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Intrinsic Specificity of DNA Binding and Function of Class II Bhlh

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Intrinsic Specificity of DNA Binding and Function of Class II Bhlh INTRINSIC SPECIFICITY OF BINDING AND REGULATORY FUNCTION OF CLASS II BHLH TRANSCRIPTION FACTORS APPROVED BY SUPERVISORY COMMITTEE Jane E. Johnson Ph.D. Helmut Kramer Ph.D. Genevieve Konopka Ph.D. Raymond MacDonald Ph.D. INTRINSIC SPECIFICITY OF BINDING AND REGULATORY FUNCTION OF CLASS II BHLH TRANSCRIPTION FACTORS by BRADFORD HARRIS CASEY DISSERTATION Presented to the Faculty of the Graduate School of Biomedical Sciences The University of Texas Southwestern Medical Center at Dallas In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY The University of Texas Southwestern Medical Center at Dallas Dallas, Texas December, 2016 DEDICATION This work is dedicated to my family, who have taught me pursue truth in all forms. To my grandparents for inspiring my curiosity, my parents for teaching me the value of a life in the service of others, my sisters for reminding me of the importance of patience, and to Rachel, who is both “the beautiful one”, and “the smart one”, and insists that I am clever and beautiful, too. Copyright by Bradford Harris Casey, 2016 All Rights Reserved INTRINSIC SPECIFICITY OF BINDING AND REGULATORY FUNCTION OF CLASS II BHLH TRANSCRIPTION FACTORS Publication No. Bradford Harris Casey The University of Texas Southwestern Medical Center at Dallas, 2016 Jane E. Johnson, Ph.D. PREFACE Embryonic development begins with a single cell, and gives rise to the many diverse cells which comprise the complex structures of the adult animal. Distinct cell fates require precise regulation to develop and maintain their functional characteristics. Transcription factors provide a mechanism to select tissue-specific programs of gene expression from the shared genome. ASCL1, ASCL2, and MYOD are class II basic Helix-Loop-Helix (bHLH) transcription factors which play crucial roles in lineage specification in the developing embryo. In vivo, these factors bind to distinct genomic sites, and regulate distinct transcriptional programs. The mechanisms by which they select their cognate binding sites vi remain poorly defined. Here, we utilize an inducible system to express these master regulatory factors in embryonic stem cells to characterize early events in bHLH factor binding and function in a common cellular context, removed from their role as endogenous master regulators of lineage specification. Using genome-wide sequencing approaches, we demonstrate that these factors maintain distinct binding when ectopically expressed in a common context. We observe that they initiate distinct transcriptional programs, which include key regulators in lineage specification. By comparing chromatin accessibility of bHLH binding sites, we reveal a shared ability for these factors to bind nucleosome-occupied sites, and meet the criteria which define pioneer transcription factors. We further characterize epigenetic features of the empirically observed genome-wide binding sites of these factors, and compare these findings to the conventional understanding of bHLH factor function. This work represents the first comprehensive approach to direct comparison of early events in the binding and transcriptional profiles of ASCL1, ASCL2, and MYOD. ACKNOWLEDGEMENTS In preparing this work, I have reflected often on the great many kindnesses which have been visited upon me during my time at UT Southwestern. How can one begin to square the accounts of the generous efforts of so many? Regardless, this is the best opportunity to do so en masse, and I would be remiss to neglect the chance to express my appreciation. First, I wish to express my most sincere gratitude to my mentor, Dr. Jane E. Johnson. Her hard work, dedication, and collaborative spirit serve as an example for all scientists. Her kindness and generosity are without limit, and her patience, without equal. Many scientists fall prey to the illusion that ours is a field of competitors, rather than colleagues; Jane has always sought to keep friends, rather than make enemies of her peers. She has chosen to maintain her office in the center of a noisy and chaotic lab, rather than a posh and quiet executive suite, and maintains an open door for questions, feedback, approval, and support. She has earned the respect and friendship of everyone who knows her. I also gratefully acknowledge the generous guidance of my dissertation committee, which has been invaluable in the completion of this work; Dr. Helmut Kramer, who taught me to always try to test a theory, rather than try to confirm it, Dr. Raymond MacDonald, whose hand-written notes were always more valuable than my work deserved, and Dr. Genevieve Konopka, whose thoughtful and considered insight has always encouraged me to push forward, and to find the silver lining. They are all great assets to our institution, and to their respective fields of research. I am forever indebted to each for the guidance and