Summary Steroid hormone receptors are ligand-inducible transcription factors that exhibit potent effects on gene expression in living cells. Precise dissection of their mode of action at the molecular level can best be carried out in functional cell-freesystems. This article will describe the benefits of such systems and review their development up to the recent establishment of steroid receptordependent in vitro transcription. Subsequent advances in our knowledge of receptor function arising from the exploitation of this powerful experimental tool will be described. Particular emphasis will be placed upon two key problems: the role of steroid hormone in receptor action and the mechanisms by which steroid receptors activate gene transcription.

Introduct io n The processes of cell growth and differentiation are controlled by complex patterns of expression of a multitude of regulatory genes. Gene expression in any cell is subject to modulation by extracellular signals, signals which generally initiate a cascade of intracellular biochemical reactions leading ultimately to the activation of a specific set of genes. The products of at least some of these genes will, in turn, activate other genes. H Hormone

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-fi

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Hormone binding

These products can thus be termed transcription factors. which are DNA-binding (or sometimes nonDNA-binding) proteins that are associated with the flanking control sequcnces of all genes and have the capability of activating, enhancing or repressing transcription by RNA polymerase. It is the pattern of expression and susceptibility to extracellular modulation of thcse factors that ultimately accounts for the diversity of developmental- and tissue-specific gene expression. Steroid hormone receptors are paradigmatic of this whole class of ligand-inducible transcription factors and have been the subject of intensive research since long before the term ‘transcription factor’ came into use. They constitute an extendcd family of tissue-specific or ubiquitous factors known as the ‘steroid/thyi-old hormone receptor superfamily’. each member of the family being related both functionally and at the structural level. Steroid hormones exert their potent physiological effects by binding to these receptors intracellularly . Hormone binding induces a poorly understood ‘activation’ (or ’transformation‘) of the receptor. allowing it to dimerize and recognize specific binding sites in the flanking regions of hormoneresponsive gcnes. Binding to the DNA recognition site results in alteration of the rate of transcription by an unknown mechanism. This scheme (summarizcd in Fig. 1) is generally applicable to thc steroid receptor superfamily, but may not necessarily apply to all members. Certain receptors (such as that for thyroid hormones) can bind to their recognition sequences without a requirement for hormone and thereby act as gene repressors. However, this review will concentrate on more general aspects of reccptor action: readers interested in systems such as that for the thyroid hormone are referred to Ref. I and references therein. Recent technical advances have made substantial progress in filling the blank spaccs suggested by the scheme in Fig. 1. The most important advance has becn the development of steroid receptor-dependent in vitro u u

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Receptor

activation

Receptor

DNA

binding Fig. 1. Schematic vicw of the main ccllular

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events involving steroid hormone receptors. Thc occasional instancc of negative regulation by steroid receptors is not rcyresented here, or detailed in thc text. but thc basic mechanisms of positivc and negative gene activation may be similar. SRE: sleroid rcsponse element. +1: site of transcription initbation.

transcription. This has allowed analysis of the mechanisms by which receptors are activated prior to DNA binding and by which receptors control gene expression in closer detail than has previously been possible. Steroid Hormone Receptors and Steroid Response Elements: What Molecular Cloning Allowed Us to Find Out The cloning of hormone-responsive genes and the complementary DNAs encoding almost all known steroid hormone receptors has been followed by an explosion of information during the mid- to late-1980s on the structure and mechanism of action of these proteins (sec Ref. 2 for a more comprehensive review than is possible here). Comparison of amino acid sequences and the analysis of engineered receptor mutants has led to the dcfinition of discrete structural domains involved in DNA recognition and hormone binding. Other activities, notably thc transactivation function (required for activating gene expression), are not localized to separate domains but are instead associated with small stretches of sequence scattered throughout the polypeptide. All members of the steroid receptor superfamily have the same basic structural organization, but individual members differ significantly in the degrec of sequence conservation in different regions of the protein. Briefly, the typical rcceptor consists of three major domains. The most N-tcrminal is hypervariable both in size and sequence, and its exact role in receptor function is unclear, although it is at least partly responsible for determining efficient transactivation. A central DNA-binding domain is highly conserved and possesses two cysteinc-rich 'zinc fingers'(')), motifs which are required for binding to the DNA helix. A third moderately conserved ligand-binding domain occupies the C-terminus of the protein. Two wellconserved sub-regions within this domain may form nonspecific contacts with the steroid hormone or other tran,r-actink factors within the folded polypeptide structure(' . Although fruitful, further information on receptor action obtainable using this structure-function approach will probably await the development of convcnient crystallographic analytical methods, Analysis of the promoter regions of steroid-responsive genes by transient transfection assays of mutated constructs and by in vifvo binding studies has revealed the existence of sequence elements that mediate hormone inducibility and which are bound by highaffinity receptor. Consensus sequences have been defined for the steroid response elements (SREs) mediating induction by glucocorticoids (GREs), estrogens (EREs), thyroid hormones (TREs) and retinoic acid (RARE)('). SREs consist of two closely related or identical copies of a short sequence motif; the two copies are arranged either immediately adjacent to each other or with a short space between and are either directly repeated (RARE) or dyad symmetrical (all other SREs). Although no distinct elements have been

