I know that I will judge every mentor I have in the future by his standards and I hope to be half the mentor he is one day. I thank the members of my thesis research advisory committee, whose insightful comments and novel ideas helped me focus and improve my thesis research. I thank Sheila A. Stewart and Susana Gonzalo for welcoming me into their laboratories as a rotation student, serving as inspiring role models for a young female scientist, and transmitting their passion for research to me. I thank Douglas Chalker for appearing every step of the way in my graduate school experience; he interviewed me, taught me my first year, served on both my qualifying examination and thesis advisory committees, let me T.A. and even teach one of his courses, and was always an endless fount of knowledge about epigenetics. I thank Ting Wang for his patience working with a non- computational biologist and for his boundless enthusiasm about DNA methylation and repetitive elements. I thank Sarah C. R. Elgin for making time to be my chair and for her constructive criticism and advice about my science and my future. I thank the members of the Goodfellow Laboratory, past and present, for making my time in the laboratory interesting and enjoyable: Amy P. Schmidt, Dominic M. Thompson, Daniel Wilke, Akiva Novetsky, Stefanie Ludwig, Michael Joyce, Nora Kizer, Yevgeniya Ioffe, Saketh Guntupalli, Bobbie Jo Rimel, Summer Dewdney, Andrew Reinhart, Israel Zighelboim. I especially thank Amy and Nick for their patience and lab wisdom over the past four years. I thank Patricia Werner for her brilliant assistance formatting and submitting publications. I thank the many friends I was fortunate to make in graduate school, but especially Jessica H. Geahlen, who has been by my side every step of the way. I thank the members of DNA Methylation Journal Club for their skepticism and scientific thinking. I thank others at Washington University. Michael R. Brent, Brian C. Haynes, and Sarah Spencer made our collaboration pleasant and scientifically stimulating. I thank Bo Zhang for his investment and hard work on the methylome project. I thank Kathryn Miller for teaching me how to teach. I thank John Russell for his kindness and help throughout my Ph. D. program. I thank James Skeath for the countless graduate student dinners hosted at his house and for always having time for me. I thank Stacy Kiel for her assistance keeping me organized throughout my Ph. D. program. I thank Jennifer Mosher, Thomas A. Woolsey, and all of the Washington University student volunteers associated with the Young Scientist Program for making a difficult job enjoyable and for pushing me to be a better leader. I thank the hundreds of St. Louis Public School Students who reminded me every day that education was a gift and science was the most exciting thing in the world. I thank my family- my parents, my sister Alex, and both sides of our extended family- for their endless support and love. I thank my Oaks “in-laws” for their real iv interest in my work and ongoing support. I thank my wonderful housemates, especially Amy Johnson: she has taught me by example to do what I love and love what I do. Lastly, I thank my fiance Jason Oaks for everything. His endless patience and confidence in me when I lose faith have gotten me through graduate school. He is my best friend and makes me a better person and academic. This work is dedicated to my parents, Suzanne Bakshian and Vincent A. Chiappinelli, for their enthusiastic support of everything I have wanted to do in life, whether it was becoming a veterinarian, a concert pianist, or an academic scientist. v TABLE OF CONTENTS ABSTRACT OF THE DISSERTATION ................................................................... II ACKNOWLEDGEMENTS ........................................................................................ IV LIST OF FIGURES ....................................................................................................... X LIST OF TABLES ......................................................................................................XII ABBREVIATIONS USED IN THE TEXT ............................................................ XIII CHAPTER ONE: INTRODUCTION ...........................................................................2 EPIGENETIC MECHANISMS....................................................................................... 3 CANCER EPIGENETICS ............................................................................................... 6 EPIGENETIC MECHANISMS IN ENDOMETRIAL CANCER ............................... 8 REFERENCES ................................................................................................................ 15 CHAPTER TWO: THE ACTIVE MLH1/EPM2AIP1 BIDIRECTIONAL PROMOTER IS CHARACTERIZED BY MULTIPLE TRANSCRIPTS AND AN ABSENCE OF STABLY POSITIONED NUCLEOSOMES ....................................21 FOREWORD................................................................................................................... 22 ABSTRACT ..................................................................................................................... 26 INTRODUCTION........................................................................................................... 26 RESULTS AND DISCUSSION ..................................................................................... 29 Overlapping MLH1 and EPM2AIP1 Transcripts ...................................................... 29 Absence of Nucleosomes at the MLH1/ EPM2AIP1 Bidirectional Promoter Region ................................................................................... 31 MATERIALS AND METHODS ................................................................................... 35 Cell