Transcription activation by estrogen and progesterone receptors.

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1991

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ESTROGEN AND PROGESTERONE RECEPTORS Hinrich Gronemeyer Laboratoire de Genetique Moleculaire des Eucaryotes CNRS, Unite 184 de Biologie Moleculaire et de Genie Genetique de I'INSERM, Institut de Chimie Biologique, Faculte de Medecine, 11, rue Humann, Strasbourg, France KEY WORDS:

hormone action, receptor structure-function, transcription enhancer, DNA binding, agonist-antagonist

CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . .

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HORMONE RESPONSE ELEMENTS (HREs) AND DNA-BINDING DOMAINS (DBDs) OF STEROID HORMONE RECEPTORS HAVE CO-EVOLVED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .

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The DNA-binding Domains of the Estrogen and Progesterone Receptors Include Sequences outside the Postulated "Zinc Fin/ier" Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Only Few Amino Acids Located in the N-terminal Finger CI of the ER are Critical for the Discrimination between an ERE and a GRE . . . . . . . . . . . . . . Similar Sequences can act as Response Elements for the Steroid, Thyroid and Retinoic Acid Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Estrogen and Progesterone Receptors Bind as Dimers to Palindromic Response Elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Accessory Factors are Required for DNA Binding in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cooperative Binding to Tandem Response Elements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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TWO AUTONOMOUS TRANSCRIPTION ACTIVATION FUNCTIONS (TAFs) OF ER AND PR ACT IN A PROMOTER- AND CELL-SPECIFIC FASHION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .

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A Constitutive and a Hormone-dependent TAF are Located within the N-terminal Region AlB (TAF-l) and the Hormone Binding Domain (TAF-2), Respectively, of ER and PR . . .... . . . . ..... . . .... . . . . . . ............... Cell and Promoter Specificity of TAF1 and TAF2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evidence for the Existence of Transcriptional Intermediary Factors (TlFs) . . . . . . . .. Estrogen, Progesterone and Glucocorticoid Receptor TAFs Belong to Difef rent .

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ISOFORM-SPECIFIC TRANSCRIPTIONAL ACTIV ATION . . . . . . . . . . . . . . .. . . . . . . . . .. . . . . . .

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ANTIHORMONE ACTION.........................................................................

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RU486 Induces DNA Binding of PR in vitro andin viv o and Acts as an Isoform- and Promoter-specific Agonist . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . Hydroxy-tamoxifen is a "Mixed" Agonist/antagonist. because hER TAF-2 is Inactive in its Presence and TAF-J Activates Transcription in a Cell- and Promoter-specific Fashion . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . Does the Estrogen Receptor "Constitutively" Activate Transcription in the Absence of Ligand? . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... ... ... Evidence for Antihormones that do not Induce DNA Binding of the Corresponding Receptor . . . . . .. . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . OLD OPEN QUESTIONS AND NEW PERS PECTIVES.. . . . . .. . . . . .. . . . . . . . . . . . .. . . . . . . . . . . Role of the Ligand. . . . . . . . . . . . . . . . . .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TAFs. TIFs and Other Factors . . . . . . . . . . . . . .. . . . . . . . . .. .. .. A ntihormone Action . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . .. . . . . . . . .. . . . . . . . . . . Structural Basis of Receptor Activity . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONCLUSION . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,.................... . .


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INTRODUCTION In contrast to the other major signal transduction pathway that relies on the presence of receptors anchored in the cytoplasmic membrane, both the es trogen (ER) and progesterone (PR) receptors I are located in the nucleus in the absence of ligand [due to the action of specific nuclear targeting signals (55, 113, 114)] and act by modulating the transcription of target genes in response to binding of the cognate hormone (Figure 1). Although aspects of the molecular model of steroid hormone action (46, 175), such as the cytoplas mic-nuclear translocation, have been somewhat modified recently, the basic concept of signal transduction into the nucleus of target cells, where hormone receptor complexes regulate the transcription of specific gene networks , has been amply confirmed. The identification of a family of nuclear receptors , including those for steroid and thyroid hormones, and retinoids (11, 36, 48, 49) , has extended this concept of signal transduction, obviating the need for endocrine production of the ligand, and demonstrated that this signaling pathway plays a major role in embryonic development, differentiation, and homeostasis. Members of the nuclear receptor family are now known for which only artificial (66) or no ligands (the so-called "orphan receptors") have been identified (41, 61, 76, 99, 106, 129, 144, 165, 166). Several orphan receptors have been cloned from Drosophila (39, 62, 107, 110, 115, 128, 140, 141), indicating the evolutionary conservation of signal transduction via nuclear receptors. '

The following abbreviations are used: ER, estrogen. GR, glucocorticoid, PR, progesterone

and

RAR, retinoic acid receptor; HRE. hormone response element; ERE, estrogen, ORE

glucocorticoid, PRE, progesterone response element; DBD, DNA binding and HBD, hormone hinding domain; NLS, nuclear localization Signal; TAF, transcription activation function; AAD,

acidic activation domain; DBSF, DNA-binding stimulatory factor; EXAFS, extended X-ray "h",ni(lll fine ,truclurc; NMR. nuclear magnetic resonance; CD, circular dichroism.

ESTROGEN AND PROGESTERONE RECEPTORS

91

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I CVTOPlASM I ENHANCER FACTORS AS SIGNAL TRANSDUCERS Figure 1

Two major signal transduction pathways in higher organisms utilize enhancer factors as signal transducers. Growth factors , neurotransmitters, and peptide hormones act via in

termediary membrane-bound receptors and, subsequent to a cascade of events that is still not well

understood, specific gene programs are initiated by the action of specific (sets of) enhancer factors . The second pathway is a shortcut in that receptors for certain small diffusible ligands (hormones and vitamins) are located in the nucleus and act as inducible enhancer fac tors

themselves.

The differential evolutionary conservation of seven regions (denoted A through F) within the primary structure of nuclear receptors suggested a possible modular structure (Figure 2; note that some receptors, such as the progesterone receptor, do not possess a region F) . Regions C and E were found to be highly conserved and therefore likely to correspond to functions common to all members of the family (82). Indeed, analyses of in vitro mutated ER and PR revealed that these two regions correspond to the DNA and hormone-binding domains , respectively (35 , 53, 84, 85). The pleiotropic effects of estrogen or progesterone administration are due to changes in the expression of target genes . These changes can affect all steps of gene expression, from positive or negative control of transcription to the specific alteration of RNA and protein half-lives. This review focuses on the positive control of transcription, and the findings that steroid hormone recep tors act as ligand-inducible transcription factors that bind to cognate enhancer elements (termed "hormone response elements", HREs) present in target genes . I attempt to explain how the receptor recognizes its target genes, which

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structures of the receptor are responsible for transcriptional activation (includ ing differential activation of target genes by receptor isoforms), and the roles of agonists and antagonists in these processes. Finally, I address aspects of estrogen and progesterone action that are still not fully understood.


HORMONE RESPONSE ELEMENTS (HREs) AND DNA-BINDING DOMAINS (DBDs) OF STEROID HORMONE RECEPTORS HAVE COEVOLVED To understand how ER and PR recognize and interact with their cognate HREs , the following questions must be considered: What is the minimal amino acid segment of the receptor (i .e. the DNA-binding domain, DBD) required for specific binding to HREs? Which amino acids within the DBD are responsible for the specific recognition of the HRE? What are the DNA sequences of these enhancer elements? In which form (monomer, dimer, heterooligomer) does the receptor bind to HREs? What can we conclude from such DNA-binding studies about the regulation of hormonally triggered gene networks?

The DNA-binding Domains of the Estro gen and Progesterone Receptors Include Sequences outside the Postulated "Zinc Finger" Structures The 65 to 68 amino acid core of region C (Figures 2, 3) is the most highly conserved region of the nuclear receptor family [the recently described mouse peroxisome proliferator-activated receptor is unique in having only 65 amino acids in this region (66)J. Region C contains an invariant cysteine repeat and, by analogy to the Xenopus 5S ribosomal RNA transcription factor TFIIIA (15 , 8 1 , 100), has been postulated t o fold into two zinc finger structures (36, 68) ,

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Schematic illustration of the structure/function organization of estrogen and pro

gesterone receptors. The evolutionarily conserved regions C and E are indicated as boxes, a black bar illustrates regions A/B , D, and F. Domain functions are depicted above and below the scheme

(see text). NLS, nuclear localization signal; TAF, tran sc ription activation function.


