PIERRE CHAMBON, ANDRÉE DIERICH, MARIE-PIERRE GAUB, SONIA JAKOWLEV, JAN JONGSTRA, ANDRÉE KRUST, JEAN-PAUL LEPENNEC, PIERRE OUDET and TIM REUDELHUBER,     Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Unité 184 de Biologie Moléculaire et de Génie Génétique de l’INSERM, Institut de Chimie Biologique, Faculté de Médecine, Strasbourg, France

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This chapter discusses the promoter elements of genes coding for proteins and modulation of transcription by estrogens and progesterone. The modulation of gene expression by steroid hormones offers a number of useful model systems to study how the expression of a given gene or a set of genes can be positively regulated at the transcriptional level in animal cells. It is generally thought that the effect of steroid hormones is mediated by hormone-specific intracellular receptor proteins that associate with specific DNA or chromatin sites upon binding the hormone ligand. It is assumed that this receptor-genome interaction is the primary event in the induction of transcription of a gene programmed to respond to a given steroid hormone in a given differentiated responsive cell. As the first step in initiation of transcription of a given gene is the binding of the transcription machinery to the specific DNA elements that constitute the gene promoter, it is further assumed that the hormone-receptor complex facilitates this binding by interacting with a specific region located in the vicinity of the gene. Evidently, the study of the validity of such proposals requires a previous knowledge of the structural and functional organization of the promoter region of the gene in question and, more generally, of promoters of animal genes coding for proteins. The chapter presents the current knowledge of the organization of such promoters, with a special emphasis on some studies that may be particularly relevant to steroid–hormone action.

I Introduction

How is gene expression controlled in eukaryotic cells? This question is at the heart of problems and questions which are obviously not new, but their study, at the molecular level, constitutes the new “frontier” in biology. What is the molecular basis for cell determination which leads to the development of embryos? Why is a gene active in one cell, and inactive in another? And how is this imprint of genes maintained stably even after cell division? What are the molecular explanations for differential regulation of gene expression in the differentiated cells? What is turning on the expression of “committed” genes? How could variations in hormonal, nutritional, and even environmental parameters control gene expression? There is no doubt that the answers to these fundamental questions will be of great interest to all biologists, but in addition, it is certain that they will have important consequences in medicine.

It is clear that our picture of the molecular anatomy of genomes of higher eukaryotes, and of how it functions, has changed dramatically over the last 10 to 15 years. In this regard it is interesting to note that of the many new, and sometimes amazing, findings, few have been the result of elaborate theories. Rather, as has been the case in many scientific pursuits, the discoveries have been made possible by the development of powerful new methods. This should not surprise us, because not knowing the molecular logics of evolution, it is impossible to predict how the genome is organized and how it functions. Who could have predicted the existence of split genes? Thanks to the advent of recombinant DNA technology, DNA sequencing, and a number of other related techniques, genes from almost any organisms can now be isolated and made accessible for direct manipulation. A new approach has become possible, aimed at purifying genes of known function, and ultimately at reconstructing the necessary molecular environment in a test tube. Classical in vivo genetics has been replaced by in vitro genetics (also called genetic biochemistry, surrogate or substitutive genetics), in which the study of the function of a given gene involves first its cloning, then its modification by site-directed mutagenesis, and finally its introduction into a proper cellular environment by DNA transfer with the possible help of appropriate vectors.

Transcriptional control is clearly the principal mechanism for regulation of gene expression in prokaryotes (Rodriguez and Chamberlin, 1982). Although it is today widely accepted that control of most, if not all, eukaryotic genes occurs at the transcriptional level, it is worth recalling that not too long ago the opposite view, namely that of no stringent control of gene expression at the level of transcription, was dominant. Because of the results of some DNA–nuclear RNA hybridization experiments, some people in the field thought that gene expression does not need to be as precisely regulated at the level of transcription in eukaryotes as in prokaryotes! Although I share the general belief that in eukaryotes most, but not all, of the regulation in gene expression is transcriptional, it is important to stress that there are only a few genes for which it has been unequivocally demonstrated that their regulation is indeed at the transcriptional level, and there are well-known examples of posttranscriptional regulation at the RNA processing and translational levels. In this respect, there may be surprises. We certainly should keep an eye open and not be too dogmatic.

Control of the expression of genes transcribed by RNA polymerase class B (II) is obviously of prime importance (which does not mean that other genes, such as tRNA, 5 S, and ribosomal genes are not important), because most of them code for proteins and some of them must play key roles in development. If we believe that the regulation of transcription is one of the crucial mechanisms operating during development, the first step in its study is clearly to determine how genes are turned on and off, and in particular to study how initiation of transcription is controlled at the molecular level. Investigating how a given gene functions in a differentiated cell will eventually lead to the identification of molecules important for its action and, then, by tracing them back during development, to the elucidation of the molecular nature of this process.

The modulation of gene expression by steroid hormones offers a number of useful model systems to study how the expression of a given gene or a set of genes can be positively regulated at the transcriptional level in animal cells (for refs. see Anderson, 1983). It is generally thought that the effect of steroid hormones is mediated by hormone-specific intracellular receptor proteins that associate with specific DNA or chromatin sites upon binding the hormone ligand (Yamamoto and Alberts, 1976; Mulvihill et al., 1982; Schrader et al., 1981). It is assumed that this receptor–genome interaction is the primary event in the induction of transcription of a gene programmed to respond to a given steroid hormone in a given differentiated responsive cell. Since the first step in initiation of transcription of a given gene is the binding of the transcription machinery to the specific DNA elements which constitute the gene promoter, it is further assumed that the hormone–receptor complex facilitates this binding by interacting with a specific region located in the vicinity of the gene. Evidently, the study of the validity of such proposals requires a previous knowledge of the structural and functional organization of the promoter region of the gene in question, and more generally, of promoters of animal genes coding for proteins.

The purpose of this article is to first review the current knowledge of the organization of such promoters, with a special emphasis on some of our studies which may be particularly relevant to steroid hormone action, and then to describe some of our recent work directly pertinent to the problem of regulation of transcription by estrogens and progesterone.

II Promoter Elements of Genes Coding for Proteins in Eukaryotes

In prokaryotes initiation of transcription is controlled at specific DNA regions, called promoters. Promoters were first defined by Jacob et al. (1964) primarily on genetic evidence, as cis-acting initiating regions indispensable for the expression of bacterial structural genes. Subsequent in vivo and in vitro biochemical studies have demonstrated that prokaryotic promoters are DNA regions, 5′ to the structural genes, which can be comprised of multiple functional elements (Rosenberg and Court, 1979; Losick and Chamberlin, 1976; Rodriguez and Chamberlin, 1982; Siebenlist et al., 1980; Fig. 1). A prokaryotic promoter region must contain a basic element required for recognition, binding, and RNA chain initiation by RNA polymerase. This element includes the mRNA startsite (consensus sequence CAT), the AT-rich Pribnow or Schaller box sequence located approximately 10 bp upstream from the startsite (consensus sequence 5′−TATAAT−3′), and very often a third region of homology, the “recognition”...