Evolutionary and Functional Impacts of Short Interspersed Nuclear Elements (SINEs) Revealed via Genomic Assessment of Felid CanSINEs By Kathryn B. Walters-Conte B. S., May 2000, University of Maryland, College Park M. S., May 2002, The George Washington University A Dissertation Submitted to The Faculty of Columbian College of Arts and Sciences of The George Washington University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy May 15 th , 2011 Dissertation Directed By Diana L.E. Johnson Associate Professor of Biology Jill Pecon-Slattery Staff Scientist, National Cancer Institute . The Columbian College of Arts and Sciences of The George Washington University certifies that Kathryn Walters-Conte has passed the Final Examination for the degree of Doctor of Philosophy as of March 24 th , 2011. This is the final and approved form of the dissertation. Evolutionary and Functional Impacts of Short Interspersed Nuclear Elements (SINEs) Revealed via Genomic Assessment of Felid CanSINEs Kathryn Walters-Conte Dissertation Research Committee: Diana L.E. Johnson, Associate Professor of Biology, Dissertation Co-Director Jill Pecon-Slattery, Staff Scientist, National Cancer Institute, Dissertation Co-Director Diana Lipscomb, Ronald Weintraub Chair and Professor, Committee Member Marc W. Allard, Research Microbiologist, U.S. Food and Drug Administration, Committee Member ii Acknowledgements I would like to first thank my advisor and collaborator, Dr. Jill Pecon-Slattery, at the National Cancer Institute of the National Institutes of Health, for generously permitting me to join her research group. Without her mentorship this dissertation would never have been possible. I would also like to express gratitude to my advisor at the George Washington University, Dr. Diana L. E. Johnson, who provided invaluable moral and scientific support over the years. I have had a dynamic committee who deserve my thanks: Dr. Marc Allard, my first advisor, for bringing me to the University and introducing me to the world of phylogenetics; Dr. Warren Johnson for teaching me all about cat ecology and Dr Diana Lipscomb, for ensuring that I could see this endeavor through till the end. I would also like to thank Carrie McCracken, Nicole Crumpler, Clare Holleley, Carlos Driscoll, Victor David, Joan Pontius, Sher Hendrickson and everyone else in the Laboratory of Genomic Diversity and the NCI “Core” Lab at the National Cancer Institute who gave invaluable scientific support, provided world class resources and stirred insightful discussions. I also extend a special thanks to Dr. Stephen O’Brien for including me in his extended family of conservation geneticists. I would not have been able to complete my doctorate without the support of my friends and family especially: my father, Edward Walters, a pioneer stay-at-home Dad who taught me to read when I was two-years old; my mother, Wanda Walters, who introduced me to science with a microscope at the age of five and my brother, Ryan, who continues to defy the odds. Lastly I thank my husband, Matthew Conte, for his never-ending patience). iii Abstract of Dissertation Evolutionary and Functional Impacts of Short Interspersed Elements (SINEs) Revealed via Genomic Assessment of Felid CanSINEs Short interspersed nuclear elements (SINEs) are a type of class 1 transposable element that comprise over 10% of mammalian genomes, and play a vital role in genome structure and gene function. SINEs have also been promoted as valuable evolutionary markers at the population, species, genus and familial strata. While SINEs are prolific throughout Mammalia, historically SINE-based inquiry has primarily occurred amongst primates, rodents and cetaceans. Recent developments in genomic resources enable high-throughput investigations of SINEs within a variety of mammalian lineages. Publication of domestic dog, ( Canis familiaris ), and domestic cat ( Felis catus ) whole genome sequences in 2005 and 2007 respectively provide references sequences for comparative investigations of the Carnivora order specific SINE family, CanSINEs. This dissertation explores the evolutionary implications CanSINES within the Felidae (cat) family, a charismatic carnivoran clade of 38 species that includes biomedical model organisms, companion animals, and ecologically imperiled species. Capitalizing on the relative conservation of chromosome arrangements among Felidae species, comparative genomics methods were used to find CanSINE insertions that were initially located in domestic and exotic species across the entire Felidae family and in other Feliform suborder representatives. Comparative analyses of CanSINEs elucidate many aspects of SINE biology including: characterization of two Feliform specific SINE subfamilies, the