93

I ER DNA BINDING DOMAIN (DBD) GR 421 ER

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ro�f�9iIJI�G---j I----- NON SPECIFIC DNA BINDING -------l r-- DIMEAISATION DOMAIN? -------1 f--- HSP90 BINDING ----l f---"COAE" OF REGION C(66AA) -I- NUCLEAR LOC. SIGNAL-I REGION AlB Figure 3

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C (hER/CER 100% CONSERVED)

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Hypothetical structure of the hER DBD. R egion C encompasses the sequence that is

entirely conserved between the human and chicken ER (amino acids 1 85-262), The 66-amino acid core of region C (amino acids 1 85-250) , which is highly conserved between the different nuclear receptors, is shown as two subregions CI (amino acids 1 85-215) and ClI (amino acids

216-250), Each subregion contains four highly conserved cysteine residues that may tetrahedrally coordinate zinc to form a zinc finger. Regions CI and cn are separated b y an intron located within the codon for Gly-2 1 5, Two other introns are located within the codons for Arg- 1 5 1 and Gly-254, Those amino acids that differ between the hER and hGR in the 66-amino-acid core region are marked with an asterisk and those residues common to all nuclear receptors are shown in bold. Basic amino acids are over- and underlined. Three amino acids (E, G and A) located in the N-terminal "knuckle" of

CI confer the specificity of recognition of an ERE.

Their substitution

by G, S, and V (boxed) is sufficient to change the specificity of recognition of the core of region C to that of the GR (see text). The regions implicated in specific and nonspecific DNA-binding ,

HSP90-binding, dimerization and nuclear targeting are indicated at the bottom (modified from

ref. 50) .

each resulting from the coordination of one Zn++ to four cysteine residues. In fact, EXAFS, NMR, and CD studies of peptides containing the ER or the glucocorticoid receptor (GR) region C have provided evidence for the exis tence of such structures (3, 40, 59, 60, 139). Furthermore, peptides compris ing individual GR fingers w ere shown to bind, albeit rather weakly, to glucocorticoid response elements (GREs) in a zinc-dependent fashion (3). These putative fingers represent an essential component of the human ER (hER) DNA-binding domain, since mutant forms, e. g. HEll (83, 84), lack ing the fingers do not bind to DNA in vitro or in vivo. The core of region C (amino acids 185 to 250; Figure 3) is, however, not sufficient for high-affinity


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binding to an estrogen response element (ERE). While the N-terminal AlB region of the ER can be deleted without impairing DNA binding, analysis of a series of C-terminal truncations revealed that ER mutants lacking a basic region (amino acids 256 to 270; Figure 3) located C-terminal of the zinc fingers do not efficiently bind to DNA in vitro. In gel shift experiments, a retarded band was observed with the transiently expressed ER mutant HE1 5 , which lacks the sequences C-terminal o f amino acid 281, but not with the HE 1 6 ER, which terminates after amino acid 261 ( 1 9, 83) . Selective deletions within the full-length or partial ER of this basic region further supported its role in stabilizing ERiERE complexes, since a transiently expressed ER mutant lacking amino acids 250 to 264 was unable to bind to the ERE. A deletion of residues 261 to 272 generated an ER mutant that bound to DNA in gel shift assays, but dissociation kinetics revealed a significantly reduced half-life of this DNA-protein complex when compared to that of the wild-type (wt) ERiERE complex (our unpublished results) . A similar analysis demon strated that the ERE complex with the ER mutant HEI5, which contains the basic region but lacks the HBD, was less stable than the corresponding complex with the entire ER (83). These results indicate that the ER HBD stabilizes the ERiERE complex and partially compensates for a deletion in the C-terminal (amino acids 261 to 271 ) but not the N-terminal half of the basic region. On the other hand, ER mutants lacking the HBD require both the zinc fingers and the entire basic region located between amino acids 256 and 271 for efficient DNA binding, as assayed by gel retardation (Figure 3). The stabilizing effect of the HBD is discussed further below. Interestingly, two lysine/arginine-rich motifs (amino acids 256 to 260 and 263 to 271 , respectively) located in the basic region serve a triple function , since they are also involved in the binding in vitro of the 90-kd heat-shock protein (hsp90) ( 1 9) and correspond to two proto-nuclear localization signals (proto-NLSs) of the ER (T. Ylikomi, M. T. Bocquel, M. Berry, H. Gronemeyer, P. Chambon , submitted) . The hER amino acid 1 79 to 271 stretch of hER appears to be sufficient for DNA binding, not only in vitro but also in vivo, since ER mutants expressing this sequence and one of the two transcription activation functions (T AFs , see below) activated transcription in transient transfection assays in cultured cells (84). Moreover, the mutant HEI 5 , comprising hER amino acids 1 to 28 1 , also activated transcription in yeast (12, 170) . The binding of the progesterone receptor to its cognate response element (PRE) differs from that of the ER in two respects: the PR/PRE complexes appear less stable than the ERiERE complexes as judged from gel retardation assays; in contrast to hER, no PRiPRE complex could be observed with chicken PR (cPR) mutants lacking either the HBD or sequences C-terminal of amino acid 288 in region AlB ( 1 57; M . E. Meyer, unpublished results) .



95

However, DNase I nuclease footprinting and methylation interference assays demonstrated that a bacterially expressed fusion protein between beta galactosidase and the cPR region C (amino acids 405 to 496) specifically bound to natural and synthetic PREs in vitro, although the corresponding PRE complex was less stable than that formed with the entire PR (35). Thus, the PR region C contains the minimal amino acid sequence necessary and suf ficient for specific PRE binding, but high-affinity binding requires additional amino acid sequences located both in the AlB region and the HBD . That bacterially expressed ER (1 50) and PR (35) DBDs bind specifically to their cognate HRE indicates that no eukaryote-specific posttranslational mod ifications are required for specific ER-ERE and PR-PRE interactions. In conclusion, the zinc finger-containing region C, which has been highly conserved during evolution, is both necessary and sufficient for specific binding by ER and PR to their cognate palindromic response elements . It is likely that, as for the GR (3) , the individual zinc-fingers within region C represent the smallest DNA-binding structures of ER and PRo However, for the ER, the HBD and a basic motif located C-terminally of the zinc fingers and the HBD significantly stabilize DNA binding, while both the HBD and sequences located N-terminally of region C are important for stabilizing binding of the PR to the PRE.

O nly Few Amino Acids Located in the N-terminal Finger CI of the ER Are Critical for the Discrimination between an ERE and a GRE The two putative zinc fingers of ER and PR are similar to, but clearly distinguishable from, the proposed zinc fingers of the 5S ribosomal RNA transcription factor TFIIIA (15 , 8 1 , 100). In TFIIIA and other putative transcription factors that contain TFIIIA-like zinc fingers, the finger se quences can be aligned to give a conscnsus sequence . However, the two ER and PR fingers are quite distinct from each other. The N-terminal finger (CI in Figure 3 ) contains several hydrophobic amino acids and four invariant cys teines, whereas the C-terminal finger (CII in Figure 3) contains five invariant cysteines and is richer in basic amino acids. These observations suggested that the structure and function of the CI and CII fingers may be different. Chimeric receptors with altered DNA-binding specificity were created by swapping of the 65- to 68-amino-acid core of region C among the various members of the nuclear receptor family (47, 66 , 1 1 1) . An hER chimera in which this core region was replaced by the corresponding one of hGR, activated a glucocorticoid response gene in the presence of estrogen (47). Further analysis in which the CI and ClI fingers of the ER and GR were individually swapped, revealed that the amino acids critical for the specificity of GRE versus ERE recognition were located within the CI N-terminal half of


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the core of region C (50). ER mutant in which only a few amino acids of region CI were mutated to the corresponding ones of the GR, indicated that three amino acids (E-203 , G-204, A-207; see Figure 3) located at the C terminal end of CI were critically involved in discriminating between estrogen and glucocorticoid response elements (88). However, ER mutants that exhib ited a change in HRE specificity (e.g. E-203 , G-204, A-207 of hER to G, S , V o f hGR, respectively) interacted less efficiently with a GRE than did the wild-type hGR, since their affinity for a GRE was lower and they were less efficient at activating transcription from glucocorticoid responsive reporter genes . Thus, additional amino acids of the GR region C must be involved in stabilizing GR/GRE complexes. Although substituting the three critical amino acids of ER (EGA) by the corresponding ones of GR (GSV) completely changed the specificity of RE recognition, exchanging any two, but not one, of these residues was sufficient to gain GRE recognition. The mutations EG to GS and EA to GV produced ER mutants that recognized the GRE (and not the ERE), but the mutation GA to SV yielded a mutant with reduced specificity that recognized both the GRE and ERE . It was, however, a poor transcriptional activator of both estrogen and glucocorticoid target genes. Results similar to those described above have been obtained when the specific HRE recognition of the GR was changed by mutagenesis to that of the ER (25 , 159) .

