Genetic and Epigenetic Interactions in in Vivo and in Vitro Reprogramming Margaret Ashley Young Washington University in St
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Washington University in St. Louis Washington University Open Scholarship All Theses and Dissertations (ETDs) Spring 3-5-2013 Genetic and Epigenetic Interactions in in vivo and in vitro Reprogramming Margaret Ashley Young Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/etd Part of the Immunology and Infectious Disease Commons Recommended Citation Young, Margaret Ashley, "Genetic and Epigenetic Interactions in in vivo and in vitro Reprogramming" (2013). All Theses and Dissertations (ETDs). 1060. https://openscholarship.wustl.edu/etd/1060 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 Immunology Dissertation Examination Committee: Timothy Ley, Chair Kyunghee Choi Tom Ellenberger Mark Sands Barry Sleckman Matthew Walter Genetic and Epigenetic Interactions in in vivo and in vitro Reprogramming by Margaret Ashley Young 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 May 2013 St. Louis, Missouri TABLE OF CONTENTS Page List of Figures iii List of Tables vi Acknowledgements vii Dedication ix Abstract of the Dissertation x Chapter 1 Introduction 1 References 24 Chapter 2 Canoncial and non-canonical HOX expression patterns in 35 acute myeloid leukemia References 59 Chapter 3 Genetic heterogeneity of iPS clones revealed by whole 85 genome sequencing References 113 Chapter 4 Future Directions 139 References 151 Resume 155 ii LIST OF FIGURES Chapter 2: Canoncial and non-canonical HOX expression patterns in acute myeloid leukemia Figure Legends 64 Figure 2-1. Heat map of expression data for MEIS1 and the HOX cluster genes 72 from 45 de novo AML patient samples for which there is whole genome sequencing data. Figure 2-2. Raw data from Affymetrix U133 Plus 2 array for 190 patients 73 de novo AML patient samples. Figure 2-3. Correlation of patient characteristics and HOX Expression. 74 Figure 2-4. A. Heat map of expression data for MEIS1 and the HOX cluster 75 genes from 190 de novo AML patient samples by cytogenetics. Figure 2-5. A. Heat map of expression data for MEIS1 and the HOX cluster 76 genes from 190 de novo AML patient samples by recurrent mutations. Figure 2-6. LC-MS data of total methylcytosine content for 70 de novo AML 77 patients. Figure 2-7. Methylation array data of the HOXA locus from 190 de novo AML 78 patient samples by HOXA9 expression. Figure 2-8. Methylation array data of the HOXA locus from 190 de novo AML 79 patient samples by HOXB3 expression. Figure 2-9. Methylation array data of the HOXA and HOXB loci from 190 80 de novo AML patient samples by recurrent mutations. iii Figure 2-10. HOX expression levels in healthy hematopoietic cells. 81 Figure 2-11. Comparison of HOX expression pattern in AML vs. healthy 82 hematopoietic cells. Figure 2-12. HOX expression pattern in mouse hematopoietic cells. 83 Figure 2-13. HOX expression pattern in mouse acute promyelocytic leukemia 84 samples. Chapter 3: Genetic heterogeneity of murine induced pluripotent stem (iPS) clones revealed by whole genome sequencing Figure Legends 116 Figure 3-1. Unsupervised cluster analysis of Mouse Exon 1.0 ST array data. 125 Figure 3-2. Bisulfite sequencing of the Oct4 and Nanog promoters. 126 Figure 3-3. Cystic teratoma histology. 127 Figure 3-4. OSK lentivirus insertion sites prove iPS clonality 128 Figure 3-5. Homozygous SNVs in fibroblast lines compared to B6 reference 129 genome. Figure 3-6. SNVs in iPS clones compared to parental fibroblast lines. 130 Figure 3-7. Circos plots illustrating genomic distribution of SNVs in each iPS 131 clone. Figure 3-8. Variant allele frequency plots of iPS clones. 132 Figure 3-9. Common and private mutations in the 4 iPS clones from 133 experiment 3. Figure 3-10. Analysis for common SNVs in an additional MPSVII iPS clones 134 iv from an independent reprogramming event. Figure 3-11. Common structural variant in the 4 iPS clones from experiment 3. 135 Figure 3-12: Detection of common variants in rare proportion of parental MEF 136 population. Figure 13. Comparison of common vs private variant allele frequencies 137 Figure 14. Model of selection in iPS reprogramming. 