Differences in the Abilities of Estrogen Receptors to Integrate Activation Functions Are Critical for Subtype-Specific Transcriptional Responses

Ping Yi, Sumedha Bhagat, Russell Hilf, Robert A. Bambara and Mesut Muyan

Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642

Address all correspondence and requests for reprints to: Dr. Mesut Muyan, Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642. E-mail: mesut_muyan{at}urmc.rochester.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen receptors (ER) {alpha} and ß are members of a superfamily of nuclear receptors and mediate estrogen [17ß-estradiol (E2)] signaling. ERß has considerably less transcription potency than ER{alpha} in heterologous expression systems that use E2 response elements (ERE) in tandem as the trans-acting unit. We show here that despite similar intracellular characteristics, ERß, in contrast to ER{alpha}, fails to induce gene transcription synergistically in response to E2 from tandem EREs. Moreover, our results indicate that ER{alpha}-specific partial agonistic activity of antagonists occurs additively. Although synergy contributes, it is not sufficient for differences in the transcription potencies between the ER subtypes. We demonstrate here that differences in the abilities of ERs to integrate activation functions through functional interactions between amino and carboxyl termini are critical for the transcriptional strength of ER subtypes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGEN [mainly 17ß-estradiol (E2)] signaling is mediated by estrogen receptors (ER) {alpha} and ß. ERs are members of a superfamily of nuclear receptors and function as E2-dependent transcriptional regulators. The transcription of E2-responsive genes is primarily modulated by the interaction of the E2-ER complex with specific DNA sequences, estrogen response elements (EREs). A minimal ERE consists of a palindromic arrangement of two core recognition sequences separated by three nonspecific nucleotides, as exemplified by the vitellogenin A2 gene sequence: 5'-GGTCAnnnTGACC-3' (1, 2).

Previous studies have established that although the transcriptional response from a single ERE sequence is minimal, adjacent EREs can act synergistically to produce a high ER{alpha}- and E2-dependent transcription activation (3). Synergy is defined as the integrated regulatory potential of discrete response elements such that the total regulatory capacity is greater than the sum of the effects with each element independently (3, 4, 5, 6, 7). Using tandem EREs in experimental systems, recent studies have indicated that the magnitudes of transcriptional responses mediated by ERß are substantially lower than those induced by ER{alpha} in transfected mammalian cells (8, 9). We therefore wanted to address whether differences in the extent of transcriptional regulation from multiple EREs mediated by ERs result from the differences in the mode (synergistic vs. additive) of transcription induced by ER subtypes.

Although many previous experimental approaches have used the consensus ERE as the response unit to ER, most of the natural E2-responsive genes contain imperfect EREs that deviate from the consensus by one or more nucleotides (10, 11, 12, 13). These EREs have less potency in enhancing transcription than the consensus ERE (3, 4, 5, 6, 7). As ER-mediated transcriptional responses depend on ERE sequences, ligands, promoter, and cell context (14, 15, 16), we also examined whether the integrated influences of ERE sequences and ER ligands alter the extent or mode of transcription induced by ERs in a promoter- and cell context-dependent manner.

The mechanism by which ER{alpha} induces transcription synergistically from tandem ERE sequences remains unknown. Studies addressing the structural features of ER{alpha} responsible for gene activation have indicated that functional interactions between activation function-1 (AF-1) and AF-2 are required for the transcription ability of ER{alpha} (17, 18, 19, 20). Consistent with these findings, we further suggested in a recent report that AF-1 is critical for the ability of ER{alpha} to induce synergy (21). As AF-1 of ERß has a weak trans-activation capacity (22, 23, 24, 25), we also addressed whether structural differences in domains are manifested as receptor subtype- specific differences in transcription by using mutant and/or chimeric receptor variants and mammalian two-hybrid approaches.