Similar Sequences Can Act as Response Elements for the Steroid, Thyroid and Retinoic Acid Receptors Analysis of the promoter regions of estrogen- and glucocorticoid-inducible genes led to the identification of their cognate hormone-responsive enhancer elements (HREs) . The consensus sequence of the estrogen response element (ERE:5' -GGTCAnnnTGACC) is palindromic; that of the glucocorticoid re sponse element [GRE:5' -GGTACAnnnTGTYCT, in which Y T or C; with T found in 65% of GREs (11)] is an "imperfect" palindrome; both elements contain a three-nucleotide nonconserved spacer between two halves of the palindrome (11, 78-80, 89) . The 3' half-site of the consensus GRE is more conserved among the various GREs than the 5' counterpart and a palindromic GRE maintaining the 3' motif (5'-AGAACAnnnTGTTCT) activated GRE dependent transcription, as did a consensus GRE (80, 108) . Considering only the 3' motifs , an ERE (5'-TGACC) and a GRE (5'-TGTYCT) differ at positions 3 and 4 and in the GRE with an additional T at position 6. A systematic study on the effects of point mutations in the GRE revealed that T is the only possible nucleotide in position 3 and that position 4 is entirely unconstrained, whereas position 6 must be occupied by a pyrimidine ( l 08). Note that a T is present in the same position in some EREs; since no systematic study has been performed, it is unclear whether there is any =



97

nucleotide preference for position 6 of a palindromic ERE. Thus, the major difference between the two response elements is at position 3, which must be a T in GREs and is usually an A in EREs. However, the nature of the base present at position 3 of an ERE is less restricted than in the case of a GRE, e.g. the PS2 gene ERE, which is less efficient than the consensus ERE, has 5'-TGGCC as a 3' motif (13, 109). In conclusion, GREs and EREs may differ by only one base in each half of the palindromic recognition sequence, as was in fact demonstrated with the corresponding reporter genes in transient transfection assays (80, 89) . The two or thref! amino acids identified above in the CI finger are probably central to the discrimination between these bases when this finger is interacting with the major groove of the DNA, as predicted (60, 139). The conserved amino acids within the regions CI of ER and GR may be important for specific interactions with the conserved bases of the palindromic response element. Alternatively, they might be involved in nonspecific interactions with the DNA backbone. A similar role in the nonspecific stabilization of DNA binding has also been hypothesized for the zinc finger region CII and the adjacent basic sequences (50). In fact, synthetic peptides encompassing re gion CII of GR bound to both a GRE and a ERE, whereas a peptide comprising region CI bound only to the GRE (3). Note also that the solution structure of the ER DBD shows the basic residues in ell in a suitable position to interact with the phosphate backbone of the minor groove (139). A given HRE can in some cases be recognized by several receptors. For example, the consensus GRE can also mediate induction of transcription by progestins, androgens, and mineralocorticoids (58, 80, 96). This promiscuity is not restricted to palindromic GREs, since the complex MMTV-LTR GRE can also be activated by all these steroids receptors (4, 96, 143). Although systematic studies are lacking, no difference has been detected between the four receptors in their specificity of GRE recognition. Thus, regulatory mechanisms must exist which guarantee receptor-specific activation of target genes in cells where more than one of these receptors is expressed (see below). A similar promiscuity as for GREs was also observed for EREs, since the ERE half-site motif 5' -TGACC is recognized also by thyroid hormone (TR) and retinoic acid (RAR) receptors (44; our unpublished results) . Furthermore, the vitamin D response elements (VDREs) described thus far show a striking similarity to a consensus ERE (77, 102). Even the invertebrate ecdysone response element (EcdRE) of hsp27 can be mutated to a palindrome with ERE half-sites spaced by one nucleotide, while still retaining its function (90). However, the ER activates transcription only from palindromic EREs with a fixed spacing of three nucleotides between the half-sites. In contrast, TR and RAR can recognize variably spaced palindromic half-sites (27, 44 ,

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1 59, 1 60). Interestingly, RAR, but not ER, also activates transcription from target genes with RAREs consisting of direct repeats of TGACC-related motifs (26, 1 45 , 1 47, 1 6 1 , 1 62). If RARs bind as dimers to response elements made up of either direct or inverted repeats, this dimerization domain must be very different from those of steroid receptors that bind as dimers only to palindromic elements exhibiting a two-fold rotational symmetry (see below) .


Estrogen and Progesterone Receptors Bind as Dimers to Palindromic Response Elements The finding that estrogen and progesterone receptors bind to palindromic elements supported the suggestion made almost 20 years ago that steroid receptors bind to DNA as dimers ( 1 74) . In fact, the appearance of an intermediary retarded band in gel shift assays with palindromic EREs and in vivo coexpressed wild-type and receptor mutants truncated for the AlB region indicated that two receptor molecules bound to a single ERE (83, 96) . That no ER binding could be detected in gel shift assays using half-site EREs (83) indicated that one of two mechanisms increased the affinity in vitro of ER to a palindromic site: either the binding of a second ER molecule increases the stability of the complex due to an alteration of DNA structure (DNA alteration model) or protein-protein interactions between the two ER molecules are required to achieve high-affinity binding (dimer model). Supporting the possible existence of a dimerization domain in the region containing the HBD, it was found by gel shift assays that one HBD-truncated ER molecule could not cobind with a wild-type (wt) molecule to a single ERE, whereas two such HBD-truncated hER mutants could cobind. In addi tion, HBD-truncated ERs bound considerably more weakly to a palindromic ERE than the wild-type receptor (83) . These experiments suggest that the HBD of the wt ER may stabilize ER homodimers , which dissociate less rapidly than those of mutants lacking the HBD (see below), and that dimeriza tion may stabilize ERiERE complexes. Indeed, the presence in the ER HBD of a dimerization domain was demonstrated by identifying amino acids within this domain that were critically involved in both high-affinity DNA binding in vitro and dimerization in solution, as demonstrated by gel retardation and coimmunoprecipitation assays, respectively (37). Although most of the amino acids were required not only for dimerization and high-affinity DNA binding , but also for hormone binding, hormone binding was not an absolute require ment for specific DNA binding in vitro (37) . Moreover, a 22-amino-acid peptide of the mouse ER HBD conferred high-affinity DNA binding onto a HBD-truncated mouse ER, and may therefore correspond to the dimerization domain (86). Results obtained by controlled proteolysis and exposure to chaotropic salt also indicate that the ER exists as a dimer in solution ( 1 3 l ) . The finding that ER mutants lacking the HBD did not bind to ERE half-sites (83) but generated intermediary bands in gel shift assays with



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palindromic EREs, suggested that a second dimerization domain in region AlB or C may stabilize the corresponding ERE/protein complexes. Alterna tively, the DNA alteration model may be operating. Since mutants lacking both the N-terminal AlB and the HBD regions produced similar results, the AlB region of ER does not appear to contribute to the stability of HBD truncated ERiDNA complexes, in contrast to GR (34) and PR (see below). Thus , the stabilization must originate from region C itself. Although the existence of dimers of HBD-truncated ERs has not yet been demonstrated, the solution structures of the ER and GR DBDs are in agreement with the existence of a dimerization interface consisting of the N-terminal portion of the ClI finger (60, 139). Evidence has been presented to support the existence of protein-protein interactions between GR DBDs bound to a GRE (23) and, further, that the substitution of the 5' N-terminal amino acids of the GR fingers Cll by the corresponding amino acids of the beta-thyroid receptor abrogated cooperative binding to a GRE (24). Interestingly, this region appears to be responsible for the differential binding/activation of ER and GR versus TR and RAR to palindromic H REs with differently spaced ERE half-sites (159; our unpublished results) . S ince RARs bind to direct repeats of ERE-related half-sites (see above), it will be interesting to determine whether the RAR DBD and HBD possess similar dimerization domains as the ER. Note that crosslinking did not reveal the existence of RAR dimers in solution (43). It has been reported that estrogen is required for dimerization and binding to ERE in vitro of hER expressed from the cDNA HEO that was initially cloned (51, 1 64). This ER cDNA was found to contain a mutation (Gly400Val) that decreases the stability of the corresponding receptor protein in the absence of estrogen , thus resulting in hormone requirement for DNA binding in vitro (152). In fact, estrogen does not appear to be required for DNA binding in vitro and for dimerization of the Wild-type ER (HEGO; see 152) irrespective of the salt and temperature conditions at which the ER-containing cell extracts were prepared (D. Metzger, M. Berry , P. Chambon, submitted) . This finding suggests that the ligand-free "8S complex", which is presumed to be a heterooligomer with hsp90 (19) , contains ER dimers (131). In vitro DNA binding of wild-type mouse ER also occurred independently of prior hormone binding (37). However, in the absence of genomic footprinting data, it remains unclear whether this is also true in vivo. Nevertheless, the ER appears to contain two dimerization interfaces, a strong one located in the N-terminal part of the HBD and a second one located in the DBD , perhaps encompassing the 5' N-terminal amino acids half of the finger ClI. Human and chicken PR are expressed as two isoforms, A and B (135) , that are translated from two mRNA classes. For human PR, these are transcribed from two promoters (53 , 69 , 73 , 75) . For chicken PR, it has also been proposed that the two isoforms arise from a single class of mRNAs by