found for progestins or androgens, both of these hormones are capable of induction via the G R E (also called GRE/PREs for this reason)(@;the receptors for glucocorticoids and progesterone make similar contacts with individual nucleotides in the response element"). While the experimcn tal approaches mentioned above, particularly those based upon in vivo analysis in cultured cells, have yielded a great deal of information on receptor function, their applicability to the study of more detailed mechanistic aspects is limited. For example, questions Such as the role of post-translational modification in receptor function or the way in which receptor cooperates with other transcription factors to alter gene expression are difficult to address within the compartmentalized environment of the living cell. An in vitro approach, utilizing cell-free extracts or purified proteins, simplifies such problems and allows one to analyze the role of individual components in a reconstituted system. The first useful cell-free transcription systcms were established in the late 1970s. Since then intensive efforts have been made in a number of laboratories to develop a steroid receptor-dependent soluble transcription system. These efforts have just recently proven successful. Developing Receptor-Dependent Cell-Free Transcription Systems Earliest attempts to reproduce accurate transcription from eukaryotic promoters in the test tube utilized purified preparations of RNA polymerase derived from sources ranging from yeast to man(8). Although these preparations yielded RNA transcripts upon incubation with purified DNA templates. none of these transcripts rewlted from specific initiation at a cellular promoter. Instead they arose from random initiation events occurring following binding of the purified polymerase to nicks in the DNA template, an activity that has been found to be characteristic of this enzyme. This indicated that the determinants of in vivo specificity were lacking in purified systems, and that cruder extracts would have to be employed in order to obtain transcripts from deproteinized DNA. It was hoped that the initial establishment of such systems would then allow systematic reconstitution of specific transcription using more purified components. The major breakthrough was made in 1978 with the demonstration by Wu that 5.5s R N A could be synthesized from purified adenovirus DNA in the presence of a crude extract from human KB cells("). The in virvo-produced transcript was identical to that made in vivo and its synthesis was dependent upon RNA polymerase 111. The establishment of this powcrful technical tool allowed study to begin on the general and gcne-specific factors required for the transcription of eukaryotic gcnes. Within two years, specific transcription of a number of class 111 genes (genes which are transcribcd by RNA polymerase I11 and encode small nontranslated RNAs, including SS and tRNA genes) had been reproduced in Brown and

Roeder's laboratories using extracts from Xenopus laevis (frog) oocytes and mammalian cells@).Fractionation of Xenopus extracts led to the identification of a number of factors required for transcription of the SS gene, one of which, TFIIIA (for 'class 111 transcription factor A'). was the first transcription factor to be extensively purified and characterized("). The establishment of cell-free transcription of protein-coding (class 11) genes soon followed. Weil et al. (I1) demonstrated specific transcription of adenovirus 2 genes in crude KB cell extracts supplemented with purified RNA polymerase 11. Subscqucntly. Manley et aZ.(") showed that class TI in vitro transcription could be supported by the endogenous RNA polymerase present in whole-cell extracts of human HeLa cells. Nuclear extracts from these cells were even more potent in transcribing purified DNA templates("). The availability of this simple and rapid technology (see Fig. 2) at last made it feasible to reconstitute the cellular regulation of any gene in the test tube. Similar crude extracts were developed for the transcription of genes derived from lower eukaryotes such as Drosophilu, fungi and yeast (see Ref. 14 and references therein). Numerous viral and cellular genes have been DNA template

Whole cell extract Crude system Nuclear extract Purified proteins

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1 Transcript detection: (1) Direct (run-off or G-free)

(2) Indirect (SI nuclease analysis, primer extension or RNase protection)

Fig. 2. Design o f a typical in vitvo transcription experiment. Purified DNA templates (circular or linear) are incubated at 20 to 30°C under the appropriate buffer and salt conditions with RNA polymerase. the general transcriptional machinery and any specialized gene regulatory factors (such as steroid receptor), all of which are provided in a crude cellular extract and/or ac: a purified preparation. Transcription, which requires ribonucleotides, is allowed to proceed for 4S to 60 min. Following termination of the reaction. transcripts are purified, elcctrophoresed under denaturing conditions. and detected either directly (if radiolabelled) or indirectly (using radiolabelled probcs).

