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Identification and Characterization of Barrier Insulator Activity in the T-Cell Receptor Alpha Locus Control Region

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Identification and Characterization of Barrier Insulator Activity in the T-Cell Receptor Alpha Locus Control Region City University of New York (CUNY) CUNY Academic Works All Dissertations, Theses, and Capstone Projects Dissertations, Theses, and Capstone Projects 2-2019 Identification and Characterization of Barrier Insulator Activity in the T-Cell Receptor alpha Locus Control Region Gayathri Devi Raghupathy The Graduate Center, City University of New York How does access to this work benefit ou?y Let us know! More information about this work at: https://academicworks.cuny.edu/gc_etds/3005 Discover additional works at: https://academicworks.cuny.edu This work is made publicly available by the City University of New York (CUNY). Contact: [email protected] IDENTIFICATION AND CHARACTERIZATION OF BARRIER INSULATOR ACTIVITY IN THE T CELL RECEPTOR α LOCUS CONTROL REGION by GAYATHRI DEVI RAGHUPATHY A dissertation submitted to the Graduate Faculty in Biology in partial fulfillment of the requirements for the degree of Doctor of Philosophy, The City University of New York 2019 © 2019 GAYATHRI DEVI RAGHUPATHY All Rights Reserved ii IDENTIFICATION AND CHARACTERIZATION OF BARRIER INSULATOR ACTIVITY IN THE T CELL RECEPTOR α LOCUS CONTROL REGION by Gayathri Devi Raghupathy This manuscript has been read and accepted for the Graduate Faculty in Biology in satisfaction of the dissertation requirement for the degree of Doctor of Philosophy. Date [Ben Ortiz] Chair of Examining Committee Date [Cathy Savage-Dunn] Executive Officer Supervisory Committee: Laurel Eckhardt Shubha Govind Diana Bratu Fei Li THE CITY UNIVERSITY OF NEW YORK iii ABSTRACT IDENTIFICATION AND CHARACTERIZATION OF BARRIER INSULATOR ACTIVITY IN THE T CELL RECEPTOR α LOCUS CONTROL REGION by Gayathri Devi Raghupathy Advisor: Dr.Ben Ortiz Genes of different spatiotemporal expression profiles are often juxtaposed in the genome. This organization raises risks of cross-regulatory influences from neighboring genes; for instance heterochromatin can spread over euchromatin or long-range acting enhancers can inappropriately activate genes. Gene regulatory elements such as Locus Control Regions (LCR) and Insulators prevent such cross-communications and allow for normal gene expression patterns. In transgenic systems, LCRs limit influences from surrounding chromatin by providing site-of-integration independent and specific spatiotemporal expression upon a linked transgene. The field’s understanding of the ability of an LCR to overcome chromatin influences and allow site-of- integration independent expression is minimal. Interestingly, this function of an LCR closely resembles that of barrier insulators. Barrier insulators prevent the spread of heterochromatin onto a euchromatin region and are characterized by their ability to suppress site-of-integration dependent chromatin influences upon a transgene. We hypothesize that the integration site- independence activity of LCRs is mediated by insulator-like DNA elements present within the LCR. In support of this hypothesis, we identify a novel barrier insulator activity within the mouse T-cell receptor (TCR)-α LCR. A 4.0-kb compilation of TCRα LCR sub-elements insulates a linked transgene in barrier assay- a long-term culture of stably transfected T cell lines. TCRα LCR-derived insulators enable maintenance of euchromatin and prevention of heterochromatin at a linked transgene. We find one element within the TCRα LCR that interacts iv with the USF1 transcription factor, which has been shown to have an important role in barrier insulation. In contrast to previously identified barrier insulators, the function of TCRα LCR- derived insulators does not require them to bi-laterally flank a gene. These data suggest that the TCRα LCR-derived elements may support both known and novel mechanisms of barrier insulation. v Acknowledgements I would like to thank the CUNY Graduate Center for accepting me into the PhD program. My sincere thanks to Dr.Ben Ortiz for giving me an opportunity to work in his laboratory, where I evolved as a scientist. Thank you Ben for being available to answer my questions and bearing with me through my struggles with experiments. I would like to thank my committee members- Dr.Eckhardt, Dr.Bratu, Dr.Li and Dr.Govind for guiding me through my research at various stages. My sincere thanks to Dr.Eckhardt, whose brilliant advice to focus on certain projects, helped me to not only save time and resources but to also learn to prioritize. A big thanks to Dr. Li and his lab members, Dr.Qianhua Dong and Dr.Haijin He for supporting me throughout the collaboration. Dr.Li thank you for being very encouraging and appreciative of my efforts. This project would not have existed if not for Armin Lahiji, who laid the groundwork for this research. My first day in the Ortiz lab, Armin was my teacher and since then he has been supportive and helpful through these years. I thank