Gene Regulation By Steroid Hormones Ii

Receptors and Gene Expression It is now more than three years since the last Meadow Brook Conference on Hormones, and a great deal has happened in the meantime. We have become comfortable with the totally unanticipated fact that the coding sequences of genes are in discontinuous arrangements and that the RNA transcribed from them must be extensively processed to form messenger RNA. We have also learned about the strategy of "mixing and matching" of genetic segments so that a small amount of DNA can go a long way in producing a huge variety of different proteins, as in the immunoglobulin system. The explosive effort directed toward DNA sequence analysis has led us to the conclusion that there are signals within the DNA that specify sites of transcription initiation and possibly sites for interacting with regulatory molecules such as hor mones and their receptors. The current intense interest in the structure of chromatin beyond the nucleosome-that is, the superstructural characteris tics of the genetic material-is finally yielding meaningful results that give promise for understanding the regulation of gene activity. ROBERT F. GOLDBERGER Preface Research on the molecular mechanism of steroid hormone action continues at an extraordinary pace and a great deal of progress has been made. Steroid hormones have been localized on target genes providing the long awaited evidence for the concept of a direct effect of the steroid-receptor complex on gene regulation. Purified steroid receptors have been dissected to identify different functional domains.

The first Meadowbrook Symposium was held in 1978 and during the intervening ten years our knowledge concerning how steroid hormones function at the level of gene expression has advanced by leaps and bounds. In this volume, which sum marizes our fourth meeting, these advances are very evident. What seemed like science fiction ten years ago has become commonplace science. Who would have imagined that we could synthesize a nucleotide sequence that binds a specific steroid receptor and acts as a controlling element for gene expression? No one; but as is evident from the results reported in several chapters, this technique is yielding a wealth of information. Using these and other techniques it has become apparent that gene transcription is controlled by interactions between transacting factors and DNA recognition sequences (response elements). These transacting factors appear to be members of a large gene family that includes steroid hormone receptors, transcription factors, protooncogenes and homeobox proteins. Thus a great deal has been learned, but as usual, questions remain. Many of these questions are posed by the findings and observations found in several chapters in this volume. Non hormone binding forms of steroid receptors and their relevance to receptor down regulation, recycling and biological response remain a mystery. The quantitative relationship between receptor binding and biological response still presents agonizing problems. These and many other intriguing questions are discussed in this volume and set the stage for what should be a most rewarding time in endocrinology. Winter 1989 ARUN K.

Within the last two decades endocrinological research has taken a definite tum toward biochemistry and molecular biology. This has resulted in a new discipline called' 'molecular endocrinology. " Studies on the mechanism of hormone action have continued to make headlines with fundamental discov eries in receptor action and gene regulation. Recently the insect endocrino logists have also begun to explore the molecular mechanism of steroid hor mone action taking advantage of the vast number of Drosophila mutants, the library of Drosophila gene, and several well-characterized cell-lines. The availability ofthe recombinant DNA technology has provided a truly revolu tionary tool in the hands of the molecular endocrinologists. "Gene Regula tion by Steroid Hormones" is compiled and presented in this frontier spirit, and we hope that this volume will serve not only the active investigators in the field but will also be very useful to students and researchers with a gen eral interest in regulatory biology. The book is an offshoot of the Conference on Molecular Mechanism of Steroid Hormone Action held at the Meadow Brook Mansion of Oakland University in the fall of 1978. We wish to acknowledge the financial assist ance from the National Science Foundation and Oakland University. The conferees will never forget the warmest hospitality of Dr. LOWELL EKLUND and his staff at the Meadow Brook center and we also wish to express per sonal gratitude to many of our students and colleagues for helping us to make the conference a great success.