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initiation of translation at two in-frame ATGs (20, 2 1 ) . Functional differences between these isoforms are described further below. In contrast to the wild type ER, no retarded PR/PRE complex could be seen in the absence of hormone in gel shift assays when using cell extracts containing "cloned" PR. However, in the presence of progestins or of the antagonist RU486, the PR isoforms formed homo- and heterodimers (6, 28, 33, 96); no retarded com plexes were seen with half-palindromic elements (our unpublished data) . When purified from target tissue, neither GR ( 1 72), chicken, or rabbit PR (8, 126) required hormone for specific DNA binding. However, in these ex periments the cPR was purified by DNA-affinity chromatography after expo sure to high salt at a previous step of the purification, and cPR prepared in low salt did not bind to DNA (126, 1 27). The salt exposure during purification may alter the structure (heterooligomeric , see below) of the PR, thus mimick ing hormone exposure in the above studies. An additional complication arises in these PR DNA-binding studies from the possible effect of DNA-binding stimulatory factors (DBSFs, see below) (32; our u npublished results) , which was, however, not seen by Rodriguez et al ( 1 26). Interestingly, no "mixed ligand" isoform heterodimers (i.e. dimers containing, for example, form A bound to the progestin R5020 and form B bound to RU486) could be detected, suggesting that hormone and antihormone form incompatible dimerization interfaces or, alternatively, inhibit cobinding of differently liganded PR iso forms to the palindromic PRE (96). Thus, as with ER, dimerization may stabilize PR/PRE complexes, but in contras t to ER, the hormone or the antihormone may be required for dimerization. Immunoprecipitation of hPR form B with form B-specific antibodies using extracts of the human breast cancer cell line T47D, which expresses the two PR isoforms A and B , resulted in the coprecipitation of hPR form A, provided that high salt nuclear extracts of hormone-treated cells or cytoplasmic extracts treated in vitro with high salt and hormone were used (28) . Coisolation of form A could not be demon strated with cytosolic extracts of nonhormone-treated T47D cells containing the 8S "unactivated" PR (28) , indicating that the hormone is required for dimer formation. In contrast, crosslinking experiments have suggested that both isoforms of the cPR assemble into dimers in solution even in the absence of hormone (127) . Purified receptor preparations have been used in this latter study and may not therefore provide meaningful information about the ligand requirement for dimerization (see above). These data indicate that the salt and/or hormone-activated PR spontaneously dimerizes in solution. Although the role of the ligand cannot be easily deduced from these studies, strong evidence suggests that hormone is required for PR dimerization in vivo. While studying the nuclear localization signals of the rabbit PR, Guiochon-Mantel et al (55) identified its constitutive nuclear localization signal (NLS) and observed that a mutant receptor with a deleted NLS and a nonfunctional DBD but with an intact HBD was cytoplasmic. both in the



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absence and i n the presence of hormone. However, this mutant was cotranslo cated into the nucleus in a hormone-dependent fashion when coexpressed with a second mutant that lacked both the epitope used for immunocytochemical detection and a functional DBD but not the NLS , and was located in the nucleus i n the presence of hormone (55). Cooperative binding of the isolated GR DBD (which differs by 6 amino acids from the PR DBD) expressed in E. coli has been reported to an "imperfect palindromic" GRE/PRE (5' -TGTACAggaTGTTCT) derived from the tyrosine amino transferase gene (155) . Methylation interference studies indicated that only the half-site TGTTCT was bound at low concentrations of GRE/PRE. At higher concentrations another DBD molecule bound to the second "imperfect" TGTACA site in a cooperative manner. That this was due to the formation of GR DBD dimers on the DNA was supported by ex periments showing that cooperativity was dependent on the distance and relative orientation of the two half-sites, but not on the integrity of the DNA backbone (23). Moreover, cooperativity, but not DNA binding, was lost upon mutating the five N-terminal amino acids (AORND in both OR and PR) of the ClI finger (24), suggesting that this region corresponds to a dimerization interface in accordance with the solution structure model of the GR DBD (60). A similar dimerization interface may also be present in the PR DBD . There are, however, regions located outside of region C that are essential to the binding of PR to a PRE. Gel shift studies with transiently expressed wild-type and mutated PRs have demonstrated that both the HBD and se: quences within region AlB are essential for the stability of PR/PRE complexes in vitro (35 , 157; our unpublished results) . In conclusion, in the presence of progestins and certain antihormones, the PR has the capacity to dimerize in solution in the absence of a cognate response element both in vitro and in vivo. This dimerization is important for high-affinity binding to a PRE in vitro. In view of the sequence conservation between the PR and the GR, a dimerization domain may be present in the ell finger of the DBD . However, efficient DNA binding of the PR requires additional sequences located in the HBD and in the AlB region, both of which most likely provide additional dimerization interfaces.

Accessory Factors Are Required for DNA Binding in Vitro Purified human ER or PR, overexpressed using the vaccinia virus or baculo virus vectors, does not efficiently form complexes with a cognate palindromic HRE in gel shift assays unless whole-cell or nuclear extracts of mammalian (receptor- or nonreceptor-expressing), invertebrate, or yeast cells are added (104; R. Knauthe , unpublished results) . Under identical conditions, such complexes are readily formed with crude extracts of HeLa cells transiently expressing the corresponding receptors. These results indicate the existence of DNA-binding stimulatory factors (DBSFs), which are required for efficient


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binding of these receptors to cognate HREs in vitro. These stimulatory activities are sensitive to proteases and to protein denaturing conditions. An ER DBSF has been purified from yeast cells ( 1 04); it is a single-stranded DNA-binding protein that did not associate with double-stranded DNA. Various cellular extracts have recently been shown to stimulate the binding of the progesterone (32) and thyroid hormone receptors ( 1 6, 1 05), the retinoic acid receptor (43 , 1 61), and the protein products of the proto-oncogenes c10s and c-jun (1) to their cognate DNA responsive elements. None of these activities has been purified and it is unclear whether they correspond to single-stranded DNA-binding proteins similar to the ER DBSF. It is not yet known how DBSFs act in vitro or if they are implicated at all in the binding of nuclear receptors to their response elements in vivo. H owever, it is tempting to speculate on the existence of a family of DBSFs that modulates the DNA binding of their cognate receptors. perhaps in a cell specific manner (43), and thus introduces a novel regulatory step into this nuclear signal transduction pathway.

Cooperative Binding to Tandem Response Elements Hormone response elements of target genes may be present as either perfect, e.g. the vitellogenin A2 ERE from Xenopus (79), or imperfect palindromic elements, e.g. the pS2 ERE ( 1 3 , 1 09), or as tandem arrangements of such elements , e.g. the MMTV LTR GRE (11). Alternatively, multiple different hormone response elements may occur in some promoter regions, e . g. in the chicken vitellogenin II gene containing both an ERE and a PRE (2). The functional analysis of such natural and synthetic promoters has revealed that several HREs may act synergistically in the presence of the cognate receptors, thus providing a level of transcription greatly in excess of the additive effects of the single elements. The mechanisms underlying this synergism are not fully understood. However, precedent exists for cooperativity at two distinct levels, (a) binding of regulatory proteins to DNA and (b) activation of transcription subsequent to binding. A paradigm for the first level is the cooperative binding of the lambda repressor to its regulatory region (119, 1 22). The cooperativity is mediated through protein-protein interactions be tween repressor dimers-the dimers are stable in solution since the energy of interaction between monomers is sufficient to hold them together in the absence of DNA. The second cooperative mechanism that generates synerg ism occurs even under conditions where the DNA binding sites are saturated and involves the cooperative stimulation of the transcription machinery by the transcription-activating functions (TAFs) of the activators (17,120, 121, 149 , 1 53; see also below). Only mechanism (a) is considered here, while mech anism (b) will be discussed further below. Cooperative binding of the GR DBD to a GRE can be monitored by gel shift assays because the protein-protein interaction between DBD monomers



