BIOCHEMICAL AND DEVELOPMENTAL CHARACTERIZATION OF A SNF2-LIKE ATPASE AMPLIFIED IN LIVER CANCER 1 (ALC1) BY Aaron J. Gottschalk Submitted to the graduate degree program in Biochemistry and Molecular Biology and the Graduate faculty of the University of Kansas Medical Center in partial fulfillment for the degree of Doctor of Philosophy. _______________________ Chairperson Committee members* _______________________* _______________________* _______________________* _______________________* Date defended: ________________ The Dissertation Committee for Aaron J. Gottschalk certifies that this is the approved version of the following dissertation: BIOCHEMICAL AND DEVELOPMENTAL CHARACTERIZATION OF A SNF2-LIKE ATPASE AMPLIFIED IN LIVER CANCER 1 (ALC1) Committee: _______________________ Chairperson* _______________________ _______________________ _______________________ _______________________ Date defended: ________________ 2 ACKNOWLEDGEMENTS First and foremost, I would like to express my deepest thanks and gratitude to my mentors Joan and Ron Conaway. During my tenure in their lab as a graduate student I have been afforded an amazing opportunity to learn and grow as a scientist while independently developing this project. It is their extreme kindness, steadfast encouragement, guidance, and humor that have made even the toughest of problems both scientific and in life seem very surmountable, and for that I thank them profoundly. Next, I would like to thank my committee members Drs. Glen Andrews, Jennifer Gerton, Leslie Heckert, and Ken Peterson for all their useful advice and suggestions throughout the course of my graduate career. Not only did my committee members provide great scientific direction but they also made bi-annual meetings fun and engaging. I would like to thank all of the members of the Conaway Lab including: Charles Banks, Margaret Banks, Yong Cai, Jingji Jin, Stephanie Kong, Nawel Mahrour, Ana Pedraza, Chieri Sato, Shigeo Sato, Henrietta Szutorisz, Hide Takahashi, Tingting Yao, Dotan Sela, Lu Chen, and Merry McClaird. The members of the lab provided me with an infinite amount of support and advice all while promoting a wonderful work environment that was full of fun times and much laughter. I will forever treasure the long hours spent with my labmates. I would like to particularly thank the core faclities here at Stowers Institute including Mike Washburn, Laurence Florens, and Selene Swanson in Proteomics; Tari Parmely, Maria Katt, and Valerie Neubauer in Tissue Culture; and countless members of the molecular biology and aquatics core facilities who have through their collaborative services allowed my research to flourish. I would like to thank Andreas Ladurner and 3 Gyula Timinzsky (EMBL), and Lee Kraus and Raga Krishnakumar (Cornell University) for collaborative efforts on this project. I would particularly like to thank Jim and Virginia Stowers for providing such an amazing institute. Lastly, and perhaps most importantly, I would like to thank my parents and family members for their unconditional love and support. 4 ABSTRACT Post-translational modifications play a key role in recruiting chromatin remodeling and modifying enzymes to specific regions of chromosomes to modulate chromatin structure. Alc1 (Amplified in Liver Cancer 1), a member of the SNF2 ATPase superfamily with a carboxy-terminal macrodomain, is encoded by an oncogene implicated in the pathogenesis of hepatocellular carcinoma. Using a variety of biochemical techniques we show that Alc1 interacts transiently with chromatin- associated proteins, including histones and the poly-(ADP-ribose) polymerase Parp1. Alc1 ATPase and chromatin remodeling activities are strongly activated by Parp1 and its substrate NAD and require an intact macrodomain capable of binding poly-(ADP-ribose). Alc1 is rapidly recruited to nucleosomes in vitro and to chromatin in cells when Parp1 catalyzes PAR synthesis. We propose that poly-(ADP-ribosyl)ation of chromatin- associated Parp1 serves as a novel mechanism for targeting a SNF2 family remodeler to chromatin. Using zebrafish as a model organism, we aimed to study possible roles of ALC1 in early organismal development. We found through qPCR and whole mount in situ analysis, that ALC1 is expressed ubiquitously within the blastomere prior to gastrulation, with peak expression observed during the Dome stage, and later at 24hpf that ALC1 is expressed within the anterior central nervous system. The injection of embryos with morpholinos targeting ALC1 resulted in pleiotropic phenotypes that were partially rescued by co-injection of either human or zebrafish mRNA. These findings suggest that Alc1 and its associated enzymatic activities are most likely required for proper organismal development. 5 TABLE OF CONTENTS CHAPTER I. INTRODUCTION...............................................................................................................................9 Chromatin Structure and Nuclear Architecture .....................................................................................9 Histone Modifying enzymes and their Modifications............................................................................13 Conservation and diversification of SNF2 ATPase superfamily...........................................................19 SNF2 ATPases and their influence on nuclear processes.....................................................................22 SNF2 ATPases: Fine-tuned Motors for specific functions....................................................................28 Poly(ADP-ribosyl)ation in the Nucleus ................................................................................................33 The PARP Family of Proteins...............................................................................................................38 Parp1 and its role in DNA Damage Repair..........................................................................................42 Parp1, NAD+ metabolism and Cell Death............................................................................................47 Modulation of Chromatin Structure by Parp........................................................................................49 Parp and its roles in transcriptional regulation...................................................................................52 Alc1 and its role in pathogenesis of Hepatocellular Carcinoma..........................................................57 Alc1: A SNF2 ATPase with an interesting accessory domain .............................................................60 CHAPTER II. BIOCHEMICAL STUDIES OF ALC1.........................................................................................65 Investigation of Alc1 Protein-Protein interactions...............................................................................65 Expression and Purification of wild-type and mutant Alc1 ..................................................................67 Alc1 Macrodomain is a PAR binding Motif..........................................................................................68 Studies on Alc1 ATPase activity ...........................................................................................................70 Studies on Alc1 chromatin remodeling activities..................................................................................75 Co-fractionation of Enzymatic Activities with Alc1..............................................................................78 Parp automodification is the critical event required for Alc1 enzymatic activation ............................80 Alc1 recruitment to chromatin is mediated by PARylated Parp1.........................................................83 CHAPTER III. BIOLOGICAL STUDIES OF ALC1............................................................................................89 Alc1 conservation amongst species ......................................................................................................89 Studies on Alc1 expression during zebrafish development ...................................................................92 ALC1 and its potential role in early development ................................................................................99 CHAPTER IV. DISCUSSION................................................................................................................................110 Alc1 Enzymology: Mechanism and Implications................................................................................110 Alc1: biological roles under normal physiological circumstances.....................................................120 Role of Alc1 in proper organismal development ................................................................................128 Role of Alc1 in Carcinoma Pathogenesis ...........................................................................................132 Future Directions ...............................................................................................................................135 CHAPTER V. MATERIALS AND METHODS...................................................................................................138 Biochemical studies of Alc1................................................................................................................138 Biological studies of Alc1...................................................................................................................142 APPENDIX ..............................................................................................................................................................147