TRANSCRIPTIONAL REGULATION OF THE MOUSE ADENYLYL CYCLASE TYPE 4 (Adcy4) IN Y1 ADRENOCORTICAL TUMOR CELLS By Xianliang Rui A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Pharmacology and Toxicology University of Toronto © Copyright by Xianliang Rui 2010 Library and Archives Bibliothèque et Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l’édition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre référence ISBN: 978-0-494-67725-4 Our file Notre référence ISBN: 978-0-494-67725-4 NOTICE: AVIS: The author has granted a non- L’auteur a accordé une licence non exclusive exclusive license allowing Library and permettant à la Bibliothèque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par télécommunication ou par l’Internet, prêter, telecommunication or on the Internet, distribuer et vendre des thèses partout dans le loan, distribute and sell theses monde, à des fins commerciales ou autres, sur worldwide, for commercial or non- support microforme, papier, électronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. 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Transcriptional Regulation of the Mouse Adenylyl Cyclase Type 4 (Adcy4) in Y1 Adrenocortical Tumor Cells Xianliang Rui Degree of Doctor of Philosophy, 2010 Department of Pharmacology and Toxicology University of Toronto ABSTRACT Adenylyl cyclase (Adcy) is an important early effector of adrenocorticotrophin (ACTH) on the adrenal cortex; however, this enzyme consists of ten isozymes in mammalian cells and the factors governing the expression of different Adcy isozymes have not been well defined. The aim of this study is to investigate the regulation of mouse Adcy4, one of ten isozymes, in Y1 adrenocortical tumor cells and in mutant subclones derived from the Y1 cells. Adcy4 is expressed at a high level in brain but at lower levels in many other tissues including the Y1 cells. Moreover, this isozyme is specifically deficient in Y1 mutants with impaired steroidogenic factor 1 (SF1) activity. These observations support a hypothesis that Adcy4 expression is influenced by both ubiquitously expressed and tissue-specific transcription factors. My sequencing results indicate that mouse Adcy4 is highly homologous to the human and rat counterparts; its gene is located less than 1 kb downstream of Ripk3 and contains 26 exons. Primer extension and in silico analyses suggest that Adcy4 contains a TATA-less promoter and initiates transcription from multiple sites. Luciferase reporter gene assays indicate that Adcy4 promoter activity is mainly stimulated by the proximal GC-rich region but is inhibited by the first intron. This 124 bp GC-rich region is well conserved among several mammalian species and exhibits strong promoter activity in Y1 cells, which is functionally compromised in the Adcy4-deficient mutant. Within this region, three Sp1/Sp3- and one SF1-binding sites have been identified which bind the corresponding proteins Sp1 and Sp3 or SF1 in electrophoretic mobility shift assays (EMSAs). Site-directed mutagenesis reveals that the 5’-most Sp1/Sp3 site enhances Adcy4 promoter activity, ii whereas the middle Sp1/Sp3 and SF1 sites each repress this activity. In Y1 mutant cells, mutating the SF1 site restores Adcy4 promoter activity and knocking down SF1 with shRNA increases Adcy4 expression. All these data demonstrate that Adcy4 expression is under the control of the ubiquitous factors Sp1 and Sp3 and the tissue-specific factor SF1 and establish that SF1 is a repressor for Adcy4 promoter activity. This study is the first to demonstrate a repressor function for SF1 in certain promoter contexts. iii ACKNOWLEDGMENTS I would like to thank my supervisor, Dr. Bernard Schimmer, for providing me the precious opportunity to do my Ph. D. research in his lab. Dr. Schimmer has inculcated me with valuable instruction on scientific writing skills and rigorous logical thinking that will certainly benefit me for the rest of my life. His exact research style and perseverance in pursuing perfection have set up a high standard for me to be a good scientist and prompted me to strive for nothing less than high-quality research. Without his excellent supervision, enthusiastic support, as well as his