103

is weak enough to allow the formation of monomer/GRE and dimer/GRE complexes (23, 24, 1 55). With intact ER and PR receptors , however, due to additional dimerization domains located outside the DBD , the dissociation constants of the corresponding dimers is low and hence they also exist as dimers in the absence of DNA (see above) . Consequently, gel shift assays reveal only one complex, comprised of a receptor dimer and a palindromic HRE. Contradictory results have been reported concerning the existence of cooperative binding of such ER dimers to tandemly repeated "perfect" or "imperfect" palindromic EREs (9 1 , 116). Martinez & Wahli (91) observed the formation of monomer (i .e. one ER dimer on a tandem ERE) and dimer (i .e. two ER dimers on a tandem ERE) complexes and presented dose response curves that are in agreement with a cooperative binding mechanism. In contrast, Ponglikitmongkol et al (116) observed no such cooperativity for the ER, whereas they did observe cooperative binding of hPR to tandem PREs. The reason for this discrepancy is unclear, but both groups observed that tandem "imperfect" palindromic EREs synergistically activated transcrip tion. "Perfect" palindromic EREs, however, acted synergistically in a stereo alignment-dependent fashion only when positioned at a great distance from the activated promoter elements (116) . The GR binds cooperatively to tandemly repeated "imperfect" palindromic GREs ( 1 34), thus leading to synergistic activation of transcription (136, 146). Moreover, GR and ER synergistically induced transcription from promoters linked to a region of the chicken vitellogenin II gene that contains both a GRE and an ERE. A comparison of the estrogen and dexamethasone dose dependency of transcriptional activation from such reporter genes showed that half-maximal activity with one steroid was obtained at lower concentrations when the other steroid was present in saturating amounts , suggesting that the synergy was based on an increase in affinity of the receptors to their binding sites (2). Finally , several transcription factors have been found to syn ergistically activate transcription with the GR or PR (136, 137), but it is unclear whether this cooperativity occurs at the level of DNA binding or transcriptional activation. TWO AUTONOMOUS TRANSCRIPTION ACTIVATION FUNCTIONS (TAFs) OF ER AND PR ACT IN A PROMOTER- AND CELL-SPECIFIC FASHION

A Constitutive and a Hormone-dependent TAF are Located within the N-Terminal Region AlB (TAF-l) and the Hormone Binding Domain (TAF-2), Respectively, of ER and PR Two types of experiments demonstrated the existence within the PR of two domains responsible for the transcriptional activation of target genes. On the one hand, PR mutants lacking either the N-terminal region AlB (or fragments


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thereot) or the HBD activated transcription much less than did the wild-type receptor, but, significantly, not all of their activity was lost (53, 96) . These experiments suggested the possible existence in the PR of more than one autonomous TAF. Indeed, when region AlB or the HBD of PR was linked to the (transcriptionally inactive) DNA-binding domain of the yeast transcription factor Ga14, the resulting chimerae efficiently activated transcription from Gal4-responsive genes (96). Similarly, the existence in the ER (and GR) region NB and the HBD of an autonomous TAF- l and TAF-2, respectively, was also demonstrated (153, 167) . The existence of a TAF-l was not obvious from experiments conducted with ER-deletion mutants (84) , since TAF-l i s only weakly active in some cell-types (see below) . The nature of the two PR and ER TAFs is unclear. In contrast to a report showing that an acidic 30-amino-acid peptide (designated "tau-2") of the GR HBD fused to the Gal4 DBD was able to activate transcription from Gal4 reporter genes (64), no such sequence could be identified in the ER TAF-2 when individual or com binations of exons constituting the HBD were assayed for their ability to activate transcription (168). Also, a Gal4 chimera containing hPR sequences corresponding to the GR tau-2 did not measurably activate transcription (our unpublished results) . Short sequences containing (the core ot) TAF- l of ER and PR have been delineated (our unpublished studies) , but they do not exhibit common characteristics, nor do they seem to resemble prototypic acidic activating domains (AADs) (121).

Cell and Promoter Specificity of TAF1 and TAF2 Initial investigations i nto the location and number of TAFs within steroid receptors produced apparently contradictory results. While it was reported that a deletion of the HBD within the rat GR created a constitutively acting "super-receptor", which activated transcription at least as well as the full length GR (45) , such a deletion in hER or cPR abolished 95% of the transcriptional activity (53, 84) . Furthermore, the deletion of hER region AlB had no effect on the induction of the vit-tk-CAT reporter gene (84) , while a cPR lacking the corresponding region lost most of its activation capacity (53) . Finally, one group found that the cPR truncated for the HBD lost most of its transcriptional activity (53) , while another group ( 18) reported that the same deletion had no effect. Consequently , it was concluded that the HBD of the GR (45) and of the PR (18) has a repressor type of function in the absence of ligand . A systematic study of the different parameters involved in the various experimental protocols used by the different groups finally explained these discrepancies (14) . It was demonstrated that two TAFs could indeed be observed in both the PR and GR, provided that the transfections were done in HeLa cells. In CV1 cells, TAFs-2 were weakly active relative to TAFs- l



1 05

when high amounts of receptor expression vectors were transfected, such that both wild-type and HBD-Iess GR or PR activated transcription equally well. A similar cell specificity was demonstrated for ER TAF- I and TAF-2, since chimeric receptors containing the Gal4 DBD and the ER region AlB activated transcription from Gal4-responsive promoters in chicken embryo fibroblasts (CEF) but not in HeLa cells ( 1 2 , 153). In contrast, ER TAF-2 activated transcription in HeLa and CEF cells, but not in yeast, where TAF-l appears to be the only functional TAF of hER when using minimal promoter-based reporter genes ( 12, 94, 170) . Collectively, these data indicate that each of the TAFs of a steroid receptor may have a distinct pattern of cell specificity . It is possible that the TAFs of various receptors exhibit different cell specificities, which may be related to their function in vivo . The nature of the promoter context of a given target gene may also influence ER TAF activities. Transcription from reporter genes (e . g . vit-tk CAT, ERE-tk-CAT), which contain complex promoters with several up stream elements (such as the HSV tk), was strongly enhanced by TAF-2 in the presence of estradiol. In contrast, TAF-2 very weakly stimulated a minimal promoter composed of the adenovirus major late TATA box region placed downstream of a palindromic ERE. While transcriptional activation by TAF-2 strongly depends on the promoter context, the activity of TAF- l is, III general, less affected by such variations ( 1 2, 153).

Evidence for the Existence of Transcriptional Intermediary Factors (TIFs) What could be the basis for cell-specific activation of transcription by the steroid receptor TAFs? One possibility is that TAFs may interact, directly or indirectly, with other cell-specific transcription factors necessary to mediate TAF activity to the basic transcriptional machinery. We have termed these hypothetical coupling proteins, which do not have to bind on their own to the target gene promoter and may interact with multiple cognate (classes of) TAFs, transcriptional intermediary factors (TIFs) . Initial evidence for the existence of TIFs came from the so-called "squelch ing" or "transcriptional interference" experiments performed with the yeast transcription factor Gal4 and steroid hormone receptors ( 1 4, 42, 95, 1 49). Gill & Ptashne (42) observed that the expression of high levels of Gal4 inhibits ("squelches") transcription from other genes that do not contain cognate Gal4 binding sites. The Gal4 TAF was found to be responsible for this inhibition, which was proposed to result from the interaction of the trans-activator with a titratable non-DNA-binding (i .e. expressed in limiting amounts and interacting with the activator off the template) intermediary factor (TIF), thus resulting in a decrease of transcription from promoters whose enhancer factor TAFs require the same TIF as Gal4 ( 1 20). Moreover,


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if expressed at high concentrations under conditions where the template is limiting, an activator may actually auto-inhibit transcription from its reporter gene, since it will sequester its cognate TIF in solution. Consistent with the TIF hypothesis, expression of increasing amounts of hER or hER TAF2 (mutant HEI9) in HeLa or CVl cells resulted in bell shaped dose-response curves of transcriptional activation (14). The mediating factor(s) presumably titrated off the template in this experiment may be commonly "used" by different steroid receptors, since both hER TAF- l and TAF-2 acted as efficient squelchers of transcriptional activation by PR and OR, with PR and OR reporter genes that lacked estrogen response elements (95 ) . Conversely, both PR and OR expression inhibited activation of transcription by the hER from a reporter gene lacking PRIOR binding sites (95 ) . In all these experiments, squelching occurred only when the particular TAF was in its "active" state, i . e . able to activate transcription when a cognate reporter gene was provided. While TAF- l was constitutively active, transcriptional activation and squelching by the full-length receptor or mutants/chimera containing the HBD required the presence of the cognate hormone. In the absence of steroid agonist or in the presence of some steroid antagonists (devoid of any agonistic activity), squelching by the full-length receptor or by TAF-2 present in HBD-containing mutant receptors/chimerae was severely reduced or abolished (95). It is important to stress that cell specifically expressed TIFs introduce additional steps into the nuclear recep tor signal transduction pathway, and, therefore, additional combinatorial possibilities for regulating the transcription of the target genes.

