THE ROLE OF LAMIN ASSOCIATED DOMAINS IN GLOBAL CHROMATIN ORGANIZATION AND NUCLEAR ARCHITECTURE By Teresa Romeo Luperchio A dissertation submitted to The Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland March 2016 © 2016 Teresa Romeo Luperchio All Rights Reserved ABSTRACT Nuclear structure and scaffolding have been implicated in expression and regulation of the genome (Elcock and Bridger 2010; Fedorova and Zink 2008; Ferrai et al. 2010; Li and Reinberg 2011; Austin and Bellini 2010). Discrete domains of chromatin exist within the nuclear volume, and are suggested to be organized by patterns of gene activity (Zhao, Bodnar, and Spector 2009). The nuclear periphery, which consists of the inner nuclear membrane and associated proteins, forms a sub- nuclear compartment that is mostly associated with transcriptionally repressed chromatin and low gene expression (Guelen et al. 2008). Previous studies from our lab and others have shown that repositioning genes to the nuclear periphery is sufficient to induce transcriptional repression (K L Reddy et al. 2008; Finlan et al. 2008). In addition, a number of studies have provided evidence that many tissue types, including muscle, brain and blood, use the nuclear periphery as a compartment during development to regulate expression of lineage specific genes (Meister et al. 2010; Szczerbal, Foster, and Bridger 2009; Yao et al. 2011; Kosak et al. 2002; Peric-Hupkes et al. 2010). These large regions of chromatin that come in molecular contact with the nuclear periphery are called Lamin Associated Domains (LADs). The studies described in this dissertation have furthered our understanding of maintenance and establishment of LADs as well as the relationship of LADs with the epigenome and other factors that influence three-dimensional chromatin structure. I provide evidence that LAD patterns from DNA adenine methyltransferase identification (DamID)-derived molecular contact maps are reflective of higher order chromatin structure in both ensemble population measure ii and single cells. Importantly, this work provides the first in situ visualization of chromosome-wide molecular data in a single cell. These data, showing LAD and nonLAD organization, indicate that there is a speicifc and reproducible organization of sub-chromosomal domains. In addition, this work has furthered our understanding of the influence of chromatin state on both LAD and overall chromosome organization—demonstrating that higher-order chromatin structure and epigenetic signatures are closely linked. This work has contributed to the finding that LAD formation can be sequence driven, which was uncovered by examining variable LADs (vLADs) where LAD patterning differs between cell types. Also, examination of LADs across multiple cell types has uncovered genomic characteristics that can define LADs and may have a functional role in the process of genome organization iii DISSERTATION REFEREES Graduate Advisor: Karen L. Reddy, Ph.D., Assistant Professor, Department of Biological Chemistry and Center for Epigenetics, Johns Hopkins University School of Medicine Dissertation Reader: Sean D. Taverna, Ph.D., Associate Professor, Department of Pharmacology and Molecular Sciences and Center for Epigenetics, Johns Hopkins University School of Medicine THESIS COMMITTEE Chair: Jef Boeke, Ph.D., Professor, Department of Biochemistry and Molecular Pharmacology, Institute for Systems Genetics, New York University School of Medicine Policy Chair: Carolyn Machamer, Ph.D., Professor, Department of Cell Biology, Johns Hopkins University School of Medicine Sean D. Taverna, Ph.D., Associate Professor, Department of Pharmacology and Molecular Sciences and Center for Epigenetics, Johns Hopkins University School of Medicine Xin Chen, Ph.D., Associate Professor, Department of Biology, Johns Hopkins University Karen L. Reddy, Ph.D., Assistant Professor, Department of Biological Chemistry and Center for Epigenetics, Johns Hopkins University School of Medicine iv ACKNOWLEDGEMENTS I would like to express my gratitude to Dr. Karen Reddy for accepting me into her lab and granting me her guidance, support, and patience throughout my doctoral work. Karen has provided me a great opportunity to train as a scientist on a project that is both meaningful and exciting, while also taking a sincere interest in fostering my professional and personal strengths. I am grateful to have worked with Karen throughout my graduate degree and know I will always be able to reflect on my time with her when searching for direction in years to come. Thank you to all the faculty who have provided insight and encouragement during my Ph.D. work. Thank you Drs. Sean Taverna, Jef Boeke, Xin Chen for serving on my thesis committee and your support and suggestions. A special thank you to Sean for his humor and constant support, and for working with me to complete this dissertation. Thank you Drs. Barbara Sollner-Webb and Kathy Wilson, who along with Karen have led by example and shared their opinions on how to succeed as a woman in science. Thank you to Dr. Carolyn Machamer for helping us navigate the path to graduation, especially in the final stages of my degree. Also, thank you to Drs. Trina Schroer, Andy Hoyt, Joel Schildbach, who were key players in my decision to pursue a doctoral research degree I also wish to recognize our generous collaborators Drs. Roger Reeves, Andy Feinberg, and especially Agilent Technologies and Dr. Alice Yamada who provided the novel LAD and nonLAD sub-chromosome paints. Their material contributions and guidance were critical to the success of this project. I must thank my colleagues in the lab and the Epigenetics Center for their skills, advice, and friendship. Specifically I would like to thank all the members of v the Reddy lab Drs. Xianrong Wong, Marie-Cecile Gaillard and Jennifer Harr, Mohammad Heydarian and Jevon Cutler, for their scientific collaboration during my graduate work. I would especially like to thank Jevon Cutler and Dr. Xianrong Wong for their friendship in the lab, which has been crucial for my personal and professional well-being. I especially want to thank my colleagues from the Epigenetics Center who have doubled as very close friends, Bridget O’Brien, Carolina Montano, and Drs Romeo Papazyan, Annie Cieniewicz, and Tonya Gilbert for all the fun, love, and support without which I would not have made it through. Thank you to the Federation of American Societies for Experimental Biology and the Office of Public Affairs where I spent a three month internship working on science policy and Congressional affairs, for the opportunity to expand my scientific world-view beyond the bench, and develop my professional skills. The experience was invaluable and I hope to continue to advocate for science throughout my career. Specifically, I would like to thank Dr. Howard Garrison and Dr. Anne Deschamps for the opportunity to work in the office and their continued guidance and friendship. I want to thank my personal support team, my friends and family, for encouraging me to never give up. Thank you Rachel Cylus and Dr. Kate Szarama for standing by me regardless of the time or distance between us. Thank you to all my family members who cheered me on and always met me with love and kindness. I would especially like to thank my parents Angela and Domenic Romeo, and my siblings Francesca Romeo and Frank Romeo for their unwavering support. I would like to thank my grandmothers Francesca Dattola and Teresa Romeo, who I miss very much, for their love, and for knowing they would be very proud of me. vi Lastly, I give the biggest thank you to my amazing husband Dan and sweet daughter Clara. You both are my everything and I would be nothing without you. Thank you for loving me and never letting go. vii Table of Contents Abstract ......................................................................................................................... ii Dissertation referees .................................................................................................... iv Thesis Committee ........................................................................................................ iv Acknowledgements ........................................................................................................ v List of Figures .............................................................................................................. xi Chapter 1: Introduction ................................................................................................ 1 1.1 Epigenetics and Chromatin ................................................................................. 1 1.2 Nuclear Structure and the Nuclear Periphery ................................................... 4 1.3 LADs in regulation .............................................................................................. 7 Chapter 2: LAD definition ...........................................................................................11 2.1 Introduction ........................................................................................................11 2.2 LADetector Algorithm ........................................................................................13 2.3 Methods ..............................................................................................................19 2.3.1 Preparation of DamID library .....................................................................19 2.3.2 Hybridization