138 v LIST OF TABLES Chapter 2: Canoncial and non-canonical HOX expression patterns in acute myeloid leukemia Table 2-1: Clinical characteristics of 190 de novo AML patients 67 Table 2-2: Somatic mutations within HOX clusters identified by whole genome 69 sequencing of 45 de novo AML patients Table 2-3: Expression correlation values for pairs of HOX genes 70 Table 2-4. Orthagonal validation of AffyMetrix U133 Plus 2 Array data with 71 custom NanoString codeset Chapter 3: Genetic heterogeneity of murine induced pluripotent stem (iPS) clones revealed by whole genome sequencing Table 3-1. Generation and characterization of iPS clones 118 Table 3-2 Whole genome sequencing coverage and lentivirus insertion sites 118 of all 10 iPS clones Table 3-3 Validated coding region SNVs 120 Table 3-4 Common indels in all 4 Experiment 3 iPS clones 122 Table 3-5 Pools tested for presence of Apaf1 and Sbno2 variants 123 Table 3-6 Pathways identified by MUSIC suite as being enriched for in genes 124 with SNVs vi ACKNOWLEDGEMENTS There are many people who have helped me along the way to the completion of my thesis. Over the past four years, I have had the privilege to work with an incredible group of people in the Ley lab. First, I must thank my mentor, Tim Ley. There have been a few bumps along the way, but Tim has always had a positive attitude and an ability to turn a negative result into an unexpected finding. I have learned an incredible amount of science from Tim, but more importantly I have learned what it means to be a great mentor. To appreciate Tim’s passion for training the next generation of scientists, one must just walk down the hallway of the 6th floor of the Southwest Tower to see his successful trainees. I look forward to following in their footsteps with Tim’s continued support along the way. My colleagues in the Ley lab, both past and present, have been an integral part of my graduate experience. In particular, I would like to thank Dan George and Tami Lamprecht for their help completing this work; it would not have been possible without them. In addition, I would like to thank Sheng Cai, Cynthia Li, David Germain, Lukas Wartman, John Welch, and Jeff Klco for their advice, support and most of all friendship. I also want to thank my extended lab family- the entire 6th floor as well as the ES core and the HSCS core. Working here has been a truly unique experience of collaboration and I thank all of them for their help over the years. I would also like to thank the members of my thesis committee- Tom Ellenberger, Mark Sands, Matt Walter, Barry Sleckman and KC Choi. My initial thesis proposal is barely recognizable in the work presented here and they have been a great source of advice and vii support along its evolution. I especially want to thank Mark Sands for lending his expertise in the MPSVII model, along with Marie Nunez from his lab. Their reagents and advice have been indispensible. I thank the MSTP for administrative and financial support of my work along with the Division of Hematology training grant. Most importantly, I would like to thank the patients who decided to make the most of a horrible situation and participate in the Washington University tumor-banking program. Although I will never know who they are, this work would not be possible without their contributions. My graduate experience would not have been complete without the friends I have made along the way. I am extremely grateful to have a great group of friends that have been on the MSTP journey with me since I first came to St. Louis. Toni, Matt, Brian and Mark, you have all been a source of motivation and encouragement for me over the past 6 years, which I know will continue as we move along in our careers. In addition, St. Louis would not have been the same without Jess, Dena, LeRoy, Gabe, Diego and all of the amazing people I have met through medical and graduate school. I especially thank Steve for keeping me sane through the writing process. I am lucky to be a part of a large family and they have always been my biggest supporters, especially my siblings, Jessie, Becky and Danny. Most importantly, I would like to acknowledge my mom, Lori, for the sacrifices she has made to get me here. viii DEDICATION As a tribute to my own genetics, I thank my grandpa, Walter Queen, for my work ethic, thirst for knowledge and somewhat obsessive-compulsive traits, which have made me the scientist I am today- I dedicate this thesis to his memory. ix ABSTRACT OF THE DISSERTATION Genetic and Epigenetic Interactions in in vivo and in vitro Reprogramming by Margaret Ashley Young Doctor of Philosophy in Biology and Biomedical Sciences Immunology Washington University in St. Louis, 2013 Professor Timothy Ley, Chairperson In cancer pathogenesis and induced pluripotent stem (iPS) cell production, an essential step for reprogramming is acquisition of self-renewal. In hematopoietic cells, HOX genes are partially responsible for self-renewal, and HOX gene dysregulation commonly occurs in acute myeloid leukemia (AML).