Estrogen, Progesterone, and Glucocorticoid Receptor TAFs Belong to Different Classes of Transcription-Activating Domains Although the structures of steroid hormone receptor TAFs and the mech anism(s) by which they operate are not understood, their distinct abilities to synergize (Figure 4) and to squelch (Figure 5) have allowed different func tional categories of TAFs to be defined ( 1 49, 153) . The pattern observed for the ability of the various TAFs to homosynergize and to heterosynergize when bound in the vicinity of a minimal promoter (i.e. containing only a TATA box and a cap site) is summarized in Figure 4 (149) . ER TAF- I and TAF-2 are different from one another and distinct from acidic activating domains (AADs). ER TAF- l did not homosynergize, nor did it heterosynergize with the upstream element factor (UE) of the adenovirus-2 major late promoter (Ad2MLP) . It did, however, hetcrosynergize with ER TAF-2 or with the VP 1 6 AAD ( 1 32 , 1 54). Exactly the opposite pattern of homo- and heterosynergism was observed with ER TAF-2, while the two ER TAFs, though quantitatively different, similarly heterosynergized with OR TAF-l


GR

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AAO

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Figure 4

Homosynergism and heterosynergism between the hER activation functions TAF-J

and TAF-2, the hGR activation functions TAF- I and TAF-2, and the AAD of VP1 6 . The results

are expressed as fold stimulation of transcription, taking one for additive stimulation (from ref. 149).

and TAF-2. The synergistic properties of the OR TAFs clearly distinguished them from the hER TAPs and from the VP 16 AAD, while the OR TAPs themselves could not be clearly distinguished from each other by this approach ( 1 49). The two hPR TAFs can heterosynergize with each other, but only weakly homosynergize, if at all (our unpublished results). Thus, clear differences exist in the synergistic properties of the TAFs of ER, OR and PR, suggesting that they may be functionally distinct, and also distinct from AADs. A comparison of the cell specificity of hER and hPR TAF-l and TAF-2 further supported that the two ER TAFs are different from one another, and that ER TAFs are distinct from PR TAFs . With the same reporter gene, hER TAF-l activated transcription efficiently in CEF but not in HeLa cells, while ER TAF-2 was active in both cell types ( 1 2, 153) . ER TAF-2 could not activate transcription in yeast ( 1 2) , whereas both hPR TAF-l and TAF-2 could. Moreover, the two PR TAFs were also active in CEF and HeLa cells (our unpublished results). In contrast, TAF-2 of PR is weak in CVl cells (such that TAF-l is responsible for most of the transcriptional activation in these cells), while both PR TAFs are similarly active in HeLa cells. No such difference was seen between HeLa and CVI cells for the two ER TAFs, since TAF-2 was the dominant activation function in both cell lines (14) . Finally, transcriptional interference/squelching assays, performed with the TAFs of hER, hOR, Ga14, and VP1 6 (the latter two being prototypes for activators with AADs) again demonstrated that the steroid hormone receptor TAFs are functionally distinct from each other and from AADs (Figure 5; 149), Collectively, the above studies indicate that the ER and PR TAFs belong to

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ACTIVATOR ER COMPETITOR

TAF-2

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VP16

GAL4

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+

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G

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+

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+

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+

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e

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ER


GR

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Figure 5

AAD

GR

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+/-

+++

+++

Transcriptional interference between the hER activation functions TAF-l and TAF-2,

the hGR activation functions TAF- l and TAF-2, and the AAD of VP16. Results are expressed as residual activation of transcription by the activator taking the stimulation in the absence of competitors as 100%. + + + , + + , + / - , and 0 correspond to 9%-22%, 30%-36%, 52%-70%, 750/0-77%, and 92%- 103% of residual activity, respectively (for further details see ref. 149).

different classes distinct from the class of AADs, which may activate transcription in mechanistically different ways. The existence of TAF classes that differ from AADs has also been suggested for a number of other transcription factors such as Sp1 (22), CTF/NF- 1 (92), and Oct-2A ( 1 03). In view of the different cell- and promoter context-specificities of TAFs, the presence of two TAFs in ER, PR, and GR appears to be an obvious advantage to differentially regulate target gene transcription in various tissues. In fact, steroid hormone receptors appear to be sophisticated enhancer factors, since they contain within a single molecule two different activation functions that ,

can homosynergize and heterosynergize and act in a cell- and promoter

context-specific manner. ISOFORM-SPECIFIC TRANSCRIPTIONAL ACTIVATION Some nuclear receptors exist as multiple isoforms that originate from a unique gene, but differ from one another in their N-terminal region AlB . While originally believed to be a peculiarity of the progesterone receptor system,

two PR forms of different molecular weights have been observed in the (52 , 65, 1 35), multiple isoforms are now known to exist also for RAR alpha (87), beta ( 1 79), and gamma (74) . While all these receptor isoforms bind their cognate ligand, both hormone-binding and nonbinding isoforms of the thyroid recep tor (TR) exist (67, 1 0 1 , 1 77) . RAR and TR isoforms are assembled by differential splicing and alternative promoter usage, whereas the chicken and human PR isoforms--designated A and B (135), are translated from two mRNA po pulations transcribed , in hPR, from two different promoters (69,

where

cytosol of human breast cancer and chick oviduct tubular gland cells



1 09

7 3 , 75). The hPR gene contains two in-frame ATGs (ATG l and ATG2) in its first exon and transcription is initiated either 744 nucleotides upstream of ATG 1 or downstream of ATG 1 (75). Consequently, translation of hPR transcripts having cap-sites located downstream of ATG 1 will be initiated at ATG2 and thus yield the smaner isoform A. A clue to the possible biological significance of isoforms lies in the demonstration that the two chicken and human PR isoforms differentially activate target genes. While transcription from PRE/GRE-tk-CAT was equal ly stimulated by the two isoforms , MMTV-CAT, which contains the complex PRE of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) , was preferentially activated by isoform B . In contrast, the ovalbumin or cPR promoter was activated only by form A (75, 1 5 1 , 1 56) . The mech anism by which this target gene specificity is achieved is not entirely clear. It is known that the core of PR TAF- 1 is located in the vicinity of the DBD, and that no separate autonomous TAF is present in the sequence of form B lacking in form A. However, the 1 64 N-terminal amino acids of PR form B are able to further stimulate transcription activation by the core ' of TAF-1 and pre liminary transcriptional interference/squelching experiments suggest that these N-terminal residues may interact with (a) TlFs or coactivators (63) required for TAF- l activity (our unpublished results). It is tempting to speculate that differential isoform expression would enable target cells to simultaneously control the transcription of different gene networks in re sponse to a single ligand signal , again increasing the combinatorial possibility of control of gene expression with a limited number of trans-activator genes.

ANTIHORMONE ACTION RU486 Induces DNA Binding of PR in Vitro and in Vivo and Acts as an Isoform- and Promoter-specific Agonist Progesterone receptor agonists and some , but not all (see below) , antagonists, like RU486, induce the binding of the PR to its cognate response element in gel shift experiments in vitro (7, 33, 35, 96) . Ligand binding is also a prerequisite for DNA binding in vivo, since hPR/RU486 competed out the hGRIDex complex from the common MMTV-PRE/GRE and thus inhibited transcriptional activation, whereas the ligand-free PR was unable to compete (96). hPRlRU486 also inhibited cPRlR5020-induced transcriptional activa tion of the MMTV-CAT reporter gene (note that cPR does not bind RU486). The observed inhibition was due to a "true" competition for the response element, since hPRlRU486 was unable to squelch/interfere with hGRIDex- or cPRlR5020-induced transcription. Furthermore, no evidence was obtained for the formation of hGRIDex-hPRlRU48 6 or cPRlR5020-hPRlRU486 heterodimers (96; see also above). The formation in gel shift experiments of cPRlhPR heterodimers and of hPR isoform A-B heterodimers was observed in