We show here that ERß, in contrast to ER{alpha}, fails to induce gene transcription synergistically from tandem EREs in response to E2. Moreover, our results indicate that ER{alpha}-specific partial agonistic activity of the antagonists 4-hydroxytamoxifen (4-OHT) or ICI 182,780 (ICI) is an additive response, in contrast to E2, which augments ER{alpha}-induced transcription synergistically. Although synergy contributes, it is not sufficient to explain differences in the magnitude of transcriptional responses between the ER subtypes. Our results demonstrate that the ability of ER to integrate the activation functions through a functional interaction between the amino and carboxyl termini is necessary in defining the receptor subtype-specific transcriptional responses.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Functional Differences between the ER Subtypes in Transfected Cells
Transcriptional responses from ERE-containing reporter constructs.
Studies have indicated that the magnitude of transcriptional responses mediated by ERß is substantially lower than that induced by ER{alpha} from heterologous reporter constructs bearing tandem ERE sequences in transfected mammalian cells (8, 9). As adjacent EREs can act synergistically to produce a high E2- and ER{alpha}-dependent transcription activation (3), we initially addressed whether differences in the extent of trans-activation between the receptor subtypes are due to the inability of ERß to induce gene transcription synergistically. The expression vector without (as control) or with cDNA for ERß or ER{alpha} was cotransfected into ER-negative mammalian cells together with a reporter plasmid bearing zero, one, or two ERE sequences. The simple TATA box (TATA) or the moderately strong enhancer-less thymidine kinase (TK) promoter (see Materials and Methods) drives the reporter luciferase enzyme cDNA. Normalized activity from each reporter was compared with basal activity from the reporter construct bearing no ERE in response to the parent expression vector (pM2) in the absence of ligand, a value set at 1.

Recent studies (26, 27) indicate that although both ERs show a similar binding specificity to EREs, ER{alpha} binds to an ERE with an approximately 2-fold higher affinity than ERß. Moreover, the affinity of both receptors for an ERE sequence is correlated with the number of nucleotide variations from the consensus. Two nucleotide substitutions within the consensus ERE, for example, reduce the binding affinities of the receptors 20-fold. We therefore tested the responsiveness of the optimal ERE (17) (5'-CAGGTCActcTGACCTG-3') or an ERE sequence that has two (17d2) nucleotide changes from the consensus (5'-CAGGGCTctcTGACCTG-3'; changes are underlined) to the ERs.

The mode of transcription was considered synergistic if the total effect of the unliganded or liganded ER on responses from tandem ERE sequences was greater than the sum of the effects observed with each element independently (3, 28). In the absence of E2, ERß had little effect on the reporter enzyme activity from the TATA box constructs bearing a single consensus (17) or variant ERE (17d2) in transiently transfected COS-1 cells (Fig. 1Go). Similarly, transcriptional responses to ERß in the absence of E2 were low from the tandem consensus (17-17), variant (17d2-17d2), or hybrid (i.e. one optimal ERE sequence and one variant, 17-17d2) ERE sequences. E2 with increasing concentrations (shown is 10-9 M) augmented ERß-induced enzyme activity additively from the tandem ERE sequences.



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Figure 1. Transcription from Tandem ERE Sequences Mediated by ERß or ER{alpha} in Response to ER Ligands in COS-1 Cells

A, Reporter constructs are schematically depicted. Promoter indicates the simple TATA box or the enhancer-less TK promoter bearing no ERE sequence (P). 17 and 17d2, Consensus and nonconsensus EREs with two nucleotide variations from the core, which are underlined, respectively. 17-17, 17-d2, and 17d2-17d2 depict two ERE sequences in tandem. The distance between the center nucleotide (nt) in the 13-base core of the first ERE and the TATA box in the TATA box promoter construct is 106 nt (10 helical turns). The center to center spacing between two EREs is 38 nt. These EREs with the same center to center spacing are juxtaposed to the enhancer-less TK promoter. Luc cDNA, Luciferase enzyme cDNA. B, COS-1 cells were transfected with 500 ng TATA box or enhancer-less TK promoter constructs that drive the firefly luciferase cDNA as the reporter. These reporter constructs contain zero (TATA or TK), one (17 or 17d2), or two tandem consensus (17-17), hybrid (17-17d2), or nonconsensus (17d2-17d2) EREs. The reporter plasmids were cotransfected with 300 ng expression vector bearing ERß or ER{alpha} cDNA and 2 ng internal control plasmid bearing CMV promoter that drives the expression of the Renilla luciferase cDNA. Insets show the effects of 600 ng expression vector bearing ERß cDNA on luciferase activity from the TATA box or the TK promoter containing none, one, or two consensus EREs. COS-1 cells were treated for 24 h in the absence or presence of 10-9 M E2. The cell extracts were assayed for luciferase, and the normalized firefly/Renilla luciferase activities are presented as the fold change compared with the control. Shown are the mean ± SEM of three independent experiments performed in duplicate.