Martina Kucerova Levisohn for always being available (amidst her crazy schedule) to brain storm ideas and troubleshoot my experiments. I often refer to Martina’s lab notebooks for protocols, thanks to her for leaving behind meticulously written records. I also would like to thank my undergraduate students, Valia Kostiyuk and Calvin Herman for being energetic and ready to help at all times in the middle of their classes and tests. They have helped me get through my dull days and I have learnt quite a lot from them in terms of aiming high, multi-tasking and handling pressure. I would like to thank Zayd Daruwala, for creating a fun environment in the lab and for giving me cool cloning tips. The best part of the last seven years in this program has been my best friend Jordana Lovett. She has been my classmate, lab mate, friend, family, motivator and a ‘shrink’. We have had a shared experience throughout this program, since day 1 of our classes. Jordana has helped me bring out the hidden best side in me; she has unconsciously played a huge role in daring me to vi make my dreams into reality. We both know that our journey together as friends and ‘dynamic duo’ does not end with this program; we are looking forward to doing bigger things together. I have so many more things to share and thank Jordana for; it might be way too long so I will stop here… I would like to thank the Department of Biology at Hunter College and my friends, Livia, Irena, Jyoti, Chong for being supportive and giving feedback on my work. Thanks to Inna and Razib for making teaching convenient and less laborious. Thanks to Priscillia Lhmouad, Skok Lab, NYULMC for being very helpful in optimizing in my experiments. Thanks to my parents, who have been my emotional support system throughout this program. While they did not get my experimental issues, their kind encouraging words, ‘there will be light at the end of the tunnel’ kept me going. My parents did not get to complete their education but they raised me to appreciate education and stay committed to it. My success of achieving this highest level of education truly belongs to them. I dedicate my PhD work to my parents, Mr.Raghupathy and Mrs.Padma Raghupathy. Thanks to my brother, Hari for trying to understand what was wrong with my experiments and even attempting to fix it! Thanks to my in- law family for being very understanding and supportive during my research. Last but not the least, I would like to thank my husband, Kishan without whose unconditional love and support, I would have been very stuck. Kishan, you have played a tremendous role in enabling me to get through every struggle that I had through this journey. You have supported me in small and big ways. Thanks for always believing in me and pushing me to stay positive and be my best. This would not have been possible without you! There is a very special person; I would like to thank-my Anya baby. While I feared that my pregnancy would hinder me in wrapping-up, it indeed has powered me up with positivity and energy, Thank You Biology! . vii Table of Contents CHAPTER 1: INTRODUCTION 1 1.1 LOCUS CONTROL REGION 1 1.2 BARRIER INSULATORS 5 1.3 TCRα/DAD1 LOCUS 8 CHAPTER 2: MATERIALS AND METHODS 13 2.1 CHROMATIN IMMUNOPRECIPITATION 13 2.2 DNASE I SENSITIVITY-QUANTITATIVE PCR 15 2.3 SOUTHERN BLOTTING TO DETECT TANDEM INTEGRATION OF TRANSGENES 16 2.4 POLYMERASE CHAIN REACTION TO DETECT TANDEM INTEGRATION OF TRANSGENES 16 2.5 T CELL LINE MAINTENANCE AND TRANSFECTION 17 2.6 DATA MINING METHODS BASED ON CISTROME AND UCSC GENOME BROWSER 18 2.7 YEAST INSULATOR ASSAY 18 CHAPTER 3: IDENTIFICATION AND CHARACTERIZATION OF BARRIER INSULATOR ACTIVITY IN THE TCRα LCR 21 3.1 HALLMARK EPIGENETIC EVENTS ASSOCIATED WITH BARRIER INSULATORS 21 3.2 TCRα LCR ELEMENTS PRESERVE EUCHROMATIN STATUS AND PREVENT HETEROCHROMATIN ENCROACHMENT AT THE REPORTER GENE LOCUS 22 3.3 TCRα LCR ELEMENTS MAINTAIN REPORTER GENE LOCUS ACCESSIBILITY IN CHROMATIN 25 CHAPTER 4: NOVEL FEATURE OF INSULATION BY TCRα LCR ELEMENTS 27 4.1 CLASSICAL BARRIER INSULATORS INSULATE ONLY WHEN FLANKING A TRANSGENE 27 4.2 TCRα LCR ELEMENTS INSULATE THE TRANSGENE IN A UNILATERAL MANNER 27 viii CHAPTER 5: DETERMINING THE BINDING OF TCRα LCR TO THE INSULATOR FACTOR, USF1 30 5.1 ROLE OF USF1 IN BARRIER INSULATION 30 5.2 INSULATOR FACTOR USF1 BINDS TO THE HS1’ REGION, BUT NOT THE HS4 OR HS6 REGIONS OF THE TCRα LCR 31 5.3 TESTING SHORT HAIRPIN RNAs (SHRNA) TO KNOCKDOWN USF1 33 CHAPTER 6: IDENTIFICATION OF FACTORS THAT BIND TO THE TCRα LCR35 6.1 CANDIDATE FACTORS THAT BIND TO THE TCRα LCR 35 CHAPTER 7: CHROMATIN LANDSCAPE OF THE TCRα/DAD1 LOCUS 38 7.1 CHROMATIN MARKS ASSOCIATED WITH TCRα, LCR AND DAD1 IN THYMUS AND NON-THYMUS TISSUES 38 CHAPTER 8: ASSESSING THE INSULATOR ACTIVITY OF TCRα LCR IN A FISSION YEAST MODEL 42 8.1 FISSION YEAST: TESTING A NEW MODEL TO STUDY VERTEBRATE INSULATORS 42 8.2 HS6 ELEMENT OF THE TCRα LCR IS NOT SUFFICIENT TO INSULATE A TRANSGENE IN YEAST INSULATOR ASSAY 43 CHAPTER 9: CONCLUSION 45 9.1 MODEL OF INSULATION 46 9.2 FUTURE DIRECTIONS 48 9.3 SIGNIFICANCE 49 REFERENCES 51 ix LIST OF FIGURES Figure 1.
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