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the presence of either agonist or RU486, but no "mixed ligand" heterodimers [cPRJRS020-hPR/RU486] or [hPR form B/RS020-hPR form A/RU486] could be detected (96) , suggesting that agonist and antagonist form incompatible dimerization interfaces. These results confirmed earlier data showing that in transient cotransfection experiments a RU486-liganded hGR chimera or the RU486-liganded rabbit PR inhibited transcriptional induction by activators which recognize the same response element (56, 167; note that in those experiments transcriptional interference was not excluded) . RU486-liganded activators containing TAF-2 as their sole transcription activation domain do not activate or squelch transcription (95) . Together with the observations that RU486-liganded hPR migrated differently from pro gestin-liganded hPR (33 , 96) , and that R5020- and RU48 6-liganded receptors did not form heterodimers (96), these results indicate that RU486 may induce a conformation of the HBD different from that induced by R5020, thus preventing TIFs from interacting with TAF-2. Since hPRJRU486 binds to DNA in vivo, it was expected that TAF-l would be active in the presence of RU486. RU486 induced hPR form B to activate the synthetic PRE/GRE-tk promoter, thus providing final evidence for the DNA-binding ability of the hPRJRU486 complex in v ivo (96) . This activation was similar to that induced by hPR5 , a mutant PR lacking the HBD. In terestingly, RU486-induced transcriptional activation was both isoform- and promoter-specific , since no activation of the PRE/GRE-tk was seen with hPR form A, and neither form A nor form B activated the MMTV promoter (96) . Although these data may suggest that the TAFs- l of hPR forms A and B are different from each other, and that the hPR form B TAF-l acted in a promoter-specific manner, this cannot be the entire explanation, since the two corresponding mutant PR of form A and B lacking the HBD both activated the PRE/GRE-tk promoter (our unpublished results) . That hPR form B did not activate the MMTV promoter in the presence of RU486, while its HBD truncated mutant PR activated the same promoter, is a significant indication that the RU486-liganded HBD can influence the activity of TAF- l . Collectively, the above data clearly show that RU486 induces DNA binding of the hPR in vivo and that its major antihormonal effect is on the ability of TAF-2 to activate transcription. This, of course, does not rule out the possible contribution of additional mechanisms to the abortion properties of RU486, e.g. a retarded dissociation of the heterooligomeric 8S receptor complex (9) .

Hydrox y-tamoxifen is a "Mixed" Agonist/antagonist, because hER TAF-2 is Inactive in its Presence and TAF-J Activates Transcription in a Cell- and Promoter-specific Fashion To investigate the mechanism of action of two antiestrogens [4-hydroxy tamoxifen (OHT) and ICI 1 64,384 (ICI) (72, 163)] , transcription activation by



III

the wild-type hER and various ER mutant and chimerae containing only one of the TAFs was analyzed, taking into account the cell- and promoter specificity of hER TAF- I and TAF-2 ( 1 2) . In the presence of either OHT or ICI, a chimeric receptor composed of the Gal4 DBD fused to the HBD of hER (TAF-2) was unable to activate Gal4-responsive genes in HeLa or CEF cells, whereas transcription was efficiently induced by estrogen . Similar results were obtained with the hER mutant HE1 9G , which lacks the N-terminal region AlB harboring TAF- l . Thus, TAF-2 is apparently inactive in the presence of these antagonists ( 1 2). In contrast, the full-length hER efficiently activated transcription in the presence of OHT, provided that conditions were chosen under which TAF-1 was active, i.e. when the transfection was done in CEF cells ( 1 2) . In Hela cells, where TAF- 1 is weakly active, OHT/hER only slightly increased reporter gene transcription . Finally, in yeast, where TAF- I is the only active TAF with certain promoter genes , hER, but not the mutant HE 1 9G ER, which is truncated for TAF-2, efficiently stimulated transcription in the presence of OHT, while no activity was observed in the absence of estrogen or OHT ( 1 2 , 94) . Thus, OHT may be considered as a cell type- and promoter-dependent agonist due to the ability of hER TAF- 1 to activate transcription in its presence . This raises the interesting possibility that previously observed agonistic effects of OHT may be due to the ability of hER TAF- I to stimulate transcription in a particular tissue from specific estrogen-responsive promo ters . In contrast to the experiments performed in yeast, transient transfection assays of HeLa cells with the wild-type hER expression vector HEGO re sulted in a weak "constitutive" activation of transcription (which was com pletely antagonized by ICI) even in the apparent absence of hormone ( 1 2 , 1 5 8 ; see below) . However, no transcriptional activation b y HEGO or HE 1 9G could be detected in HeLa or CEF cells in the presence of ICI, irrespective of the reporter gene chosen ( 1 2) . Thus ICI acted as a complete antagonist under all the conditions tested.

Does the Estrogen Receptor "Constitutively" Activate Transcription in the Absence of Ligand? The originally cloned hER cDNA (5 1 , 1 64) used to construct the expression vector HEO contains a point mutation that results in the substitution of a valine for a glycine at position 400 ( 1 52; see above) . The expression vector HEGO encodes the wild-type hER, which has different properties than the HEO encoded hER-val . For example, hER-gly, but not hER-val , activated transcription from the vit-tk-CAT reporter gene in a seemingly "constitutive" way, even when cells were cultured in phenol red-free medium with charcoal treated fetal calf serum ( 1 52). Similar constitutive activities as for the wild type hER were reported for the chicken ( 1 52) and mouse ERs (86, 1 7 1 ) , both

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of which contain a glycine at the position corresponding to gly400 of hER. Based on such observations, it has been argued that hER is a constitutive activator of transcription in vivo ( 1 58) . However, no constitutive activation of transcription by hER was seen in yeast where no serum is added to the growth medium (12, 94). Therefore it seems most likely that the so-called "ligand independent" activation of transcription by the human, chicken, and mouse ER results from the presence of residual estrogens in the hormone-stripped culture medium.

Evidence for Antihormones that do not Induce DNA Binding of the Corresponding Receptor In gel shift assays progestins, or the antihormone RU486, promote binding of the PR to PRE (see above) . However, some antiprogestins and anti glucocorticoids that antagonize transcriptional activation by agonists in tran sient transfection assays, prevent binding of the receptor to its cognate response element in vitro and in vivo (M. T. Bocquel, unpublished results) . Thus, at least two classes of antihormones apparently exist: type I anti hormones like RU486, and type II antihormones that do not enable the receptor to bind to its cognate HRE. Clearly, type II antihormones will always be full antagonists, whereas type I antihormones may be TAF-I -dependent agonists. The existence of these two classes of antihormones is of obvious interest to drug development, since it raises the prospect of developing antagonists that either completely block a signal transduction pathway or modify it such that the TAF- l -, but not the TAF-2-dependent activation of target gene transcription is maintained . OLD OPEN QUESTIONS AND NEW PERSPECTIVES Since the cloning of the estrogen, glucocorticoid, and progesterone receptors, much has been learned about the molecular mechanisms responsible for steroid hormone action. Although a clearer picture has emerged of the molecular architecture of the receptors and their specific interaction with target genes, old key questions remain unanswered as new questions have been raised.

Role of the Ligand Little is known about the ligand's role in the various molecular processes leading to transcription activation in vivo. There is good evidence that steroid receptors exist in the cytosolic fraction of target cell extracts as 8S heterooligomers containing hsp90 (9, 19, 70, 117, 118) and additional pro teins (123-125 , 133). It has been hypothesized (9, 10, 54, 117) that hsp90, which binds in vitro to the HBD of OR (118) and to two regions (the HBD and the basic region next to the zinc fingers, see Figures 2, 3) of ER ( 19), blocks



1 13

the receptor DBD and is released upon ligand binding due to conformational changes of the HBD. If this mechanism were operating in vivo, the receptors should, in the absence of hsp90, be able to bind to target genes and con stitutively activate transcription by virtue of TAF- l . However, experiments in yeast indicated that mostly hsp90-free GR and ER failed to enhance transcrip tion in the absence of ligand. In the presence of hormone, the receptors activated transcription, but, unexpectedly, higher concentrations of hsp90 increased the efficiency of hormonal induction of transcription ( 1 1 2) . There i s substantial evidence that progesterone induces receptor-dimerization in vivo and that stable dimers are formed in vitro, both in the absence and pres ence of PRE. Furthermore, DNA binding in vitro and in vivo requires the pres ence of the cognate hormone (or type I antihormone), suggesting that dimeriza tion may be necessary for efficient DNA binding. However, the role of the ligand in these processes has been questioned (see above). For the ER, the role of hormone binding in dimerization and DNA binding in vitro and in vivo is much less clear. No hormone is required to efficiently generate ERIERE complexes in vitro, under conditions where no PRIPRE complexes are seen without hormone. Dimerization of ER appears necessary for ERE binding in vitro but hormone binding is not (37). Moreover, stable ER dimers may be present in the ligand free 8S complex and such complexes may bind to ERE in vitro (131) . Note that in vivo crosslinked ER of MCF-7 cells was found associated with chromatin, whether or not the cells had been exposed to estrogen, or the pure antiestrogen ICIl64,384 (173). In vivo genomic footprinting studies of nuclear receptors are required for a clearer picture of the receptor DNA-binding abilities under vari ous conditions . It is also not known whether the hormone plays a direct role in inducing an active conformation of ER and PR TAF-2 or rather "unmasks" a preexisting active TAF-2.

