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COMPUTATIONAL PREDICTION AND EXPERIMENTAL VALIDATION OF NOVEL MOUSE IMPRINTED GENES A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Chelsea Marie McLean August 2009 © 2009 Chelsea Marie McLean COMPUTATIONAL PREDICTION AND EXPERIMENTAL VALIDATION OF NOVEL MOUSE IMPRINTED GENES Chelsea Marie McLean, Ph.D. Cornell University 2009 Epigenetic modifications, including DNA methylation and covalent modifications to histone tails, are major contributors to the regulation of gene expression. These changes are reversible, yet can be stably inherited, and may last for multiple generations without change to the underlying DNA sequence. Genomic imprinting results in expression from one of the two parental alleles and is one example of epigenetic control of gene expression. So far, 60 to 100 imprinted genes have been identified in the human and mouse genomes, respectively. Identification of additional imprinted genes has become increasingly important with the realization that imprinting defects are associated with complex disorders ranging from obesity to diabetes and behavioral disorders. Despite the importance imprinted genes play in human health, few studies have undertaken genome-wide searches for new imprinted genes. These have used empirical approaches, with some success. However, computational prediction of novel imprinted genes has recently come to the forefront. I have developed generalized linear models using data on a variety of sequence and epigenetic features within a training set of known imprinted genes. The resulting models were used to predict novel imprinted genes in the mouse genome. After imposing a stringency threshold, I compiled an initial candidate list of 155 genes. A subset of these genes was tested for evidence of imprinting using allele-specific restriction digests in either brain or placenta. Of the 10 genes tested in placenta, 2 showed evidence of maternal allele-specific expression. I also designed a custom microarray to test a total of 563 genes predicted as imprinted at lower stringency levels. Of these 563 genes, I experimentally tested 32 in placenta and 8 in brain, resulting in the identification of an additional 5 novel imprinted genes in placenta. This study is the first to demonstrate the utility of epigenetic marks in the prediction of imprinted genes. Furthermore, specific combinations of epigenetic marks were commonly found within particular regions relative to the transcriptional start sites of imprinted genes, implicating their placement and localization in the imprinting mechanism. BIOGRAPHICAL SKETCH Chelsea McLean was born on June 19, 1982 in Rochester, Michigan to Dara and Larry McLean. She graduated from Rochester Adams High School in 2000, where she took part in two field courses: Tropical Rainforest Ecology and Coral Reef Biology in Belize, Central America and The Ecology of Greater Yellowstone National Park in Yellowstone National Park. She credits participation in these courses for a large part of her interest in science. After high school, she continued on to the Lyman Briggs School at Michigan State University in East Lansing, Michigan. While at Michigan State, she participated in a wide variety of service organizations and taught two Honors level Biology labs: Lyman Briggs Honors Organismal Biology Lab, under the direction of Dr. Charles Elzinga, and Lyman Briggs Honors Cell and Molecular Biology Lab, under the direction of Dr. John Urbance. She also conducted research for two years in the Canine Genetics Lab of Dr. Patrick Venta and for one summer in the Small Fruit Entomology Lab of Dr. Rufus Isaacs. In 2004, Chelsea graduated with High Honor and a Bachelor of Science degree in Microbiology and Molecular Genetics and was recognized as a Michigan State University Outstanding Senior. After graduation, she moved to Ithaca, New York to attend graduate school at Cornell University and to pursue a Ph.D. in Genetics and Development under the instruction of Dr. Paul Soloway. Upon completion of her dissertation in 2009, Chelsea continued on to a postdoctoral position in the lab of Drs. Elizabeth Robertson and Elizabeth Bikoff at the University of Oxford in Oxford, U.K. iii To my family, Dara, Larry, and Danielle: for your love and support. And for always telling me that I can do anything I set my mind to. To my teachers: Todd Vince and Dave Glenn for sharing your enthusiasm for science and for being wonderful teachers. To my best friend: Nick Brideau for always being there for me. To all of my friends: For making my time in Ithaca memorable and enjoyable. iv ACKNOWLEDGMENTS There are so many people I have to acknowledge for their help, guidance, and friendship while I was at Cornell. Without these people, I would not be where I am today. First, my advisor, Paul Soloway, for his support over the last five years. From him, I learned countless scientific techniques, as well as how to think both critically and creatively. Also, the members of my special committee for their guidance throughout the many difficult phases of my various Ph.D. projects: Dr. Andrew Clark, Dr. John Schimenti, and Dr. Tudorita Tumbar. And, along the way, I was lucky to have some wonderful labmates, who taught me so much through example and through discussion. Rebecca Holmes, Anders Lindroth, and Yoon Jung Park were an invaluable part of my experience at Cornell, as was Herry Herman, who was my mentor during my rotation in the Soloway lab. And, of course Patrick Murphy for his sunny disposition and his unbridaled enthusiasm for science, Ruquian Zhao for her support, encouragememt, and phoenix good luck charm. Jim Putnam for his ever-sunny attitude and for keeping our lab running smoothly. And, of course, my friends. Karen Osorio for always listening, for making me laugh, and for her amazing desserts. Rebecca Holmes and Nathalie Nicod, who were there with me everyday and provided (decaf) coffee breaks, laughter, chocolate, and encouragement as needed. Sam and David Infanger for beautiful days on Ms. Green and fun nights of creative social events. Kim Holloway for her endless cheer, great barbecue, and love of good v fish and chips…with or without mushy peas. Amanda Larracuente for our fun dinners out and Bluestone Mojitos. And, of course, “The Girls” for our girls’ nights. I also have to thank my very best friend and future husband, Nick Brideau. Without him, I could not have made it through everything that happened to me while I was in Ithaca, both personally and professionally. Without his love and support, I quite literally would not be writing this dissertation. Finally, I would like to acknowledge my family. It is because of them that I learned the value of education and organization. From as far back as I can remember, my family managed to combine education and fun. For this I am grateful, as I never viewed education as a chore, but as a privelage. Also, because of the various after school activities that I was involved in, I learned how to multi-task and to complete projects under a deadline. I couldn’t have learned these skills without dedicated parents who not only shuttled me to and from my various activities on time, but also made sure my homework was complete at the end of each day. Even now, from across the country, my family manages to provide their unconditional support, for which I am extremely grateful, and I can only hope that I have made them proud. vi TABLE OF CONTENTS BIOGRAPHICAL SKETCH........................................................................... iii DEDICATION............................................................................................... iv ACKNOWLEDGMENTS .............................................................................. v TABLE OF CONTENTS............................................................................... vii LIST OF FIGURES ...................................................................................... xi LIST OF TABLES......................................................................................... xv LIST OF ABBREVIATIONS ......................................................................... xvi LIST OF SYMBOLS..................................................................................... xix I. INTRODUCTION I.1 Epigenetic regulation of gene expression ............................................ 1 I.1.1 Introduction to epigenetics ............................................................ 1 I.1.2 DNA methylation........................................................................... 2 I.1.3 Histone modifications.................................................................... 4 I.2 Genomic imprinting.............................................................................. 5 I.2.I Introduction to genomic imprinting .................................................. 5 I.2.2 History of genomic imprinting......................................................... 7 I.2.3 DNA methylation and genomic imprinting ...................................... 8 I.2.4 Histone modifications and genomic imprinting............................... 12 I.3 Epigenetics in development and disease............................................. 14 I.3.1 Developmental timing of methylation marks .................................. 14 I.3.2 Trans-acting DNA methylation reprogramming factors .................. 15 I.3.3 Cis-acting DNA methylation reprogramming factors ...................... 17 I.3.4 Causes and consequences
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