 
To ensure that the amount of expression vector used was not a contributing factor to the transcriptional mode, we transfected cells with increasing concentrations (0–1200 ng) of expression vector bearing ERß cDNA. Although the magnitude of transcription increased, the mode of activation from tandem ERE sequences remained additive in the absence or presence of E2 (the inset in Fig. 1Go shows transcriptional responses from the TATA box construct bearing zero, one, or two consensus EREs to 600 ng ERß expression vector).

ERß at any concentration tested had minimal effect on the reporter enzyme from the enhancer-less TK promoter activity in the absence of ligand. Although E2 augmented these responses, the pattern of enhancement was additive even when the concentration of ERß expression was increased (Fig. 1Go, inset). This effect was independent of the number and the nature of ERE sequence (Fig. 1Go).

The E2-ERß complex also increased reporter enzyme activity additively from tandem ERE sequences regardless of the promoter type in CHO or HeLa cells (data not shown).

ER{alpha}, similar to ERß, increased enzyme activity additively from the TATA box promoter construct in the absence of E2 (Fig. 1Go). Pretreatment of cells with increasing concentrations of E2 (shown is 10-9 M) augmented the ER{alpha}-induced enzyme activity dramatically and synergistically from two consensus EREs. Although the E2-ER{alpha} complex augmented luciferase activity from two variant EREs (17d2-17d2) to a smaller extent, the mode of increase remained synergistic. The hybrid E2-responsive unit also displayed a synergistic response to ER{alpha}. The increase in transcription was intermediate between those from 17-17 and 17d2-17d2.

ER{alpha} also augmented luciferase enzyme activity in response to E2 synergistically from the enhancer-less TK promoter bearing 17-17 compared with a single 17, whereas in the absence of hormone the receptor had little effect. ER{alpha} augmented luciferase activity from 17-17d2 or 17d2-17d2 additively in response to E2 independently from the amount of expression vector used (data not shown).

ER{alpha} increased reporter enzyme activity synergistically from the tandem 17-17 and 17-17d2 EREs compared with a single ERE, but it augmented enzyme levels additively from 17d2-17d2 in transiently transfected CHO or HeLa cells independently from the promoter type (data not shown).

Effects of ER ligands on ER-induced transcriptional responses.
Studies have indicated that the identity of ERE sequence and the nature of ER ligand are modulators of ER{alpha}-mediated transcription (14, 26). As ER{alpha} can modulate transcription differentially depending upon ligand and response elements (14, 15, 16), we also wanted to examine whether different ER ligands affect the responses mediated by ERs from different ERE sequences in a promoter- and cell context-dependent manner. Moreover, we tested whether the partial agonistic effect of an antagonist is due to differences in the mode of transcription from ERE-dependent reporter constructs compared with responses observed with E2. To examine these issues, we used HepG2 cells as a model system (18, 23).

In transiently transfected HepG2 cells (Fig. 2Go), E2, 4-OHT, or ICI at any concentration tested (shown are the effects of 10-7, 10-8, or 10-6 M E2, 4-OHT, or ICI, respectively) had little effect on the reporter enzyme activity induced by ERß. ERß displayed similar intracellular characteristics as ER{alpha} in HepG2 cells (data not shown; see also below). Hence, the minimal effects of ligands on ERß-induced reporter enzyme activity could be due to the absence or the presence at low concentrations of cofactors or comodulator proteins critical for the transcriptional ability of the liganded receptor.



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Figure 2. ERß- or ER{alpha}-Mediated Transcription from ERE Sequences in Response to ER Ligands in HepG2 Cells

HepG2 cells were transfected as described in Fig. 1Go. Cells were treated without or with 10-7 M E2, 10-8 M 4-OHT, or 10-6 M ICI for 24 h. Values represented as fold change are the mean ± SEM of three independent experiments performed in duplicate.

