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The Three-Dimensional Structure of the Cystic Fibrosis Locus: a Dissertation

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The Three-Dimensional Structure of the Cystic Fibrosis Locus: a Dissertation University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2014-11-18 The Three-Dimensional Structure of the Cystic Fibrosis Locus: A Dissertation Emily M. Smith University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Genetic Processes Commons, Genomics Commons, and the Structural Biology Commons Repository Citation Smith EM. (2014). The Three-Dimensional Structure of the Cystic Fibrosis Locus: A Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/M2SK51. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/744 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. THE THREE-DIMENSIONAL STRUCTURE OF THE CYSTIC FIBROSIS LOCUS A Dissertation Presented By EMILY MALINDA SMITH Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY NOVEMBER 18, 2014 BIOMEDICAL SCIENCES THE THREE-DIMENSIONAL STRUCTURE OF THE CYSTIC FIBROSIS LOCUS Dissertation Presented By Emily Malinda Smith The signatures of the ee signify completion and approval as t the Dissertation The signature of the Chair of the Committee signifies that the written dissertation meets the requirements of the Dissertation Committee Sean omm1ttee The signature of the Dean of the Graduate School of Biomedical Sciences signifies that the student has met al aduation requirements of the school. Carruthers, Ph.D., School of Biomedical Sciences Interdisciplinary Graduate Program November 18, 2014 iii ACKNOWLEDGEMENTS I would like to acknowledge a number of people who helped me personally and professionally while I completed the work for this degree. Job Dekker is a fantastic PI and mentor. He has a deep knowledge of the field and continues to remain on the cutting edge of experimental techniques. The projects that come from his lab make waves throughout the scientific community. I am grateful for his assistance throughout this process. Without Job’s vision my project would not be what it is today. I am a better scientist because of what he has taught me. The members of the Dekker Lab, past and present, have also contributed to my growth as a scientist and as a person. I would like to thank Nele Gheldof, who began this CFTR project while she was in the lab as a postdoc. Thanks to Adrianna Geldart (formerly Miele) for welcoming me into the lab and teaching me many things. Thanks to Ninke VanBerkum for teaching me 3C, and to Amartya Sanyal and Ye Zhan who helped me with 5C. I also thank Bryan Lajoie and Gaurav Jain for all their help with the analysis side of this project – it would not have been done without you and your programming skills. Thanks also to Johan Gibcus, Noam Kaplan, Natasha Naumova, Rachel Patton McCord, Jenn Phillips- Cremins and Jon Belton for your great support as colleagues. You all have made the lab atmosphere a great place to work and through our shared experiences we have all become friends. Who could ask for more? iv Additional thanks go out to my extended family at Wesley United Methodist Church, who welcomed Phil and me with open arms. You have prayed for me, celebrated with me and supported me throughout this journey. There are too many names to list. You are all special and I thank God we were able to walk together for a time. To Pastors Vicki Woods, Lisa Bruget-Cass and Shandi Mawokomatanda, who have helped guide me in my spiritual walk these past 7 years, I thank you. Special thanks is needed for all the youth that have welcomed Phil and me as their youth group leaders and mentors along the way – you have made every Wednesday night so exceptional. I continue to learn from you all: Munah, Isaac, Stacy, Elaine, Natasha, Josephine, Abby, Danielle, Will, Eleanor, Rachel, Rebecca, Gabby, Joseph, Kendra, Kristina, Akosua, Olivia and Amelia – you are all such special people. I’ve loved the trips we’ve taken, the events that we’ve hosted, the mission work we’ve done, the long rides in cars and busses, and the advice about African hair. We are God’s people – we are his hands and feet. Thank you Russell Ulbrich and Kristine Reed for your friendliness, your hospitality, your random dinner invitations, your lovely house and patio, your intriguing conversations and your companionship. It’s been so great. Thank you to my in-laws and my sister in law, Carol, John and Megan Smith. Carol, you’ve been great to talk to as I go through this process, and you’ve given me good solid advice. John, thanks for bringing the family to visit – it’s always a pleasure. I hope we will soon have a