support they have provided me over these long years. The completion of this work would not have been possible without the support of my many colleagues and friends at UT Southwestern. I have spent many late nights in the lab, and have rarely done so alone. It has been a wild ride, and I am grateful to have shared it with you. In particular, the members of the Johnson lab have selflessly shared their experience and knowledge to help see this research come to fruition, and have always provided honest feedback. Dr. Helen Lai and Dr. Tou Yia Vue have always challenged me to consider carefully my approach and results, and to find and cultivate the story that it has revealed. My fellow students Dr. Mark Borromeo and Dr. Joshua Chang always shared the wisdom of their experience, and taught me how to survive the gauntlet of the neuroscience graduate program. We oft burned the midnight oil, and our late nights and early mornings were some of the happiest and most memorable times that I spent in lab. They also taught me to play poker, which has ensured that I will never be taken to cards, as I will surely lose. Lauren Tyra, who will certainly have finished her Ph.D. by the time this is printed, has been a constant source of personal support and scientific discussion. She has worked alongside me in various volunteer efforts and leadership roles, and serves as an example of a student who has gone the extra mile to improve opportunities for those less fortunate than ourselves. Erin Kibodeaux, who has left science to follow her dream of caring for others, has been a dear friend, and brought much-needed levity to my time in the lab. Her patients will benefit greatly from her intelligence and skill, and will be comforted by her kind demeanor. I am especially indebted to Clark Rosensweig, for reasons too numerous to list here. His passion and capability for research is exceptional, his insight, invaluable, and his wit, peerless. His constant support and encouragement have been immeasurable comfort in hard times. He is indisputably the better scientist, but has steadfastly insisted that we are equal, and that I have something to contribute. The gilded words of true friends are bracing in the face of hardship, even when we know them to be too generous. I am eternally grateful for his kindness and his fraternity over the years. I also wish to thank Dr. Matthew Goldberg, who took great risk in hiring me, despite my having no significant experience in research. He entrusted me with precious resources, trained me in many techniques, and treated me as a valued member of his laboratory. I never would have imagined that I could be a successful mouse surgeon. He encouraged me to make the most of my time at UT Southwestern, and allowed me to attend lectures and conferences. He supported me when I left to pursue my education, and eagerly wrote letters of introduction and recommendation, without which I certainly would never have had this opportunity. Often neglected are the many faculty and staff which make up the University of Texas Southwestern Medical Center, and especially the College of Biomedical Sciences Graduate Program. The many people that make up the Office of the Dean spare no effort in keeping this complex operation running smoothly. I am especially grateful for the unique and exceptional opportunities to serve our students and our university, which have been some of the most satisfying experiences I have enjoyed during my tenure. I especially want to recognize the efforts of Wes Norred, who has given myself and others the opportunity to advocate for our students in many venues. He secured an audience with everyone I ever asked, listened, and supported my modest efforts to improve our fair institution. This school would not be the same without his tireless advocacy for our students. I genuinely cannot fathom why I have been allowed such liberty, and such privilege, but it has been a great honor. Outside the academe, Chad Lumley, an exceptional mathematician and physicist, has been a great friend, and our many projects, adventures, and discussions have been a welcome and satisfying distraction from my studies. Few friends will let you call them at any hour, even fewer will invite you over for dinner when you wake them up. I thank him especially for teaching me calculus, and for enduring my terrible jokes about maths. I remain certain that my definition of a polar bear is correct. Miryam Prodanovic has been a constant comfort, and despite great distance has remained my steadfast friend and confidant. Her handwritten letters have lifted my spirits when they were low, and her many drawings and photographs have provided inspiration and opportunity for reflection. To be Miryam’s friend is to drink from a hydrant of kindness, support, and creativity. I am forever in arrears for her friendship, and I will forever labor to repay her. I feel compelled to assert that I could not possibly convey my appreciation for those mentioned above, much less the many who are not mentioned herein.
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