TAFs, TIFs and Other Factors How the various TAFs function is a key to understanding how trans-activators stimulate the basic transcription machinery . In this respect the characteriza tion and purification of the putative TIFs is an absolute prerequisite. There is evidence for the existence of additional factors that may affect receptor action by apparently different mechanisms . In vitro, DBSFs appear to be required for high-affinity DNA binding by purified ER and PRo It is unclear whether these factors are also required in vivo. Yet another type of factors may affect receptor activity. It has been reported that the GR and the proto-oncogene products c-Jun and c-Fos mutually inhibit one another' s ability to activate transcription due to a mutual inhibition o f DNA binding (71, 138 , 176) . It has been concluded on the basis of in vitro experiments that the formation of transcriptionally inactive heterodimers may explain this mutual inhibition of transcription. However, experiments performed with

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various receptors indicate that not only inhibition , but also stimulation of transcriptional activation can be observed in the presence of c-Jun and that the effects of c-Jun and c-Fos on receptor-dependent transcriptional activation are not at all reciprocal . In addition , c-Fos and c-Jun affect the activity of steroid receptors in a cell- and promoter-specific fashion (our unpublished results) . Thus, the molecular mechanism(s) responsible for the possible "cross-talk" between the two signal-transduction pathways (Figure 1) remains to be eluci dated.

A ntihormone Action There are apparently two types of antiprogestins: type I antagonists induce DNA binding by the PR in vivo and in vitro and type II antagonists do not. At present, it is unclear how type II antiprogestins work, though they may interfere with the dimerization or modification of the PR required for efficient DNA binding. Despite substantial evidence that OHT is a partial agonist due to the cell and promoter-specific activity of ER TAF- l (since TAF-2 is inactive in presence of OHT) , the mechanism of action of ICI l 64 , 384 remains con troversial. It has been suggested that IC1164,384 inhibits ERE binding in vitro by preventing mouse ER dimerization (38) . However, these results could not be reproduced by others using the human or chicken ER (130). It is crucial to investigate whether the ER can bind in vivo to an ERE in the absence of estrogen and OHT, as it does in vitro, and to study the effect of ICI 1 64,384 on this binding in vivo. In this respect, recent experiments demonstrate that in the presence of antiestrogen IC1 164,384 an ER-VP 1 6 can activate transcrip tion of the cognate ERE-tk-CAT reporter gene ( l l l a) . Two important con clusions can be drawn from this experiment: firstly, ICI l64,384 does promote DNA binding in vivo and, thus, is not a pure type II antagonist. Secondly, provided that the wild-type ERlICI complex can similarly bind to DNA, the ICI-liganded ER HBD apparently inhibits the constitutive activity of ER TAF- l in yeast and chicken embryo fibroblast cells. Note that a similar mechanism is discussed above for RU486.

Phosphorylation Both ER and PR are phosphoproteins (see ref. 5 for review) . ER purified from rat uteri was found to be phosphorylated exclusively on tyrosine (97). A purified calf uterus kinase reportedly increased the efficiency of estradiol binding of in vitro synthesized hER and the ER mutant HE l 4, which ex presses the HBD and a part of region D, was phosphorylated on tyrosine by this kinase in vitro (98) . The cPR is phosphorylated exclusively on serine (31), but phosphorylation does not appear to stimulate hormone-binding, since an E. coli-expressed cPR HBD bound progesterone quantitatively with



1 15

wild-type affinity (35 ) . Hormone-induced hyperphosphorylation, which in duced a characteristic mobility shift on denaturing polyacrylamide gels, was observed for the two isoforms of the human breast cancer T47D cell PR ( 142, 1 69) . Similarly, progestin-binding induced hyperphosphorylation of cPR but different target sites have been identified by various groups . While Sullivan et al ( 1 48) identified hyperphosphorylated amino acids only in region AlB , Denner et al (29) found in addition a strong hormone-dependent phosphoryla tion on Ser-530, which is located in region D. Interestingly, a recent report from Denner et al (30) suggests that phosphorylation of cPR may be related to its ability to stimulate transcription in transient transfection assays. At present, no common role of phosphorylation of ER and PR emerges from the various reports . It is unclear why the PR has a basal level of phosphorylation, and hormone-induced hyperphosphorylation appears to be important at different steps of ER and PR action. Clearly , the analyses in vivo of mutant receptors that cannot be phosphorylated at the corresponding residues will play a key role in deciphering the role of these receptor mod ifications.

Structural Basis of Receptor Activity Several reports on the solution structure of steroid receptor DBDs have been published (see above) , but no information has been obtained on the structure of the entire receptor, or of an isolated HBD. In view of the multiple functions harbored in the HBD (see Figure 2), an elucidation of its 3D-structure, in the absence and presence of agonists and antagonists, will substantially further our understanding of receptor function . CONCLUSIONS The open questions and controversies indicate that we are far from a molecu lar understanding of steroid hormone action , partially due to the increasing complexity of the phenomena that may no longer be accurately mimicked in in vitro studies. More genetic approaches are needed in the future to evaluate, for instance, the contribution of hsp90 to the control of steroid receptor activity in vivo. In this respect, studies of steroid receptor function in yeast should be very helpful . Finally, it appears that additional regulatory mech anisms are operating in living cells, such as receptor-specific activation of target gene transcription by effects occurring at the level of chromatin struc ture (57). ACKNOWLEDGMENTS I am indebted to Pierre Chambon for his help in the conception and presenta tion of this review. I am grateful to Lirim Shemshedini for critically reading

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this manuscript, and to Fran�oise Haenel for typing it. I thank the members of the steroid receptor group at the LGME for communicating unpublished results. The work at the LGME cited in this review was supported by funds from the Association pour la Recherche sur la Cancer, the Institut National de la Sante et de la Recherche Medicale (INSERM) and the Centre National de la Recherche Scientifique (CNRS) .


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Estrogen and progesterone receptors and human conjunctiva.

Estrogen and progesterone receptors in breast cancer.

Regulation of cytosol and nuclear progesterone receptors in rabbit uterus by estrogen, antiestrogen and progesterone administration.

Endometrial estrogen and progesterone receptors in cycles treated for infertility.

Distribution of estrogen and progesterone receptors isoforms in endometrial cancer.

Estrogen and progesterone receptors in epithelial ovarian tumours.

Estrogen and progesterone receptors in cytology: a comprehensive review.

The nontransformed progesterone and estrogen receptors in gastric cancer.

Quantitation of estrogen and progesterone receptors by immunocytochemical and image analyses.

Regulation of rabbit myometrial alpha adrenergic receptors by estrogen and progesterone.

Inhibition of estrous behavior by progesterone in rats: role of neural estrogen and progestin receptors.

Simultaneous identification of estrogen and progesterone receptors by HPLC using a double isotope assay.

Endometrial stromal sarcoma expression of estrogen receptors, progesterone receptors and estrogen-induced srp27 (24K) suggests hormone responsiveness.

Modulation of estrogen receptors in four different target tissues: differential effects of estrogen vs progesterone.

Regulation of estrogen receptor replenishment by progesterone.

Effects of estrogen and progesterone on transcription, chromatin and ovalbumin gene expression in the chick oviduct.

Interference by spironolactone of radioassays for progesterone and estrogen.

Progesterone receptor-B enhances estrogen responsiveness of breast cancer cells via scaffolding PELP1- and estrogen receptor-containing transcription complexes.

Immunohistochemical detection of estrogen and progesterone receptors in the normal urinary bladder and in pseudomembranous trigonitis.

Nuclear and "cytoplasmic" estrogen and progesterone receptors in squamous cell carcinoma of the cervix.

Angiogenesis in Breast Cancer and its Correlation with Estrogen, Progesterone Receptors and other Prognostic Factors.

Progesterone-induced activation of membrane-bound progesterone receptors in murine macrophage cells.

Genomic agonism and phenotypic antagonism between estrogen and progesterone receptors in breast cancer.

The elusive and controversial roles of estrogen and progesterone receptors in human endometriosis.