shown to be activatable in these extracts. In addition, tissue-specific regulation of more specialized genes, such as those encoding fibroin, globin and albumin, has been attainable using extracts from differentiated cells. Considerable progress has been made both in delimiting the DNA regulatory regions required for cell-free gene expression and in identifying the DNA-binding transcription factors responsible for this regulation. Furthermore, the identification of genenl transcription factors required for accurate initiation by purified RNA polymerase I1 on a TATA box-containing t e m ~ l a t e ( ' ~ . ' ~ ) has led directly to the development of reconstituted transcription systems composed solely of purified

component^('^). Despite these successes, the reconstitution of steroid hormone receptor-regulated gene expression in vitro proved considerably more difficult to attain. Although the classic steroid-regulated chicken ovalbumin gene was transcribable in human cell transcription was not subjcct to modulation by exogenous steroid receptor or by hormone treatment of the homologous chick oviduct extracts (unpublished data). Given the apparent complexity of the regulation of this gene and the lack of unambiguous binding sites for steroid receptors within its promoter, this failure to reproduce its complex and tissue-specific regulation outside the cell is now perhaps not surprising. Indeed, it was the detailed characterization of a large number of steroid-responsive genes, and particularly the resulting identification of SREs, which was crucial to the eventual establishment of steroid receptor-dependent transcription in a soluble system. It had been known for some time that crude receptor preparations were capable of influencing RNA polymerase activity in isolated nuclei, as assessed by the detection of increased trichloroacetic acid-precipitable counts following incubation of both in the presence of labelled uridine monophosphate(20). The initial instance of efficient RNA synthcsis stimulated by purified receptor came in 1986 with the chicken ovalbumin upstream promoter transcription factor (COUP-TF) acting on ovalbumin control sequences(*'). although this was only realized in hindsight with the identification of COUP-TF as a member of the stcroid receptor superfamily('). The first hint of success with well-characterized receptors came in 1988, when estrogen-dependent activation of a vitcllogenin promoter in homologous Xenopus laevis liver extracts was reported(22). However, while transcription was clearly dependent upon the presence of EREs in the template, it was not rigorously demonstrated that activation was mediated by the estrogen receptor itself. Transcription based upon purified receptor preparations was first shown with bacterially ex ressed fragments of the rat glucocorticoid receptora')_ then with the native chicken progesterone receptor(24). The basic transcriptional apparatus was provided in these systems by crude extracts from heterologous Drosophilu or HeLa cells, respectively. Activation was found to be attainable both

from exceedingly simple promoter templates, consisting of SREs artificial1 linked to a TATA-containing minimal promoterc2', '): and from more complex natural promoters, such as the mouse mammary tumor virus (MMTV) promote^-(^^,^'.*^). With the almost simultaneous development of thcsc crude transcription systems in several laboratories, the way was open to addressing a number of the questions that have long eluded those working in this field. Two problems, upon which new progress has already been made, will be discussed in the remainder of this review: the role of steroid hormone in receptor function and the role of interactions between the receptor and other transcription factors during gene activation.

Y

Ligand and Receptor It has always been assunled that, in the living cell, the role of hormone in steroid-dependent gene regulation is one of conversion of the receptor from an inactive to an active form. Functionally, the distinction between these two receptor forms is that one is, and one is not, capablc of turning on (or off) target gene expression. In general terms, this would imply that receptor activation proceeds by an uncomplicated, probably one-step, mechanism. However, reproduction of this process in vitro, where its details can be finely studied, has been a far from straightforward task. In fact, probably the only commonly accepted conclusion to arise from such studies to date has been that receptor activation is not simple, but is in fact a complex multistep process. Many steroid receptors can be isolated from tissue or cells as high molecular weight complexes with a sedimentation coefficient of 8 to 10s (the so-called 8s complex). In addition to the receptor, thcse complexes contain a variety of other protein moieties, including 90 and 70 kiloDalton (kDa) heat shock proteins (hsp90 and hsp70, respectively) and other uncharacterized proteins of 60 to 20kDa(Z7p2')).This complex can be partially dissociated to a smaller, 4-5s complex by a variety of in vitro treatments, including high salt and, significantly, hormonal administration at high temperatures. Similarly, receptor treated with hormone in vivo can be isolated as a 4s Functional studies have indicated that the 4s complex is distinct from the 8s in that it is able to bind to DNA(3"). These findings have led to the suggestion that at least one role of hormone ligand is dissociation of the 8s complex and Conversion to the 4s form. Consistent with this idea, some experimental data exist showing that crude unfractionated receptor binds to its cognate res onse element in a hormonedependent manner(-n,-3'), while other studies have shown that purified or even partially purified receptor has lost hormone-dependenc and can bind to DNA in the absence of ~ t e r o i d ( " ~ Furthermore, ~~. purified progesterone receptor is able to activate gene transcription in vitro without prior hormone treatme&'). This would indicate that partial or complete removal of the associated proteins from the 8s receptor (by hormone in living cells or during purification) is sufficient for

