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Regulation of Alc1's Snf2 Atpase REGULATION OF ALC1’S SNF2 ATPASE: INTERPLAY BETWEEN THE SNF2 DOMAIN, THE MACRODOMAIN AND OTHER CONSERVED ELEMENTS BY Copyright 2015 RUSHI DIPAK TRIVEDI 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. _______________________ Co-Chair, Joan Conaway, Ph.D. _______________________ Co-Chair, Aron Fenton, Ph.D. _______________________ Kenneth Peterson, Ph.D. _______________________ Jennifer Gerton, Ph.D. _______________________ Russell Swerdlow, M.D. Date defended: February 27, 2015 The Dissertation Committee for RUSHI DIPAK TRIVEDI certifies that this is the approved version of the following dissertation: REGULATION OF ALC1’S SNF2 ATPASE: INTERPLAY BETWEEN THE SNF2 DOMAIN, THE MACRODOMAIN AND OTHER CONSERVED ELEMENTS Committee members: _______________________ Co-Chair, Joan Conaway, Ph.D. _______________________ Co-Chair, Aron Fenton, Ph.D. _______________________ Kenneth Peterson, Ph.D. _______________________ Jennifer Gerton, Ph.D. _______________________ Russell Swerdlow, M.D. Date approved: March 6, 2015 ii ABSTRACT ALC1, also known as CHD1L, was originally identified as a gene present on a human chromosome 1q21 region amplified in ~50% of human hepatocellular carcinomas (HCC). ALC1 overexpressing cells form tumors in nude mice, and transgenic mice overexpressing ALC1 develop several types of spontaneous tumors. ALC1 has also been proposed as a novel candidate gene for congenital anomalies of the kidneys and urinary tract (CAKUT). ALC1, a member of the SNF2 family of ATPases, has an N-terminal SNF2-like ATPase that is most closely related to that of ISWI, and a C-terminal macrodomain that binds selectively to poly(ADP-ribose) (PAR). Between the ATPase and the macrodomain is an evolutionarily conserved region with no clear homology to any known domains. This “linker” region can be further divided into three sub-regions of greatest conservation. It was previously shown that wild type ALC1 possesses DNA-dependent ATPase and ATP-dependent nucleosome remodeling activities that are strongly dependent on the presence of poly(ADP-ribose) polymerase PARP1 (or the closely related PARP2) and its substrate NAD +. Importantly, a point mutation in the ALC1 macrodomain that interferes with PAR binding prevents PARP1- and NAD-dependent ALC1 activation. In this work, we dissected the mechanism by which PARP1 and NAD + activate ALC1 nucleosome remodeling. We demonstrate that ALC1 activation depends on the formation of a stable ALC1·PARylated PARP1·nucleosome intermediate. In addition, by exploiting a novel PAR footprinting assay, we obtained evidence that the ALC1 macrodomain remains stably associated with PAR on autoPARylated PARP1 during the course of nucleosome remodeling reactions. Results of biochemical experiments described here argue (i) that stable binding of the ALC1 macrodomain to autoPARylated PARP1 is critical for ALC1 activation and (ii) that iii activation of ALC1 depends on formation of a stable ALC1-autoPARylated PARP1-nucleosome intermediate. In the course of this study, we also find that apart from being regulated by PARP and NAD +, ALC1 possesses and additional mode of intradomain control via conserved domains in the linker region and the macrodomain. First, we identified a region, NMAC (N-terminal to Macrodomain ATPase Coupling) domain needed to couple ATP hydrolysis to nucleosome remodeling. Deleting NMAC led to a robust PARP and NAD +-dependent ATPase, which lacked appreciable remodeling activity. In addition, we identified an additional mode of control via the macrodomain when we replaced ALC1’s PAR binding macrodomain with another macrodomain from the variant histone macroH2A1.1 and discovered this chimeric protein was constitutively active independent of PARP and NAD +. Taken together our findings suggest a model of positive control of the SNF2 ATPase via the macrodomain and an additional level of control over its remodeling activities via the NMAC and other conserved linker elements. iv ACKNOWLEDGEMENTS First and foremost, I would like to thank my parents, family members and Shachi for their unconditional love and support. My parents have always given me the freedom to think independently and chart my own course in life. It is due to their encouragement that I even embarked on this ambitious journey. They have been by my side during the toughest times of this journey. My sister Ruchi and her family have been my source of strength and have seen me grow in the last decade. Shachi- I don’t have words to express the gratitude I owe to you for being by my side. You have been a pillar of support through the toughest of the times of this journey. Your appreciation “You have arrived” shall ring in my ears forever. Your presence in my life has made the biggest contribution to my personal and professional success and made this journey enjoyable. You are an amazing woman and a beautiful human being who I am truly thankful to have in my life. This dissertation would be incomplete without thanking Drs. Joan and Ron Conaway. To them I would like to express my deepest thanks and gratitude as my academic mentors. During my tenure in their lab as a graduate student I have been able to grow as a scientist and a human being. Their extreme kindness, scientific rigor, guidance, steadfast encouragement and humor made even the toughest problems of my professional life seem very surmountable. During my stay in the laboratory as a graduate student I had the distinct pleasure of independently developing this scientific project from the bottom up. I am personally gratified with my stay in the lab by having pushed the boundaries on mankind’s horizon of knowledge just a little further. Next, I would like to thank my committee members Drs. Ken Peterson, Russell Swerdlow, Jennifer Gerton, and Aron Fenton for all their useful advice and suggestions throughout the course of my graduate career. My committee members have provided great scientific direction v and made sure I was progressing in a timely manner towards graduation. I would like to thank all of the members of the Conaway Lab including: Nawel Mahrour, Aaron Gottschalk, Hidehisa Takahashi, Chieri Sato, Shigeo Sato, Vijay Kantharia, Shawn Hall and Margaret Banks. The members of the lab provided me with tremendous 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 lab mates, inside the lab and outside as well. I would like to particularly thank the core facilities here at Stowers Institute including Julie Conkwright in Screening; Tari Parmely, Maria Katt, and Valerie Neubauer in Tissue Culture; and all the members of the molecular biology core facility who have through their collaborative services allowed my research to flourish. My friends at SIMR and KUMC made the last few years of my life immensely enjoyable. I would also like to thank Andreas Ladurner and Gyula Timinzsky (EMBL), and James Kadonaga and Mai Khuong (University of California San Diego) for collaborative efforts on this project. Most importantly, I would particularly like to thank Jim and Virginia Stowers for providing such an amazing research facility and allowing basic science research to flourish for the benefit of humanity. “Hope for Life” shall forever remain in my mind. vi To Shachi & Rhea vii TABLE OF CONTENTS CHAPTER I. INTRODUCTION………….………………………………………………..…1 Chromatin Structure and Nucleosome Architecture…………………………..2 Nucleosomal Packaging……...……………………………………………….…..8 Histone modifying enzymes and their roles…………………………..……….11 Chromatin remodeling – How and Why?....................................................16 The RecA-like ATPase of SNF2 remodelers…………………………..…...…19 SNF2 ATPases – a brief history………………………………………...….…..24 SNF2 sub families………………………………………………………………...26 SWI/SNF family remodelers……………………………………………27 ISWI family remodelers………………………..……………………….29 CHD family remodelers…………..…………………………………….31 INO80 family remodelers………………………...…………………….32 Accessory domains of SNF2 ATPase………………………………….....…….35 PAR – Poly ADP-ribose………..………………………………………………..40 PAR binding Domains………………………………..………………………….43 PAR Binding Motif (PBM)…………………………..…………………43 Poly(ADP-Ribose)-Binding Zinc Finger (PBZ)……………………..44 WWE Domain……………….....................……………………...…….45 Macro domain………………………………………...…………………48 The PARP superfamily and its roles……………………..…………………….54 PARP1 and its roles in the nucleus…………………..………………………..59 ALC1: A SNF2 ATPase remodeler with a macrodomain……...……………64 CHAPTER II. ACTIVATION OF THE SNF2 FAMILY ATPASE ALC1 BYPOLY(ADP- RIBOSE) IN A STABLE ALC1∙PARP1∙NUCLEOSOME INTERMEDIATE................….70 Abstract……………………………………………………………………………71 Introduction……………………………………………………………….………72 Results………………………………………..……………………………………74 ALC1 activation is preceded by rapid formation of a benzamide resistanceintermediate………………………………………………….74 Cooperative binding of PARP1 and ALC1 to nucleosomes……….77 ALC1 protects PAR chains from ~3 to more than 20 ADP ribose units from digestion by poly(ADP-ribose) glycohydrolase (PARG)............................................................................................80 Discussion…………………..…………………………………………………….83 CHAPTER III. THE MACRODOMAIN OF HISTONE MACROH2A1.1 IS AN ACTIVATOR OF THE ALC1 CHROMATIN REMODELER..............................…………86 Abstract……………………………………………….…………………………..87 Introduction……………………………………………………………………...88
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