University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2017 Developing Models To Investigate Mechanisms Of Genomic Imprinting Stellar Kim Hur University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Developmental Biology Commons Recommended Citation Hur, Stellar Kim, "Developing Models To Investigate Mechanisms Of Genomic Imprinting" (2017). Publicly Accessible Penn Dissertations. 2349. https://repository.upenn.edu/edissertations/2349 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/2349 For more information, please contact [email protected]. Developing Models To Investigate Mechanisms Of Genomic Imprinting Abstract Genomic imprinting is a conserved, essential process in mammalian development that regulates the expression of a small number of genes in a monoallelic, parent-or-origin-specific manner. Misregulation of imprinted genes is associated with imprinting disorders including Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS) that exhibit abnormal growth phenotypes. These disorders are associated with aberrant regulation of the imprinted loci in human 11p15 including the H19/IGF2 locus. Finding various alterations of 11p15 associated with BWS and SRS inspires investigation of imprinting mechanisms in human, which could provide insights into therapeutics. Mouse models have been fundamental to the study of mechanisms of imprinting, serving as a proxy for the orthologous human locus. However, elements that regulate genomic imprinting, the imprinting control regions (ICRs), often diverge across species. Thus, it is essential to first understand whether the diverged ICR has a species- specific oler in regulating imprinting. In Chapter 2, we generated a mouse in which the human ICR sequence replaces the orthologous mouse ICR at the H19/Igf2 locus. We show that the imprinting mechanism has partially diverged between mouse and human, depending on the parental origin of the human ICR sequence in mouse. Additionally, we find that this mouse model is optimal for studying specific alterations associated with BWS and SRS. The partially diverging imprinting mechanism between mouse and human suggests that entirely human models are compelling alternatives. In Chapter 3, we demonstrate the derivation of induced pluripotent stem cells (iPSCs) from BWS patient fibroblasts. We find that the iPSCs exhibit proper epigenetic and transcriptional signatures of BWS. Although we find that certain aspect of epigenetic perturbation is inevitable in our iPSCs, the consequence of this perturbation remains unknown. Therefore, we propose that the iPSCs can be differentiated into clinically-relevant cell types to elucidate molecular mechanisms leading to BWS. Overall, the work in this dissertation underscores the versatile and complementary use of different model systems in investigating imprinting mechanisms. In addition to serving as platforms to model imprinting disorders, these models provide insights into the evolutionary perspective of imprinting as well as the significance of ariousv epigenetic mechanisms that regulate imprinting. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Marisa S. Bartolomei Keywords Beckwith-Wiedemann syndrome, Genomic imprinting, H19, IGF2, Silver-Russell syndrome Subject Categories Developmental Biology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/2349 DEVELOPING MODELS TO INVESTIGATE MECHANISMS OF GENOMIC IMPRINTING Stellar K. Hur A DISSERTATION in Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2017 Supervisor of Dissertation Dr. Marisa S. Bartolomei, Professor of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania Graduate Group Chairperson Dr. Daniel S. Kessler, Associate Professor of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania Dissertation Committee Dr. Montserrat C. Anguera, Assistant Professor of Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania Dr. Klaus H. Kaestner, Professor of Genetics, Perelman School of Medicine, University of Pennsylvania Dr. Paul J. Gadue, Associate Professor of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia Dr. Zhaolan Zhou, Associate Professor of Genetics, Perelman School of Medicine, University of Pennsylvania This dissertation is dedicated to the ones who have inspired me to: accept, understand, and appreciate. ii ACKNOWLEDGMENT I would like to thank my mentor Dr. Marisa Bartolomei for providing intellectual support and having faith in me in carrying out research. She has always respected and cared about my research and “never led me astray,” as she once