 
E2, 4-OHT, or ICI, on the other hand, augmented ER{alpha}-induced reporter enzyme activity. E2 enhanced the responses synergistically from the TATA box promoter construct bearing the 17-17 or 17-17d2 response unit, but not 17d2-17d2, compared with a single ERE. 4-OHT or ICI augmented ER{alpha}-induced luciferase activity additively from two optimal (17-17) EREs. There was no further augmentation of transcriptional response to either 4-OHT or ICI from the TK promoter constructs, whereas E2 enhanced ER{alpha}- induced transcription synergistically from 17-17 and 17-17d2 and additively from 17d2-17d2.

These results collectively indicate that ERß, in contrast to ER{alpha}, lacks the ability to induce gene transcription synergistically from tandem EREs in response to E2 independently from the identity of ERE sequence, the nature of promoter, or the cell context. Consistent with previous observations (24, 29), our results further show that mediation of the partial agonistic effects of not only 4-OHT but also ICI by ER{alpha} occurs in an ERE sequence-, promoter-, and cell-specific manner.

Although widely considered as a pure antagonist, studies both in situ (30, 31, 32, 33, 34) and in vivo (35, 36) revealed that ICI, like tamoxifen, can act as a partial agonist depending upon promoter, cell, and tissue context. Our results further show that the augmentation of the ER{alpha}-induced transcriptional response by 4-OHT or ICI is an additive response compared with that by E2, which induces synergy. This could relate to the mechanism by which the partial agonistic activity of an antagonist is achieved in the heterologous expression systems employed here.

The receptor-specific partial agonistic effects of 4-OHT and ICI are specific to HepG2 cells. Neither 4-OHT nor ICI at various concentrations (10-11–10-6 M) had an effect on the transcriptional responses induced by ERß or ER{alpha} independently from promoter, identity, or the number of ERE sequences in transfected COS-1, CHO, or HeLa cells. Both compounds with increasing concentrations, as expected, effectively antagonized the E2-induced increase in reporter enzyme activity in response to either ER (data not shown).

Intracellular Characteristics of ERß
The differences in the extent and mode of transcriptional responses to ERs could also be due to the distinct intracellular characteristics of the receptors. We initially addressed whether differences in the intracellular levels of ERs contribute to the functional differences between the subtypes using Western blot analysis of transfected cells. We observed that the level of ERß synthesis was similar to that of ER{alpha} (data not shown). Moreover, we observed, using whole cell extracts of transfected cells, that ERs bind similar amounts of [125I]E2. These results suggest that the intracellular levels of ERs do not contribute to the functional differences between the receptor subtypes.

Intracellular Distribution of ER{alpha} and ERß
Although ER{alpha} is localized constitutively in the nuclei of both native tissue of origin and transfected cells (37, 38), the intracellular distribution of ERß appears to be dependent upon the tissue of expression and the endocrine status of the donor. Recent immunohistochemical studies provided evidence that in addition to nuclear localization, ERß-like immunoreactivity is detected in the cytoplasm of cells from corpus luteum, endometrial luminal epithelia, and epithelial layer of the cervix of the human (39). The hinge region (D domain) of ER{alpha} contains multiple signals that regulate cooperatively the nuclear targeting of the receptor (38). One of the least conserved regions in ERß compared with ER{alpha} is the hinge domain (40, 41). The unique structural characteristics of the D domain of ERß may lead to the differential intracellular distribution of the receptor in response to ER ligands, consequently altering transcription of the target gene. To examine whether the intracellular compartmentalization of the ERß is affected by ER ligands in transfected cells, we comparatively assessed the intracellular localization of ERs by immunocytochemistry, using an antibody specific to the Flag epitope (M2) or the carboxyl terminus of either ERß (PA1-313) or ER{alpha} (HC-20). We observed no intracellular staining with the ERß, ER{alpha}, or Flag (M2) antibody in cells transfected with the expression vector lacking cDNA (data not shown). When expressed, ERß or ER{alpha} was localized in the nucleus in the absence of ER ligand (Fig. 3Go, A and B).