phylogenetic iv consistency of CanSINE insertion loci, the evolutionary distribution of loci in divergent species following rapid speciation, a non-random retrotransposition process wherein new inserts occur at specified DNA motifs, and the utility of both presence/absence data and sequence data derived from SINE inserts for de novo phylogenetic reconstruction. The methods employed in this dissertation allow prior assumptions regarding the functional and evolutionary activity of mammalian SINEs to be evaluated empirically, ultimately providing a framework for the study of transposable elements in all mammals. v Table of Contents Acknowledgments............................................................................................................. iii Abstract of Dissertation..................................................................................................... iv List of Figures...................................................................................................................vii List of Tables......................................................................................................................ix General Introduction.............................................................................................................. ..x Chapter 1: Carnivores specific SINEs (Can-SINEs): Distribution, Evolution and Genomic Impact (written for publication in Journal of Heredity).......................................…...............1 Chapter 2: Comparative SINE analyses reveal feliform specific CanSINE subfamilies and the complexities of Felidae evolution (written for publication in Genome Research)……28 Chapter 3: Phylogeny and Rapid Speciation of the Felidae Illuminated by CanSINE Insertion Analysis (written for publication in Molecular Phylogenetics and Evolution).............................................................................................................................64 Chapter 4: Computational Comparative Analyses of CanSINEs in Felidae (written for publication in BMC Biology)………… .............................................................................110 General Conclusions..........................................................................................................154 vi List of Figures Figure 1.1…………………………………………………………………………………16 Figure 1.2…………………………………………………………………………………18 Figure 2.1…………………………………………………………………………… ……44 Figure 2.2…………….…………………………………………………………………...46 Figure 2.3…………………………………………………………………………………48 Figure 2.4…………………………………………………………………………………49 Figure 2.5…………………………………………………………………………………50 Figure 2.s1……………………………………..………………………………………….62 Figure 2.s2……………………………………..………………………………………….62 Figure 3.1…………………………………………………………………………… ……84 Figure 3.2…………………………………………………………………………… ……86 Figure 3.3…………………………………………………………………………… ……88 Figure 3.4…………………………………………………………………………… ……91 Figure 3.5…………………………………………………………………………… ……93 Figure 3.6…………………………………………………………………………… ……95 Figure 3.s1……………………………………………………………………………….104 Figure 3.s2……………………………………………………………………………….105 Figure 3.s3……………………………………………………………………………….107 Figure 4.1………………………………………………………………………………..126 Figure 4.2………………………………………………………………………………..127 Figure 4.3………………………………………………………………………………..128 vii Figure 4.4………………………………………………………………………………..129 Figure 4.5………………………………………………………………………………..131 Figure 4.6………………………………………………………………………………..133 Figure 4.s1………………………………………………………………………………142 Figure 4.s2………………………………………………………………………………147 Figure 4.s3………………………………………………………………………………148 Figure 4.s4………………………………………………………………………………149 Figure 4.s5………………………………………………………………………………150 Figure 4.s6………………………………………………………………………………151 Figure 4.s7………………………………………………………………………………152 Figure 4.s8………………………………………………………………………………153 viii List of Tables Table 1.1………………………………………………………………………………….14 Table 2.1………………………………………………………………………………….43 Table 2.s1…………………………………………………………………………………59 Table 2.s2…………………………………………………………………………………60 Table 2.s3…………………………………………………………………………………61 Table 3.1………………………………………………………………………………….81 Table 3.s1………………………………………………………………………….. …...102 Table 3.s2………………………………………………………………………………..103 Table 4.1…………………………………………………………………………………125 Table 4.2…………………………………………………………………………………125 Table 4.s1………………………………………………………………………………..141 ix GENERAL INTRODUCTION Central to our understanding of mammalian diversity is an assessment of how genome composition and structure has evolved throughout time. Recent publication of multiple genome-sequencing projects allows ascertainment of the abundant repetitive DNA sequences known as retrotransposable elements, which can comprise up to 42% of the mammalian genome. Long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs) are classes of retrotransposable elements that play significant