house that you can build all v sorts of fun trinkets for. Meg, I’m glad we’ve gotten to know each other in the brief visits we’ve had, and I hope there are many more in the future. I wish you could stamp my thesis for me. Thanks to my mom and dad, Becky and Tom Wisnieski, for supporting me and paying for me to go to Purdue, even though it was expensive and far away. I know when I decided to come to Massachusetts for graduate school you weren’t sure it was the best decision, but you continued to support me. You understood when I couldn’t always come home for as long as we both wanted, but we had fun anyway, visiting on Cape Cod and in Vermont too. We made great memories together. Thanks again for loving me like you do. Mom, I love your determination to understand my work and explain it to other people. You taught me to love learning, and more importantly you taught me to love teaching others. Dad, you taught me the importance of good hard work, which has been so important in this process. Mark, you’re a great brother. I wish we lived closer so we could hang out as adults, which I hope we’d enjoy just as much as playing together when we were younger. Mary Rosso, you are a wonderful person and a fantastic grandma. To Bruce Rosso and Bill and Sylvia Wisnieski, you will always be in my heart. Thank you so much Meredith Edwards, my true friend and confidant. It’s been really hard living so far away from you. I’ve cherished our friendship despite the distance. You’ve got my back forever. I love you. Phil Smith, you are the best husband I could ever have. You have been so patient with me as I struggled through this PhD. You have always given me vi space when I need it. You have put up with my frustrations, my tendency to not cook, my need to escape into literature, and my random desires for creativity. You’ve never complained. I am delighted we have come through to the other side, and I can’t wait to see where life takes us next. No matter what we do, we’re going to do it together, and that thought comforts me more than I can say. I love you forever. You are my perfect companion. vii ABSTRACT The three dimensional structure of the human genome is known to play a critical role in gene function and expression. I used chromosome conformation capture (3C) and 3C-carbon copy (5C) techniques to investigate the three-dimensional structure of the cystic fibrosis transmembrane conductance regulator (CFTR) locus. This is an important disease gene that, when mutated, causes cystic fibrosis. 3C experiments identified four distinct looping elements that contact the CFTR gene promoter only in CFTR-expressing cells. Using 5C, I expanded the region of study to a 2.8 Mb region surrounding the CFTR gene. The 5C study shows 7 clear topologically associating domains (TADs) present at the locus, identical in all five cell lines tested, regardless of gene expression status. CFTR and all its known regulatory elements are contained within one TAD, suggesting TADs play a role in constraining promoters to a local search space. The four looping elements identified in the 3C experiment and confirmed in the 5C experiment were then tested for enhancer activity using a luciferase assay, which showed that elements III and IV could act as enhancers. These elements were tested against a library of human transcription factors in a yeast one-hybrid assay to identify potential binding proteins. Element III gave two strong candidates, TCF4 and LEF1. A literature search supported these transcription factors as playing a role in CFTR gene expression. Overall, this work represents a model locus that can be used to test important questions regarding the role of three dimensional looping on gene expression. viii TABLE OF CONTENTS Signature Page Page ii Acknowledgements Page iii Abstract Page vii Table of Contents Page viii List of Tables Page x List of Figures Page xi List of Symbols, Abbreviations or Nomenclature Page xiii Preface Page xviii Chapter I Introduction Page 1 Chapter II Chromosome Conformation Capture Page 21 Methods Abstract Page 23 Introduction Page 24 Technical Overview of the Methods Page 33 Experimental Design Considerations Page 40 3C Experimental Protocol Page 59 5C Experimental Protocol Page 71 Analysis of 3C Data Page 84 Analysis of 5C Data Page 91 Troubleshooting Page 94 Chapter III Identifying Functional Long-Range Interactions of the Cystic Fibrosis Gene Page 98 ix Abstract Page 100 Introduction Page 101 Materials and Methods Page 106 Results Page 114 Discussion Page 144 Chapter IV Three-dimensional Study of the Cystic Fibrosis Locus Reveals Cell-Type Specific Chromatin Looping
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