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Figure 3. ERß, as ER{alpha}, Is Localized in the Nuclei of Transfected Cells Independently from ER Ligands

COS-1 cells were transfected with the expression vector bearing Flag-ER{alpha} or Flag-ERß cDNA. Cells were treated without or with various concentrations (10-11–10-6 M) of E2, 4-OHT, or ICI for 24, 3, or 1 h. Shown is 10-9 M E2, 10-6 M 4-OHT, or 10-7 M ICI for 24 h. Proteins were probed with Flag (M2), ER{alpha}-specific HC-20, or ERß-specific PA1-313 antibody and visualized by fluorescein-conjugated secondary antibody (FITC). 4',6-Diamido-2-phenylindole hydrochloride (DAPI) staining shows the nucleus. The Flag-ER{alpha} variant ({alpha}EF) contains a Flag epitope at the amino terminal of the carboxyl-terminal E/F domain. There was no detectable protein reacting with Flag or receptor-specific antibodies in cells transfected with the parent vector, pM2, alone (data not shown).

 
The nuclear localization of ERs depended on the structural integrity of the receptor proteins. The truncated ER{alpha} variant ({alpha}EF) that contains only the carboxyl-terminal E/F domains showed a diffuse intracellular staining encompassing both nucleus and cytoplasm (Fig. 3CGo). This is due to the size of the protein and the lack of a hinge domain (38).

At all E2 or 4-OHT concentrations (10-9–10-6 M) and time points (1, 3, and 24 h) tested, both ER subtypes were always localized in the nucleus (Fig. 3Go, D and E), as shown previously for ER{alpha} (38, 42, 43, 44). ICI treatment, on the other hand, at high concentrations (10-7 and 10-6 M; shown is 10-7 M) and only at 24 h altered the intracellular distribution of both receptors (Fig. 3Go, F and G). The majority of cells continued to show nuclear staining for both receptors (Fig. 3FGo). However, less than 20% of the cells also displayed a cytoplasmic as well as a diffuse, i.e. encompassing both cytoplasm and nucleus, localization (Fig. 3GGo).

Thus, ERß, like ER{alpha}, is localized in the nucleus in transfected cells, and ICI, but not E2 or 4-OHT, can alter intracellular distribution of ERß depending upon the concentration of and the duration of exposure to the compound, as suggested previously for ER{alpha} (38).

Binding of ER Subtypes to DNA
To exclude the possibility that ER ligands differentially affect the abilities of ERs synthesized in cells to bind to an ERE, we assessed the in situ DNA binding abilities of ERs in response to ER ligands using an interference assay (45). The assay is based on the ability of ER to bind to the two consensus ERE sequences in tandem placed between the TATA box of the strong cytomegalovirus (CMV) promoter and the transcription initiation site of the bacterial chloramphenicol acetyltransferase (CAT) cDNA (45). If ER binds to the test EREs, CAT enzyme activity should decrease as a result of the physical interference with transcription of the reporter enzyme gene by the ER-ERE complex. We transfected mammalian cells (shown is COS-1, Fig. 4Go) with increasing concentrations (50–2000 ng) of the expression vector bearing receptor cDNA together with the reporter plasmid (100 ng). We observed that increased levels of expression vector bearing either ER cDNA gradually suppressed reporter enzyme activity. Shown are the effects at a 1:10 reporter plasmid to expression plasmid ratio. The treatment of cells with various concentrations of E2 (shown is 10-7 M), 4-OHT, or ICI (data not shown) had minimal effects on enzyme activity. However, when the ratio of the expression vector to the reporter plasmid was increased to 1:20, we observed a ligand effect that enhanced the ability of ERs to further suppress the reporter enzyme activity (Fig. 4Go, inset). The mutant ERs that lack the DNA-binding domains (DBDs; ER{alpha}{Delta}C or ERß{Delta}C) did not repress enzyme activity in the absence or presence of ER ligands, nor was there suppression of enzyme levels from the control constructs bearing no ERE sequences (pCMV-CAT).



