University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Fall 12-16-2016 Regulation of Alteration/Deficiency in Activation 3 (ADA3) by Acetylation and its Role in Cell Cycle Regulation and Oncogenesis Shashank Srivastava University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Part of the Biochemistry Commons, Cancer Biology Commons, Cell Biology Commons, and the Molecular Biology Commons Recommended Citation Srivastava, Shashank, "Regulation of Alteration/Deficiency in Activation 3 (ADA3) by Acetylation and its Role in Cell Cycle Regulation and Oncogenesis" (2016). Theses & Dissertations. 156. https://digitalcommons.unmc.edu/etd/156 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. REGULATION OF ALTERATION/DEFICIENCY IN ACTIVATION 3 (ADA3) BY ACETYLATION AND ITS ROLE IN CELL CYCLE REGULATION AND ONCOGENESIS By SHASHANK SRIVASTAVA A DISSERTATION Presented to the Faculty of The Graduate College in the University of Nebraska In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy Genetics, Cell Biology and Anatomy Under the Supervision of Dr. Vimla Band University of Nebraska Medical Center Omaha, Nebraska December 2016 Supervisory Committee Hamid Band, M.D., Ph.D. Rakesh Singh, Ph.D. Andrew T. Dudley, Ph.D. Lt. Runqing Lu, Ph.D. DEDICATION To my loving parents ACKNOWLEDGEMENTS Firstly, I would like to express my sincere gratitude towards my advisor Dr. Vimla Band for giving me the opportunity to pursue doctoral research in her laboratory. I joined Dr. Band’s laboratory as a master student however, after a while working under her supervision I realized that her laboratory is perfect for doctoral my studies and I ended up converting into Ph.D. program. She gave me the freedom to take up different projects without any objection and it was her expertise and experience in the field that played a major role in the successful accomplishments of my thesis projects. I cannot thank Dr. Band enough for her for providing me with constant support and encouragement during past five years. I deeply thank my doctoral committee members, Dr. Hamid Band, Dr. Rakesh Singh, Dr. Andrew Dudley and Lt. Dr. Runqing Lu for their constructive criticisms and valuable suggestions during the committee meetings. Dr. Hamid Band has been our long-standing collaborator and I would like to thank him not only for helping me in writing manuscripts but also for giving me the access to his laboratory reagents whenever I needed them in my research. Indeed his vast theoretical and technical knowledge in the field has allowed me to conduct my research in more comprehensive manner. Dr. Rakesh Singh is the most student friendly faculty that I came across in my graduate school. He has been serving as my supervisory committee member since I was a master student in the department and he, along with Dr. Band, encouraged me to convert into a Ph.D. student, which eventually turned out to be righteous decision. I thank him for directing me on correct career path. I am indebt to Dr. Dudley for his guidance throughout my graduate studies. He also served as our journal club instructor and the insightful questions asked by him during committee meetings and journal club have immensely nurtured my scientific skills. In addition to supervising in my research he also gave many valuable critiques in one of our manuscripts writing. I must say that his dedication towards students’ academic fosterage is remarkable. Dr. Lu, who sadly passed away recently, was the one who initially made me II comfortable in the department. Besides supervising in my committee, he also taught me in a couple of courses including journal clubs and his easygoing attitude always gave me huge confidence. I will always miss him and be thankful to him. My doctoral research would have not been possible without wonderful laboratory colleagues. Shakur was my mentor in many ways, right from training me new laboratory techniques to help design and troubleshooting experiments. I thoroughly enjoyed working with him in the laboratory for these many years. I extend my thanks to Riyaz a senior postdoctoral fellow and Divya, a fellow graduate student of the laboratory, for being such nice colleagues. A good support system is vital to survive during Ph.D. research and I was very fortunate to have this in the form of good friends. I thank all of them from the bottom of my heart for being there out for me. Finally, I express my deepest gratitude to the almighty, my parents and my family. Like every other parents, my parents also made huge sacrifices for me and despite of being away from me, gave me the persistent motivation and confidence to pursue doctoral studies. III ABSTRACT The ADA3 (Alteration/Deficiency in Activation 3) protein is a transcriptional adaptor protein that was initially discovered as a component of several HAT (Histone Acetyltransferase) complexes, the enzyme complex responsible for histone acetylation, which is a prerequisite for transcription. Earlier the studies from Dr. Band’s laboratory and that of others’ have deciphered a crucial role of ADA3 in cell cycle regulation (both through G1/S and G2/M phase transitions) and in maintaining the genomic stability. While our laboratory investigated the mechanism behind the role of ADA3 in G1/S transition, the same remained unknown for G2/M phase transition. Based on this prior knowledge about ADA3, I started out my Ph.D. thesis work in Dr. Band’s laboratory directed towards examining the role of ADA3 in mitosis. During my doctoral research, I demonstrated that ADA3 governs the recruitment of a key centromeric protein CENP-B on to the centromeres and regulates the chromosome segregation during mitosis. ADA3 protein has the potential to undergo posttranslational modification, including acetylation, and in the course of my Ph.D. research, I became interested in how these modifications might regulate the function of ADA3. I showed that ADA3 acetylation is regulated by coordinated actions of its associated HATs, GCN5, PCAF and p300, and a new partner I discovered, the deacetylase SIRT1. We used mass-spectrometry and site-directed mutagenesis to identify major sites of ADA3 acetylated by GCN5 and p300 and found that acetylation defective mutants were capable of interacting with HATs and other components of HAT complexes but deficient in their ability to restore ADA3-dependent global or locus-specific histone acetylation marks and cell proliferation in Ada3 deleted MEFs. A parallel focus of my studies was to define the role of ADA3 in HER2+ breast cancers, which basically emanates from a clinical study from our laboratory that revealed that ADA3 is overexpressed/mislocalized in these types of aggressive tumors. By using cell culture models I IV have established a link between ADA3 and HER2 signaling pathways. In these cell lines, I found that ADA3 is a downstream target of HER2 and discovered a novel phospho-AKT-phospho- p300-Ac-ADA3 signaling pathway. Importantly, ADA3 knockdown in these cells recapitulates the cell cycle inhibitory effects of a tyrosine kinase inhibitor lapatinib such as accumulation of CDK inhibitor p27 and reduced mitotic index. Taken together these results highlight the importance of ADA3 as a marker for treatment efficacy and a promising therapeutic target. Given the key importance of ADA3-containing HAT complexes in the regulation of various biological processes, including cell cycle, my thesis work provides an insight for the regulation of the function of these complexes through dynamic ADA3 acetylation. V Table of Contents LIST OF FIGURES .................................................................................................................................. IX LIST OF TABLES ...................................................................................................................................... X ABBREVIATIONS .................................................................................................................................. XI CHAPTER 1 ............................................................................................................................................... 1 INTRODUCTION ...................................................................................................................................... 1 ADA3, AS A COMPONENT OF SAGA COMPLEX .......................................................................................................... 2 ADA3 AS A P300 INTERACTING PROTEIN ................................................................................................................. 8 THE NON-KAT INTERACTOME OF ADA3 ............................................................................................................... 10 PHYSIOLOGICAL AND PATHOLOGICAL ROLES OF ADA3 ...................................................................................... 12 REGULATION OF ADA3 BY POST-TRANSLATIONAL MODIFICATIONS ................................................................ 14 CHAPTER 2 ............................................................................................................................................. 21 ALTERATION/DEFICIENCY IN ACTIVATION 3 (ADA3) PROTEIN, A CELL CYCLE REGULATOR, ASSOCIATES