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activation. The role of hormone would thus be localized to some early step prior to DNA binding. The nature of this step is unclear. Although dissociation from XS to 4s is necessary for receptor activation, it is probably not sufficient. This is because some steroid receptors (including the vitamin D and thyroid hormone receptors) are not likely to be associated with heat shock proteins or even present in an 8s form prior to exposure to hormone. More significantly, recent data obtained using the ccll-free transcription system established in this laboratory has shown the existence of an intermediate 4s complex prior to receptor activation(33). We have shown that crude progesterone receptor isolated from salt-treated human breast cancer cell nuclear extracts exists in a 4s form. This 4s receptor is unable to bind to GRE/PREd3") and is incapable of activating a GRE/PRE-containing promotcr in the in vitro transcription system unless it is treated with progesterone. Therefore, although it remains possible that hormone dissociates the 8s complex in vivo, this alone is not sufficient for receptor transformation. One current hypothesis is that the critical part played by the hormone lies further downstream, possibly in further dissociation of inhibitory proteins from the 4s complex (Fig. 3(A)). Alternatively, or simultaneously, the hormone may be required to place the receptor in a conformation amenable to modification by other factors, such as protein kinases. Steroid receptors are known to be phosphoproteins, although the functional relevance of phosphorylation has yet to be determined. It is likely that the primary mechanism of activation could also be an allosteric one, with hormone binding itself inducing a conformational change in receptor structure that leads to the active form. From this viewpoint, the other processes described above would be of secondary relevance to activation, or would have alternative functions. The resolution of thcse separate, not necessarily mutually exclusive mechanisms awaits further study. Cooperation between Steroid Receptors and Other Transcription Factors Once activated and bound to the control region of a hormone-responsive gene, how do steroid receptors regulate the expression of that gene? Of the possible mechanisms, the most direct, and best studied, would be transcriptional control, whereby the rate of initiation of RNA synthesis by thc polymerase would be modulated by the receptor dimer located some distance away. Finding out how this might occur is one possible application of the receptor-driven cell-free transcription system. A direct consequence of the initial establishment of RNA polymerase II-dependent cell-free transcription was the identification and purification of a number of general transcription factors (namely TFIIA, TFIIB , TFIID, TFIIE and TFTIF) which are essential for RNA synthesis by the p ~ l y m e r a s e ( ' ~ Each ~ ' ~ ) .of these factors

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Fig. 3. Proposed model for (A) receptor activation by hormone and (B) transcriptional activation by activated receptor. (A) The inactive XS receptor complex is transformed to an inactive 4s complex through the loss of some associated proteins, a process that may rcquire hormone. Subbequent activation ol the I S complex (Lo I S * ) is mediated by hormone, and may involve further dissociation of inhibitory proteins and/or receptor phosphorylation. (B) Steroid receptors promotc cnhanccd transcription (right-anglcd arrow) by facilitating assembly of or stabilizing the pre-initiation complex. Interactions are based upon protein-protein contacts (curved arrows) between rcccptor and target domains. Interactions may be ‘bridged’ by other bound transactivators, located for instance at the distal promoter (D.P.).

assembles at the gene promoter with the polymerase in an ordered step-wise manner to form a so-called preinitiation complex(35).The complete formation of this complex is a prerequisite to subsequent initiation, and at least some of the general factors are likely to remain associated with the polymerase during elongation. Naturally, any mechanism which enhances the rate of assembly of the initiation complex or increases its stability on DNA would bc likely to result in an increased rate of transcription, and hence in increased gene expression. Steroid receptors, then, could activate by interacting with other transcription factors in order to promotc or stabilize complex assembly. Such interactions would presumably be mediated by protein-protein contacts between particular regions of the receptor (most likely the transactivation domains identified by mutational

