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1 Class I Lysine Deacetylases Facilitate Class I Lysine Deacetylases Facilitate Glucocorticoid Receptor-Mediated Transcriptional Activation Item Type text; Electronic Dissertation Authors Kadiyala, Vineela Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 05/10/2021 20:26:03 Link to Item http://hdl.handle.net/10150/311669 1 CLASS I LYSINE DEACETYLASES FACILITATE GLUCOCORTICOID RECEPTOR-MEDIATED TRANSCRIPTIONAL ACTIVATION By Vineela Kadiyala ____________________________ Copyright © Vineela Kadiyala 2013 A Dissertation Submitted to the Faculty of the DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY WITH A MAJOR IN CHEMISTRY In the Graduate College THE UNIVERSITY OF ARIZONA 2013 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Vineela Kadiyala, titled Class I Lysine Deacetylases Facilitate Glucocorticoid Receptor-mediated Transcriptional Activation and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy. _______________________________________________________________________ Date: (12/12/2013) Catharine L. Smith _______________________________________________________________________ Date: (12/12/2013) Roger Miesfeld _______________________________________________________________________ Date: (12/12/2013) Tshusen-Tsao _______________________________________________________________________ Date: (12/12/2013) Bernard Futscher _______________________________________________________________________ Date: (12/12/2013) Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. ________________________________________________ Date: (12/12/2013) Dissertation Director: Catharine L. Smith 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of the requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that an accurate acknowledgement of the source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the copyright holder. SIGNED: Vineela Kadiyala 4 ACKNOWLEDGEMENTS There are a number of people who have supported me in this journey and this statement is to recognize them. First of all, I thank my mentor Dr. Catharine L. Smith. Her passion for science and research inspired me to strive harder and reach higher. Her encouragement and guidance has taught me to look at a problem from all possible angles and to figure out the answer. Her understanding nature provided me the flexibility needed for being a graduate student and a mother for which I am extremely grateful to her. I also thank my committee members for their advice, guidance and encouragement. I am grateful for my current and past lab members for their support throughout the years and for bringing fun into this journey. I would like to thank Dr. Ana Tula-Sanchez for being an amazing friend and colleague, Nina Patrick and Rosa Jamie-Frias for their friendship and their help with some of the projects. Thank you to Wana Mathieu for her assistance in running some of the experiments. I would also like to thank Mary Klein, Maryam Gilpatrick, and all the other smith lab members for becoming an amazing family, I will cherish all the days that I spent in the lab with them. I want to thank Dr. Paul Severson for his help in optimizing the ChIP assays. I want to thank Dr. Swati Kushal for believing in me when I lost belief in myself. I would never have been able to achieve this degree without my husband, Subbarao Bellamkonda. His unconditional love and support helped me to balance family and work without which I couldn’t have raised our children and achieved my PhD. My amazing daughters Neha and Nidhi who have taught me to look at the world with curiosity and who have been patient with me when work was my priority, I am grateful for them. I am also indebted to my parents, Lakshmi and Panakalarao, who have always encouraged me to be the best that I could be and the rest of my family and friends for believing in me and for being there for all my ups and downs. Finally I want to give thanks to GOD for providing me with such wonderful support system that helped me achieve my goals. 5 DEDICATION To my husband, Subbarao My daughters, Neha and Nidhi And My parents, Lakshmi and Panakalarao 6 TABLE OF CONTENTS LIST OF TABLES ............................................................................................................ 9 LIST OF FIGURES ........................................................................................................ 10 ABSTRACT .................................................................................................................... 13 CHAPTER 1: INTRODUCTION ................................................................................... 15 Glucocorticoids and their functions ......................................................................... 15 Glucocorticoid receptor ............................................................................................. 17 Structure of GR ........................................................................................................ 17 Isoforms of GR ......................................................................................................... 19 GR signaling ................................................................................................................ 22 Transcriptional regulation by GR ............................................................................. 22 Factors regulating GR transcription......................................................................... 25 Glucocorticoid response elements (GREs) .............................................................. 25 Location of GBS ....................................................................................................... 28 Ligand Binding ........................................................................................................ 28 Interaction with other transcription factors ............................................................ 29 Corepressors and coactivators ................................................................................. 30 KATs and KDACs ....................................................................................................... 35 KATs ......................................................................................................................... 36 KDACs ...................................................................................................................... 39 KDAC inhibitors ....................................................................................................... 43 Role of KATs and KDACs in GR-regulated transcription ..................................... 43 KDAC6 facilitates glucocorticoid receptor processing ........................................... 46 KDACs facilitate GR mediated transrepression ...................................................... 46 GR acetylation .......................................................................................................... 47 KDACs facilitate GR mediated activation of MMTV promoter ............................. 48 CHAPTER 2: MATERIALS AND METHODS ........................................................... 53 Materials ...................................................................................................................... 53 Cell Culture .............................................................................................................. 53 Antibodies and Reagents .......................................................................................... 53 Methods ....................................................................................................................... 54 7 RNA Analysis ........................................................................................................... 54 Nascent RNA analysis .............................................................................................. 55 Co-Immunoprecipitation ......................................................................................... 56 Western Blotting ....................................................................................................... 57 siRNA Mediated KDAC Knockdown ....................................................................... 57 Expression Profiling ................................................................................................ 57 Chromatin Immunoprecipitation ............................................................................ 58 CHAPTER 3: VALPROIC ACID IMPAIRS ACTIVATION OF GLUCOCORTICOID TARGET GENES THROUGH
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