confidence in my ability as an independent researcher, this thesis would not have been possible. I would also like to thank my thesis advisory committee, Dr. David Riddick and Dr. Denis Grant, for their suggestions, guidance and generous help throughout my Ph. D. research. Their expertise, patience and support greatly enhanced both my research and course study. I am exceedingly grateful for many past and current members in Dr. Schimmer’s Lab for their friendship, scientific discussion, and technical help. More specifically, I would like to express my sincere appreciation to Jenny Tsao, whose excellent technical assistance and generous help have made my whole graduate research much easier. I also owe my gratitude to Martha Cordova for her constant technical advises and frequent help with many experiments. This work has been generously supported by the funding from NSERC, Department of Pharmacology and Toxicology, and University of Toronto. Finally, I am deeply indebted to my parents and my brothers for their love, their encouragement and unfaltering support over these years. I would like to thank my lovely daughter, Ting, and my little angel, Rosie, who always give me joys and inspiration. Most iv importantly, I would like to thank my wife, Chunhui, for her unconditional love and support, which give me the strength to finish this work. v TABLE OF CONTENTS TITLE PAGE……………………………………..………………………………….….......i ABSTRACT………………………………………………………………………………....ii ACKNOWLEDGMENTS…………………...………………………………………….….iv TABLE OF CONTENTS…………………...…………………………………………..….vi LIST OF TABLES…………………………...…………………………………………..…ix LIST OF FIGURES………………………………………………………………..………..x LIST OF ABBREVIATIONS……………………………………………………………..xii LIST OF PUBLICATIONS……………………………………………………………….xv SECTION I. INTRODUCTION……………………………...…………………..……1-56 1. Adenylyl Cyclase……………………………………………………………………...1 1.1 Isozymes and Structure………………………………………………....…..….1 1.2 Regulatory Properties………………………...…………………………….….6 1.3 Tissue Distribution and Physiological Function…………………………..….11 1.4 Gene Structure and Transcriptional Regulation………...……………………15 2. Adrenal Steroidogenesis……………………………………………………………..17 2.1 Steroidogenesis in the Adrenal Cortex……………………………………….19 2.2 Regulation of Adrenal Steroid Biosynthesis………………………………….24 3. Essential Role of cAMP Signaling in Adrenal Steroidogenesis………….……….....28 4. Essential Role of SF1 in the Regulation of Steroidogenesis……….………………..33 4.1 SF1 and LRH1 ……………………………………………………………….36 4.2 SF1 Expression Profiles and its in vivo Function…………………………….39 4.3 SF1 Target Genes…………………………………………………………….41 4.4 SF1 Structure and its Functional Regulation………………...…………….…43 5. The Roles of Transcription Factors Sp1 and Sp3 in Steroidogenesis……………..…48 6. Rationale, Hypothesis, and Objectives………………………………………………52 SECTION II. MATERIALS AND METHDODS…………………………………….57-85 1. Synthetic Oligonucleotides………………………………………………………..…57 2. Plasmids…………………………………….…………………………………….….61 2.1 Plasmids………………………………………………………………………61 2.2 Reporter Gene Constructs…………………………………………………….61 2.2.1 Reporter Plasmids with Progressive Truncation of the 5’-Flanking Region of Adcy4…………………………………………62 2.2.2 p-631/-290 Adcy4 Reporter Plasmids………………………….……..64 2.2.3 p-404AdcyLuc Reporter Plasmid with Mutations in the –404/-320 Region……………………………………………………..64 2.2.4 p-404AdcyLuc Reporter Plasmid with the Internal –135/–19 Region of Adcy4 Deleted…………………………...……...65 2.2.5 p-404AdcyLuc Reporter Plasmid with the First Intron of Adcy4 Deleted…………………………………………...…….……...67 2.2.6 pTA-Luc-Based Heterologous Reporter Plasmids…..…..……………70 2.2.7 Reporter Constructs with Sp1A, Sp1B, and/or SF1 Mutations in the Conserved Region………………………………….72 vi 2.3 Purification of Plasmid DNA………………………………………….……..74 3. Cell Culture and Transfection………………………………………………..………75 4. Preparation of Chromosomal DNA……………………………………….............…76 5. Isolation of RNA………………………………………………………………....….76 6. RT-PCR and Real-Time PCR……………………………..…………..............……..77 7. DNA Sequencing…………………………………………………….............………77 8. Southern Blot Analysis………………………………………………................……78 9. Western Blot Analysis………………………………………………...………..……79 10. Primer Extension……………………………………………………............………79