said. This has also applied in personal interactions—she listens, understands, and gives best advices. I would also like to thank the Bartolomei lab members. First and foremost, thank you to Dr. Joanne Thorvaldsen and Christopher Krapp, my amazing baymates and best mentors in many aspects. When I was secretly happy or sad for various reasons, they have always been there for me. I thank the former and current graduate students and research associates in the lab, Dr. Jamie Weaver, Dr. Lara Abramowitz, Dr. Rob Plasschaert, Martha Stefaniak, Frances Xin, Jen Myers SanMiguel, Aimee Juan, Suhee Chang, Blake Caldwell, and Tre Artis. They have set the highest standards for colleagues—they are the most warm-hearted, brilliant, and dedicated scientists. Lastly, I thank the former and current senior scientists in the lab, Dr. Folami Ideraabdullah, Dr. Sebastien Vigneau, Dr. Martha Susiarjo, Dr. Eric de Waal, Dr. Jennifer Kalish, and Dr. Lisa Vrooman. I truly appreciate all the science and soft skills I learned from them, which became especially helpful as I was preparing for the next stage of my career. I particularly thank the devoted contributors to the work in this dissertation—Dr. Joanne Thorvaldsen, Dr. Folami Ideraabdullah, Dr. Jennifer Kalish, Suhee Chang, and the former and current undergraduates Carolyn Lye and Alice Yu. I am grateful for all the fruitful collaborative experiences. I would like to thank Dr. Andrea Riccio, Dr. Lacey Luense, Dr. Catherine May, Andrea Freschi, and Angela Hines, for their help in successfully developing and completing the projects that I have undertaken. I am also looking forward to the new collaboration on the iPSC project with the CHOP human ES/iPS core led by Dr. Paul Gadue. iii I would like to thank my exemplary thesis committee, Dr. Montserrat Anguera, Dr. Klaus Kaestner, Dr. Zhaolan Zhou, and Dr. Paul Gadue. Every committee meeting was filled with cerebral discussion, and it has always guided me in the right direction. I specially thank the chair of my committee, Dr. Montserrat Anguera, for her hands-on help in deriving and characterizing iPSCs. She always greets me with a big smile and that is enough to make me feel supported. I would like to thank my friends and family. Being away from family for so long would not have worked out without my loving friends in the US. I am also lucky to have many friends living outside the US—regardless of the time zone they are in or being able to see each other only occasionally, they find a time to talk to me and are the best supporters of mine. I thank my grandparents, who inspired me to pursue the field of science and always care about my well-being. Lastly, I thank my mother, aunt, and sister, who are my best friends. They understand, trust, and love me for who I am, which has become a cornerstone in decisions I have made at every stage of my life (and I know it will continuously be this way). I feel very fortunate to be surrounded by so many incredible people. iv ABSTRACT DEVELOPING MODELS TO INVESTIGATE MECHANISMS OF GENOMIC IMPRINTING Stellar K. Hur Dr. Marisa S. Bartolomei Genomic imprinting is a conserved, essential process in mammalian development that regulates the expression of a small number of genes in a monoallelic, parent-or-origin-specific manner. Misregulation of imprinted genes is associated with imprinting disorders including Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS) that exhibit abnormal growth phenotypes. These disorders are associated with aberrant regulation of the imprinted loci in human 11p15 including the H19/IGF2 locus. Finding various alterations of 11p15 associated with BWS and SRS inspires investigation of imprinting mechanisms in human, which could provide insights into therapeutics. Mouse models have been fundamental to the study of mechanisms of imprinting, serving as a proxy for the orthologous human locus. However, elements that regulate genomic imprinting, the imprinting control regions (ICRs), often diverge across species. Thus, it is essential to first understand whether the diverged ICR has a species- specific role in regulating imprinting. In Chapter 2, we generated a mouse in which the human ICR sequence replaces the orthologous mouse ICR at the H19/Igf2 locus. We show that the imprinting mechanism has partially diverged between mouse and human, depending on the parental origin of the human ICR sequence in mouse. Additionally, we find that this mouse model is optimal for studying specific alterations associated with BWS and SRS. The partially