analysis) and one or more targct domains in the preinitiation complex. Direct evidence for such a mcchanism has been obtained by template commitment assays in the in vifro transcription system(’”). Under conditions limiting for the general transcriptional machinery, expression from a GRE/PRE-containing minimal promoter which had been preincubated with transcription factors and progesterone receptor was found to be considerably greater than transcription from a second identical template which had not been preincubated. When receptor was omitted from the preincubation, transcription was the same from both templates. This indicated that receptor promoted the assembly of all available factors on the preincubatcd template, and that, once formed, the resultant complexes were stable to challenge by the second template. This was strong evidcnce for initiation complex stabilization by DNAbound progesterone receptor, evidence which we have extended recently to other receptors(’6). The promoter used in these studies was a skeletal one containing only a TATA box adjacent to the initiation site and the SREs, and it seems unlikely that, in nature, bound receptor will be present in such close proximity to the pre-initiation complex. In those steroidresponsive genes that have been characterized, SREs are positioned (with one notable exception(’)) between 100 and 1000bp from the initiation site and are adjacent to the binding sites for numerous other transcription factors. Under these conditions, direct interaction with the pre-initiation complex would be unlikely. Instead, receptors would interact in an indirect manner via other bound transactivators, which act as ’bridges’ between the two sites (Fig. 3(B)). Experimental evidence supporting this mechanism is reflected in the numerous descriptions of functional synergism between SREs and heterologous binding sites defined by transient transfection analysis in cell and, morc recently, using receptor-dependent cell-free transcription(39). In at least one case (activation of the chicken ovalbumin promoter by COUP-TFt2’)), evidence exists for the presence of non-DNA-binding bridging proteins during receptor-dependent gene transcription. The occurrence of these protein-protein interactions depends upon both the availability of the bridging transactivator within ~ c l l s (and ~ ~ upon ) the identity of the transactivator binding to a test promoter in v i t ~ o ( The ~ ~ )control . of steroid-dependent gene expression will therefore depend not only upon the recognition of specific binding sites by the activated receptor, but also upon the complexity of the interactions the receptor makes with other gcne regulatory factors following binding. This complexity could at least partly underlie the sensitivity and potency of hormone action in nature. Conclusions and Prospects The analysis of steroid receptor function in the controlled and quantitative manner made possible by in virvo transcription has opened the door to solving a number of long-standing problems in this field. In little

ovcr a year, important advances have already becn made. In addition to future possibilities along the lines outlined in this review, other areas are now amenable to exploration. One area of concern is the role of phosphorylation in receptor action. Another relates to the part played by chromatin in mediating steroid responsiveness, a part that may be less passive than has often been assumed. Although in vitro approaches will not answer these questions by themselves, in conjunction with well-established in vivo techniques, they should allow an cxplosion of fruitful research into the molecular mechanism of steroid receptor action over the next fcw years.

Acknowledgements We are grateful to Lisa Gamble for helping in manuscript preparation. References 1 BANIAHMAD. A . , STIEINLK, C.. KOHNE,A. C. AND REKKAJVIIZ. R. (1990). Modular structure oC a chicken lysozyme sil thyroid hornionc receptor binding site. Cell 2 EVANS,R. M. (1988). The sieroid and thyroid hornionc receptor huperfamily. Science 240. 889-895. 3 MIILER. J.. McLmILm, A. D . APID KLUG,A. (1985). Repetitive zincbinding domains iii the protein transcription iactor IIIA from Xenopw o o q t e s . EMBO .I. 4, 1609-1614. 4 WANti. L.-H., TSAI.s. Y., COOK. R. G., BEATTIL. W. G.. T&\l. M.-J. AND O'MALLEY, B. W. (1989). COUP transcriptinn facior i\ a member of the steroid receptor superfamily. ,Yamre 340. 163-166. 5 DE THO.H.. DOLMARVIVANCO-RCIZ. M.. 71o1.1.m.P.. STUNYENBERG. H. .4ND DLJE.AK. A. (1990). Identitication of a retinoic acid rcsponsivc clement in the retinoic acid receptor /3 gene. rvorrire 343. 177-180. 6 HAM,J.. THOMSOK, A., NEDDHAM, M.. WEBB,P. .4ND PAKK:~X: M. (1988). Characterization of response elemenls lor androgen,, glucucorticoids and progcstins iii mouse mammary turnour virus. Niccl. Aridr Re>.16, 5263-5277. 7 CHALEPAKIS, G.. ARNEMANN. J.. SLATER, E.. BRUILER, H.-J., GROSS, B. AND UEATO, M. (1988). Differential gene activation by glucocorticoids and progestins through the hormone regulatory element of mouse mammary tumor IRCESS, R . R . (1982). Eukaryutic RNA polymerases. In The E n q w m c . vol. XV (cd. P. D. Boycr), pp. 109-1.53. Academic Press. New York. 9 WLI.C;.-J. (1978). Adenovirus DNA-directed transcription of 5.5s RNA iir vitro. Pro