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Figure 4. ERß, Like ER{alpha}, Binds to ERE in Situ as Assessed by an Interference Assay

COS-1 cells were transfected with a reporter plasmid containing the strong CMV promoter that drives the expression of CAT cDNA. The reporter plasmid bears none (control) or two consensus ERE sequences placed between the TATA box of the CMV promoter and the start site of transcription of CAT cDNA. The reporter plasmid (100 ng) was transfected together with 1000 ng expression vectors bearing no cDNA (pM2 ), ER{alpha}, ERß, or DNA-binding domain (C) mutant of each ER (ER{alpha}{Delta}C or ERß{Delta}C) cDNA into COS-1 cells together with a reporter plasmid bearing ß-galactosidase enzyme to monitor transfection efficiency. Cells were incubated in the absence or presence of ER ligands for 24 h, and cell lysates were assayed for CAT activity. The inset indicates transcriptional responses to ERs when the cells were transfected with a 1:20 ratio of the expression vector to the reporter vector. The normalized enzyme activity is expressed as the percent change from the control, which was set at 100%. The data are the mean ± SEM of three independent experiments performed in duplicate.

 
Thus, as shown previously for ER{alpha} (45, 46), ERß interacts with DNA in situ whether unliganded or liganded with ER ligands. We also observed that the unliganded or liganded ERß, like ER{alpha}, synthesized in cells binds similarly to an ERE sequence in vitro, as assessed by electrophoretic mobility shift assay (EMSA; data not shown) (22, 47). These results indicate that ERß synthesized in cells interacts with DNA in a manner similar to ER{alpha}.

Binding of ER{alpha} and ERß to Tandem ERE Sequences
Cooperative binding of ER{alpha}, as for other steroid hormone receptors, to adjacent response elements is thought to be critical for the ability of the receptor to induce transcription synergistically from tandem ERE sequences (2, 4, 5). We therefore examined whether detectable differences in the binding mode of ERs to tandem ERE sequences contribute to the receptor subtype-specific transcriptional responses. We analyzed in vitro binding of ERs to oligomers containing two ERE sequences in tandem using EMSA (Fig. 5Go). 32P-end-labeled 0.125-nM oligomers containing two consensus (17-17) or nonconsensus (17d2-17d2) ERE sequences were incubated with ER{alpha} or ERß. As shown in Fig. 5AGo, ERs with increasing concentrations gradually retarded the electrophoretic migration of the end-labeled oligomers reflected in the formation of complexes 1 (C1) and 2 (C2). C1 represents a single ER dimer bound to a single ERE site, whereas C2 represents the binding of two ER dimers to two EREs. ERs at low concentrations initially formed the C1, which was gradually converted to the C2 with increasing concentrations of ERs. At the highest concentration tested, ER formed predominantly the C2. We (26) and others (27) previously reported that ERß binds to an ERE with 2-fold less affinity than ER{alpha}. The quantitative analysis of EMSA data (2, 4), however, revealed that the relative dissociation constant of C2 is 2-fold lower compared with that of C1 (Fig. 5Go, B and C) whether the receptor is ERß or ER{alpha}. Ligands did not alter ER-ERE interaction (data not shown). As both ERE sequences in a single DNA fragment are identical, these results suggest that ER binds to an ERE with higher affinity when the adjacent ERE is occupied and that both ERs bind cooperatively to the 17-17 oligomer. Similarly, either ER bound cooperatively to the oligomer containing two adjacent nonconsensus ERE (17d2-17d2; Fig. 5CGo). Thus, it appears that the binding mode of ERs to ERE sequences in tandem is not a contributory factor for the receptor subtype-specific differences in gene activation.



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Figure 5. ERs Bind to EREs in Tandem Cooperatively

We analyzed in vitro binding of recombinant ERs expressed in insect cells to oligomers containing two ERE sequences in tandem using EMSA. 32P-end-labeled 0.125-nM oligomers containing two consensus (17-17) or nonconsensus (17d2-17d2) ERE sequences were incubated with increasing concentrations (0–20 nM) of ER{alpha} or ERß on ice for 1 h. A, Reactions were resolved on 4% nondenaturing polyacrylamide gel. C1 depicts a single ER dimer bound to a single ERE site, whereas C2 represents the binding of two ER dimers to two EREs. Shown is a representative phosphorimage of at least three independent experiments. B, Quantitative analysis of the results in A is shown. C, The ratio of dissociation constants of ER binding to one (Kd1) or two (Kd2) EREs is shown.

