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V / DISSERTATION DOCTOR of PHILOSOPHY Denton, Texas May, 1992 3~7? /V / MOLECULAR MECHANISM OF ACTION OF STEROID HORMONE RECEPTORS DISSERTATION Presented to the Graduate Council of the University of North Texas in Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY By Zafar Nawaz, B.S., M.S. Denton, Texas May, 1992 Nawaz, Zafar, Molecular Mechanism of Action of Steroid Hormone Receptors. Doctor of Philosophy (Biology), May, 1992, 136 pp., V tables, 46 illustrations, bibliography, 204 titles. A novel bacterial expression system that is capable of producing high levels of soluble, stable, biologically active human vitamin D3 and estrogen receptors has been developed. The method utilizes ubiquitin fusion technology and a low temperature nalidixic acid induction of the lambda PL promoter. This system can produce large quantities of receptor antigen, but only a small fraction displays wild-type DNA and hormone binding properties. Therefore, the use of this system to overproduce receptors for crystallization studies is not practical. To overcome these problems, a 2 um based ubiquitin fusion system which allows regulated expression of the estrogen receptor in yeast (Saccharomyces cerevisiae) was developed. This system produces the estrogen receptor to a level of 0.2% of the total soluble protein. Moreover, this protein is undegradable, soluble, and biologically active. To test the transcriptional activity of the estrogen receptor produced in yeast, a cis-trans transcription assay was developed. This assay revealed that the transcriptional activity of the human estrogen receptor expressed in yeast was similar to that observed in transfected mammalian cells. This reconstituted estrogen transcription unit in Saccharomyces cerevisiae was utilized to examine the regulation of estrogen receptor functions by antiestrogens, to develop a random and rapid approach for identifying novel estrogen response elements, to characterize estrogen receptor variants cloned from human breast tumors, and to examine the effect of estrogen receptor on the regulation of osteocalcin gene. PREFACE It has been my hope that during these years of research work, I could have help to clarify the "mystery" that surrounded the many aspects of the steroid receptor gene superfamily. I have the hope that the results of this work can serve either as the starting point for further detailed studies on the same topic, or as applications in other related or unrelated research studies. I also hope that a reader will find this thesis a useful reference. My research work has gone on for the last four years benefiting from the help of so many people that it is impossible to thank everyone concerned. But let me make a start with apologies to those unmentioned others whose involvement is also greatly appreciated. I owe a special debt of gratitude to Donald P. McDonnell who provided not only valuable guidance and the opportunity to work independently, but who also gave me financial support throughout the course of this research. I would not have embarked on and completed this endeavor had it not been for his warm support and good counsel. For most of the research work period, I am also deeply grateful to Bert W. O'Malley for access to the facilities of his laboratories at Baylor College of Medicine in Houston, Texas. I thank him also for precious scientific advice and for his assistance in part of the work. I thank Tauseef R. Butt for the valuable time spent in his laboratory at Smith, Klein, and Beecham Laboratories in Philadelphia, and for kindly providing the ubiquitin fusion technology. I am also deeply grateful to Don W. Smith for his understanding, kindness, and for having encouraged me throughout my graduate studies. As some of the work presented in this study was done with the support of other members of the laboratory in Houston, I would also like to express my thanks to them: iii West Pike for kindly providing vitamin D3 antibodies; Ahmed Usman for helping with the bandshift experiments using E. coli expressed estrogen receptor; Geoff Green for kindly providing the estrogen receptor cDNA and estrogen receptor antibodies; Charles Densmore for helping with the Scatchard analysis and calf thymus DNA-cellulose chromatography of yeast expressed estrogen receptor; Nancy Weigel for assisting with immunoprecipitation analysis of estrogen receptor; John Elliston and Salman Haider for helping with transfections and CAT assays; Tony Pham for helping with in vivo footprinting; Suzanne Fuqua for kindly providing the variant receptors cloned from human breast tumors; and Paula Howard for skilled technical assistance. I thank the members of my thesis committee for their advice and support during the course of this study: Tauseef R. Butt, John Knesek, Donald P. McDonnell, Gerard A. O'Donovan, Mark S. Shanley, Don W. Smith, and G. R. Vela. All this immense help from those mentioned and from unmentioned others is gratefully acknowledged. Houston, Texas May, 1992 iv TABLE OF CONTENTS Page LIST OF TABLES ................................................ x LIST OF FIGURES ............................................... xi Chapter I. INTRODUCTION .......................................... 1 General Aspects of the Steroid Receptors ...................... 1 Structure and Functions of Steroid Receptors ..................... 3 DNA-binding domain .............................. 5 Steroid receptor DNA-binding sites ..................... 9 Steroid-binding domain ............................. 11 Transcriptional activation domains ..................... 13 Steroid Signal Transduction ............................... 15 Effect of hormone on DNA binding .................... 16 Effect of hormone on transactivation ..................... 17 Recent Developments .................................... 17 II. MATERIALS AND METHODS .............................. 19 Materials ............................................ 19 Bacterial Strains ....................................... 20 Yeast Strains ......................................... 20 Cell Lines and Human Tumor Specimens ..................... 20 DNA Constructions ..................................... 21 Bacterial expression vectors ......................... 21 Yeast expression vectors ............................ 22 V Yeast reporter vectors .............................. 24 Mammalian expression and reporter vectors.......... ............................. 25 Induction of Recombinant Proteins in Bacteria ................... 26 Western Immunoblotting of Proteins Expressed in Bacteria ........................................... 27 Hormone Binding Determinations of Bacterial Expressed Proteins ....................... 27 Hormone-binding assays of vitamin D receptor .................. 0.....6...... .27 Hormone-binding of estrogen receptor......................... 28 DNA-binding Properties of Bacterial Expressed Proteins .............................. ....28 DNA-binding properties of vitamin D receptor......................... ......28 DNA-binding properties of estrogen receptor......................... ...0.4.0.0.0.0.29 Induction of Estrogen Receptor in Yeast and Preparation of Yeast Extracts ................ 29. ....* Preparation of MCF-7 Estrogen Receptor ..................... 30 Hormone-binding Analysis of Estrogen Receptor from Yeast...........................................30 DNA-binding Analysis of Estrogen Receptor from Yeast...........................................31 vi DNA-cellulose chromatography.......................31 Band shift assays.................................31 Western Blot Analysis of Estrogen Receptor from Yeast...........................................31 Antibody Immunoprecipitation of Estrogen Receptor ........................................... 32 Immunodot Analysis of Estrogen Receptor .................... 32 Assay of Estrogen Receptor Function in Yeast .................. 32 Construction and Propagation of (SRE)n Library and Development of the Screen for Identification of SREs .................................. 33 Transient Transfection Assays .............................. 33 III. RESULTS ............................................. 34 Development of Bacterial Expression System ................... 34 Expression and Biochemical Characterization of Vitamin D3 Receptor ................................. 34 Expression and Biochemical Characterization of Estrogen Receptor .................................... 40 Development of Saccharomyces cerevisiae as a Host System for Steroid Hormone Action Studies........................................... .. 45 Production of Mammalian Steroid Hormone Receptors in Yeast* ...................................... 46 Biochemical Characterization of Yeast Expressed Estrogen Receptor ............................. 50 v11 Reconstitution of Steroid Hormone Responsive Transcription Units in Saccharomyces cerevisiae ............................................ 59 Applications of Saccharomyces cerevisiae System............................................67 Effect of antiestrogens on the regulation of estrogen receptor ......................... 67 Identification of novel estrogen response elements.......................................75 Study of variant receptors cloned from breast tumors .................................... 84 Regulation of osteocalcin gene by estrogen receptor .................................. 99 IV. DISCUSSION.........................................107 Bacterial Expression System........................... .. 107 Development of Saccharomyces cerevisiae as an Expression System ................................. 110 Development
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