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A High-Throughput Approach to Uncover Novel Roles of APOBEC2, a Functional Orphan of the AID/APOBEC Family

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A High-Throughput Approach to Uncover Novel Roles of APOBEC2, a Functional Orphan of the AID/APOBEC Family Rockefeller University Digital Commons @ RU Student Theses and Dissertations 2018 A High-Throughput Approach to Uncover Novel Roles of APOBEC2, a Functional Orphan of the AID/APOBEC Family Linda Molla Follow this and additional works at: https://digitalcommons.rockefeller.edu/ student_theses_and_dissertations Part of the Life Sciences Commons A HIGH-THROUGHPUT APPROACH TO UNCOVER NOVEL ROLES OF APOBEC2, A FUNCTIONAL ORPHAN OF THE AID/APOBEC FAMILY A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy by Linda Molla June 2018 © Copyright by Linda Molla 2018 A HIGH-THROUGHPUT APPROACH TO UNCOVER NOVEL ROLES OF APOBEC2, A FUNCTIONAL ORPHAN OF THE AID/APOBEC FAMILY Linda Molla, Ph.D. The Rockefeller University 2018 APOBEC2 is a member of the AID/APOBEC cytidine deaminase family of proteins. Unlike most of AID/APOBEC, however, APOBEC2’s function remains elusive. Previous research has implicated APOBEC2 in diverse organisms and cellular processes such as muscle biology (in Mus musculus), regeneration (in Danio rerio), and development (in Xenopus laevis). APOBEC2 has also been implicated in cancer. However the enzymatic activity, substrate or physiological target(s) of APOBEC2 are unknown. For this thesis, I have combined Next Generation Sequencing (NGS) techniques with state-of-the-art molecular biology to determine the physiological targets of APOBEC2. Using a cell culture muscle differentiation system, and RNA sequencing (RNA-Seq) by polyA capture, I demonstrated that unlike the AID/APOBEC family member APOBEC1, APOBEC2 is not an RNA editor. Using the same system combined with enhanced Reduced Representation Bisulfite Sequencing (eRRBS) analyses I showed that, unlike the AID/APOBEC family member AID, APOBEC2 does not act as a 5-methyl-C deaminase. Finally, using a combination of biochemical, Chromatin Immunoprecipitation Sequencing (ChiP-Seq) and polyA RNA-Seq analyses I show that APOBEC2 is a (negative) regulator of gene expression (at least in muscle cells) and binds chromatin directly to inhibit transcription of genes involved in muscle cell differentiation. While the precise mechanism behind this activity is still a matter of investigation, this role of APOBEC2 in inhibiting genes involved in cell cycle exit, might have implications for its role in in cancer. ACKNOWLEDGMENTS “It takes a village to make a scientist” I would like to thank my advisor Nina Papavasiliou for giving me the opportunity to be part of the lab. Thank you for your enthusiasm for science, encouragement, and support. Thank you, for generously spending a lot of time ‘on call’, especially during the last months of graduate school. You have taught me a lot about being persistent along the setbacks and triumphs of the scientific process. The mixture of your optimism and my overly skepticism was a good match to move this project forward. I thank the members of my thesis committee: Mary Goll, Sanford Simon and Agata Smogorzewska, for their guidance and generously dedicating some of their precious time providing feedback, thoughtful comments, questions, and advice through the years. Special thanks, to Mary Baylies, for accepting to serve as the external examiner in my thesis defense, and for her thoughtful comments. I would like to thank George Cross for hosting me in his lab space during this last year at Rockefeller University. I am indebted to my undergraduate advisor Diana Bratu at Hunter College for her support through the years and inspiring me with her strength, tenacity and good-hearted nature. I would also like to thank Shirley Raps, at Hunter College who was the first one to see scientific potential in me and encouraged me to keep going on this path. I am also thankful to Gerardo Morfini and Scott Brady for their support early on in this scientific journey and for allowing me to have a fantastic first science experience in their labs at the Marine Biological Laboratories (MBL). Many thanks to Juleen Zierath and Anna Krook for the opportunity of a short research exchange at the Karolinska Institutet (KI) and to so many people at KI who allowed me to ‘pester’ them with questions and helped me learn more about the model system of my research. I am grateful for many people at the Rockefeller University and the German Cancer Research Center (DKFZ) who have helped me along the way: Diego Mourao-Sa, for the many scientific discussions, advice and thoughtful questions that helped me tremendously, and also for reviewing this thesis; Dewi Harjanto for being patient and thorough in answering many questions on bioinformatics to someone with zero coding background. I have learned