The field of steroid hormone action has continued to expand into the realm of molecular biology at a pace even faster than most of us ever imagined. techniques of molecular biology have made it possible to clone The hormone-regulated genes and to examine steroid-receptor interactions with these genes. Nucleotide sequences of these genes, which show preferential binding of steroid receptors, have been identified. These results are complemented by the identification of chromatin acceptor proteins, which also show preferential binding for steroid-receptor complexes. Thus, one can envision the day when cloned genes, purified acceptor proteins, and receptor-steroid complexes will be recombined in vitro to form a functional unit. Cellular localization of steroid receptors has undergone recent revision, and it now appears that receptors are localized primarily in the nuclear compartment. These findings, although controversial, will lead to a reassessment of many of the previous concepts of steroid-receptor interactions and regulation. The way in which these observations at the of physiology, molecular and cellular levels fit into the overall scheme development, and evolution are continuing to progress, and the future promises some very exciting syntheses of understanding at all levels of biological organization. The third Meadow Brook Conference on hormones was held in order to bring together investigators who will undoubtedly contribute heavily to this future synthesis and to permit a free exchange of ideas and concepts as they relate to the current state-of-the-art in molecular endocrinology. Spring 1986 ARUN K. Roy JAMES H. CLARK Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

This text summarizes recent progress on the regulation of target gene transcription by steroid hormone receptors. Steroid hormones travel via the blood stream to thier target cells which they enter by diffusion. Steroid hormone receptors exist in these cells in inactive forms either in the cytoplasm or nucleus. Upon binding to their respective hormone ligands, the receptors go through a transformation step and become activated. The activated receptor can then activate transcription of hormone response genes.

The Second International CBT (Center for Biotechnology) Synposium was held in Stockholm November 4-5 1988 and had the title "The steroid/thyroid hoITIDne receptor family and gene regulation". This meeting contained contributions from most leading laboratories within the field of steroid/thyroid hOITIDne receptors and gave a very exciting perspective on the dynamic development of this i:ntx>rt. ant research field. Today all known steroid hoITIDne receptors have been cloned and sequenced and novel members of this supergene family are constantly being discovered. Sane of these new receptors remain to be characterized regarding the nature of their ligand. Access to probes for steroid receptors has enabled studies on mechanisms of regulation of receptor gene expression. Deletion and II'Altational analysis of steroid receptor cDNAs followed by expression in cells together with suitable reporter genes has yielded a detailed knowledge about the functional significance of the various domains the receptors are composed of. In certain cases, steroid resistance in patients have been shown to be due to point mutations in the corresponding steroid receptor genes resulting in non-functional receptors. The availability of receptor cDNAs also makes it possible to express receptors at high levels in procaryotic and eucaryotic cells. It is e. g. possible to express the DNA-binding domain of the glucocorticoid receptor in E. coli as a fusion protein with protein A which interacts specifically with DNA. Such studies are necessary for production of sufficient quantities of receptors to allow crystallization and X-ray crystallography for detailed structural information.

The past few years have witnessed the emergence of steroid hormones as the wonder molecules which generate as much discussion in the scientific literature as they do in a typical living room. This transition has been a result of the tremendous public and scientific interest in the normal functioning of the hor mones as well their suggested involvement in several clinical conditions. In the recent past, notable scientific and technological advances have been made in the areas of contraception and regulation of fertility. Steroid receptors are the indis pensable mediators of hormonal responses and are complex protein molecules which appear to exist in association with other, yet undefined, proteins and/or factors. Receptors for vitamin D, retinoic acid and the thyroid hormones share structural similarities with steroid receptors, and the roster of this superfamily is still expanding. While our knowledge of the diversity and magnitude of steroid effects has advanced, the precise mode of steroid hormone action has alluded investigators. This volume brings together an international team of prominent investigators who discuss their most recent work on the basic and clinical aspects of steroid/nuclear receptors. The contributions represent updated versions of the invited presentations made at The Second Meadow Brook Conference on Steroid Receptors in Health and Disease. I am grateful to my colleagues on the Scientific Committee: Etienne Baulieu, Jack Gorski, Benita Katzenellenbogen, David Toft and James WittJiff, who provided the vision and guidance in formulating an out standing program.