 
Structural Features of ERs Responsible for the Receptor Subtype-Specific Transcriptional Responses
Structurally distinct AF-1 domains of ERs are critical for cell- and promoter-specific effects of the receptor subtypes in response to ER ligands (22, 23, 24, 25). As intracellular characteristics of ERs are similar, we wanted to examine how differences in structural features of ERs are reflected in differences in transcriptional responses between ER subtypes. To accomplish this, we generated truncated, mutant, and chimeric forms of ER{alpha} and ERß (Fig. 6Go). The truncated ER{alpha} or ERß ABCD variant (amino acids 1–301 or 1–243, respectively) contains the amino-terminal A/B domain together with DNA binding (C) and hinge (D) domains, but lacks the carboxyl-terminal ligand-binding domain (E/F). The CDEF receptor variant (181–595 for ER{alpha} and 178–530 for ERß) lacks the entire amino-terminal A/B domain. The AF2 mutant receptor of ER{alpha} contains a three-amino acid replacement (D538A, E542A, and D545A) that destroys AF-2 function without altering DNA and ligand binding properties (18, 21, 48) (data not shown). Similarly, the ERß AF2 mutant contains three amino acid replacements (D489A, E493A, and N496A) that are at the analogous positions with those of ER{alpha} when aligned (23).



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Figure 6. Transcriptional Responses to Mutant, Truncated, and/or Chimeric ERs

COS-1 were transfected with expression vectors bearing ER{alpha}, ERß, {alpha}CDEF, ßCDEF, {alpha}AF2m, ßAF2m, {alpha}NßC, ßN{alpha}C, {alpha}NßCAF2m, or ßN{alpha}CAF2m cDNA as described in Fig. 1Go. Cells were treated without (data not shown for clarity) or with 10-9 M E2 for 24 h. T, 1, and 2 depict the TATA box promoter constructs without (T) or with one (1) or two (2) consensus EREs, respectively. Stars depict the point mutations that destroy AF-2. Normalized luciferase values represented as fold changes are the mean ± SEM of three independent experiments performed in duplicate.

 
We found, as reported previously for ER{alpha} (17, 18, 21, 25, 46, 49, 50), that the ABCD variant of either ER is incapable of inducing transcription independently from promoter and cellular context (data not shown). The function of the A/B region became apparent when the domain was tested within the context of full-length receptor containing mutations that destroy the AF-2 (18, 21). The ER{alpha}AF2 mutant ({alpha}AF2m), only in the presence of E2, increased the transcriptional activity of the reporter enzyme synergistically from the TATA box promoter bearing 17-17 in COS-1 cells. This contrasted with the ERßAF2 mutant (ßAF2m), which had minimal effect on enzyme activity. Both truncated CDEF variants induced transcription to a similar extent and showed an additive response. The nature of transcriptional induction of the reporter enzyme activity was independent from the level of synthesis and nuclear localization of the variant receptor proteins as well as the cell type (data not shown) (21). Thus, the amino-terminal AF-1 domain of ER{alpha}, but not of ERß, contains structural features required for synergy. Moreover, the lack of synergy with the ER{alpha} CDEF variant and the restoration of synergy, but not the extent of induction, with the ER{alpha}AF2 mutant also suggest that the integrated functions of AF-1 and AF-2 domains are required for the complete receptor- specific transcriptional response.

To address this issue further, we constructed chimeric receptors. The {alpha}NßC chimera contains the entire A/B domain of ER{alpha} and the C, D, and E/F domains of ERß. The ßN{alpha}C chimera combines the A/B domain of ERß and the C, D, and E/F domains of ER{alpha}. If the unique functional features of the AF-1 domains are solely responsible for the receptor subtype-specific differences in the mode of transcription, the ßN{alpha}C chimera should show an additive response, in contrast to the {alpha}NßC chimera, which should induce gene transcription synergistically. Expression vectors bearing the cDNAs for chimeric receptors were cotransfected into mammalian cells with a luciferase reporter driven by the TATA box promoter without or with one or two consensus EREs. We found that although the extent of transcription was greatly reduced compared with that of wild-type (WT) ER{alpha}, both {alpha}NßC and ßN{alpha}C induced transcription synergistically, in contrast to ERß, which additively augmented enzyme activity. In the absence of AF-2, {alpha}NßCAF2m and ßN{alpha}CAF2m showed minimal response. As {alpha}AF2m induces transcription synergistically, in contrast to ßAF2m, these results collectively suggest that although a strong AF-1 domain is critical for the mode of transcription, this activation domain (AD) alone is not sufficient to explain functional differences between ERs. Thus, it appears that the integrated effects of both AFs are required for the receptor-specific transcriptional responses.