a lot from you; Eric Fritz who gave me the lowdown on the lab, inspired me to be more strategic and structured in my experiments; Thomas Carroll, for the expertise and instrumental input in bioinformatics analyses; Pete Stavropoulos, for the feedback with the biochemistry related experiments and together with Nicholas Economos for purifying antibodies important for my work; Danae Schulz for her guidance in the ChIP-seq experiment and along with Monica Mugnier for their thoughtful questions and comments in lab meetings and practice talks; Erik Debler and Philipp Schmiege for their help in purifying protein important in future work; Sandra Ruf and Jose (Paulo) Lorenzo for being a pleasure to work with as my virtual labmates at DKFZ and for carrying the ‘APOBEC2 torch’ forward. iii I am especially grateful to Violeta Rayon, Maryam Zaringhalam and Jason Pinger who luckily joined the lab alongside me, for their scientific discussion, friendship and much needed moral support during the many challenges. I would also like to thank Dimitrios Garyfallos for his friendship and company in the lab especially when I was working late at nights. Special thanks, to Deanna Belsky for her friendship and inspiring me with her positivity. Thanks to many other friends in New York, who I have met through the years for their support and for making the graduate school years fun and unforgettable. Importantly I would like to thank all the people and groups who have also taught me important lessons beyond bench science: The colleagues and friends of INet NYC, for being such great team players, for their support and friendship; The Science Alliance Leadership Training (SALT) of the New York Academy of Sciences; the Fundamentals of Bioscience Industry Program of the Center of Biotechnology at Stony Brook University. I am thankful for the financial and administrative support I have received through the David Rockefeller Graduate Program and the Nicholson Exchange fellowship. I am grateful to the staff in the Dean’s Office, and especially Andrea Morris for ongoing career guidance and support. I would have never completed this journey without the love, support, and encouragement from my family. I thank everyone in the Molla, Babali, and Blanco-Melo family: I thank my parents for their love, their trust in me, and for instilling in me the importance of hard work and integrity; I thank my sisters Olta and Jona for always being there for me, especially for being my support system in the challenging first years when we first moved to NY. Faleminderit mami, babi, Olta, Jona! ; My aunt Jeta and uncle Sulo for hosting my sisters and I, helping us get started in NY; Vinela and Ledion for their unconditional support and advice through the years; Elka for her warmth and affability. Faleminderit nga zemra!; Hector and Aurora, for embracing me with your support and showing me the beautiful Mexico. Gracias por su apoyo! Last but not least I would like to thank my husband Daniel Blanco-Melo. I am so happy this graduate school journey brought us together, thank you for your love, support, patience (especially when 10 more minutes of work turned into 2 hours), listening at my long rants during the tougher times and for always finding a way to make me laugh. Te quiero mucho Daniel! iv TABLE OF CONTENTS ACKNOWLEDGMENTS................................................................................................iii TABLE OF CONTENTS ..................................................................................................v LIST OF FIGURES.........................................................................................................vii LIST OF TABLES..........................................................................................................viii CHAPTER 1. Introduction...............................................................................................1 1.1 Beyond the central dogma of Molecular Biology.....................................................1 1.2 Polynucleotide Cytidine Deaminases: The AID/APOBEC family of enzymes .......3 1.2.1 The startling discovery of the AID/APOBEC family .......................................4 1.2.2 The common evolutionary origin of the AID/APOBEC family .......................5 1.2.3 AID/APOBECs are similar by structure homology..........................................8 1.2.4 AID/APOBECs are very diverse in their biological function and physiological substrates....................................................................................................................9 1.3 APOBEC2, the ‘orphan’ deaminase .......................................................................15 1.3.1 No study has demonstrated enzymatic activity for APOBEC2 ......................17 1.3.2 Many studies demonstrate that APOBEC2 has a biological function.............19 1.4 Myogenesis,
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