Functional Interactions between Amino and Carboxyl Termini of ERs
Functional interactions between amino and carboxyl termini of ER{alpha} are shown to be critical for the ability of the receptor to induce gene transcription (20, 51, 52). It is possible that the absence of a functional interaction critical for the integration of AF-1 and AF-2 of ERß could be one of the underlying reasons for the receptor-specific transcriptional responses.

To address whether differences in the abilities of AF-1 and AF-2 to functionally interact are crucial for the receptor subtype, we used a two-hybrid system that relies on stable protein-protein interactions. This approach exploits the modular nature of eukaryotic transcription factors possessing functionally distinct regions: DBD and AD domains. The Gal4 DBD binds to Gal4 response elements preceding a simple TATA box to position protein components to the promoter. The AD derived from the VP16 protein of the herpes simplex virus induces transcription by recruiting transcription factors. These independent modules, however, cannot activate gene transcription unless each module is brought into close proximity by a direct or indirect interaction between distinct proteins, or domains of the same protein, separately fused to a module. To investigate the interaction between the AF-1 and AF-2 domains of ERs, we genetically fused the carboxyl-terminal E/F domain to Gal4 DBD and the amino-terminal A/B region to the AD of VP16. The fusion proteins were then expressed in COS-1 cells. If there is a functional coupling between the AF-1 and AF-2 domains, we should observe an augmentation of reporter enzyme activity compared with levels induced by each domain alone. The results revealed that the interaction between the A/B and E/F domains of ER{alpha}, but not ERß, only in the presence of E2 augments enzyme activity (Fig. 7Go). Moreover, the amino-terminal region of ERß interacted with the carboxyl terminus of ER{alpha}, as the amino-terminal region of ER{alpha} interacted with the carboxyl terminus of ERß. Differences in the magnitude of trans-activation by the fusion constructs are reminiscent of those observed with chimeric receptors. This together with the observation that 4-OHT or ICI completely prevents the interaction of the A/B and E/F domains (data not shown) strongly suggest that the E2-mediated functional interaction between AF-1 and AF-2 is critical for the receptor subtype responses.



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Figure 7. The Amino and Carboxyl Termini of ER{alpha}, But Not ERß, Interact Functionally to Induce Transcription

The carboxyl-terminal E/F domain of ER{alpha} or ERß was genetically fused to Gal4 DBD, whereas the amino-terminal A/B region of ER{alpha} or ERß was fused to the AD of VP16. The fusion proteins were then expressed in COS-1 cells together with the reporter plasmid bearing five Gal4 response elements juxtaposed to the simple TATA box promoter that drives expression of the firefly luciferase enzyme cDNA as described in Fig. 1Go. Cells were treated without or with 10-8 M E2 for 48 h. Normalized luciferase activities represent the mean ± SEM of three independent experiments performed in duplicate.

 
Transcriptional Responses to ERs from Promoters of E2-Responsive Genes
We have shown here that ERs induce transcription differently from heterologous promoters that rely on the nature of ER ligand, the ERE sequence, or the number of EREs. However, a single nonconsensus ERE sequence within the context of a variety of cis-acting elements is sufficient to provide E2 and ER{alpha} induction from promoters of E2-responsive genes. For example, the human pS2 gene confers E2 responsiveness through a nonconsensus ERE (5'-GGTCAcggTGGCC-3', deviation from the core sequence is underlined) (53, 54). Similarly, the third component of complement (C3), a substrate for the C3-cleaving enzymes of the complement cascade, is also responsive to stimulation by both E2 and tamoxifen (18, 55, 56). The E2 mediation of transcription requires the presence of a nonconsensus ERE, 5'-GGTGGcccTGACC-3' (18, 56, 57). The expression of the lysosomal proteinase cathepsin D (CatD), is augmented, albeit modestly (0.6- to 0.9-fold compared with basal expression), by the E2-ER{alpha} complex also through a nonconsensus ERE (5'-GGCCGggcTGACC-3' (58, 59).

To assess whether the unliganded or liganded ERß compared with ER{alpha} can display a high transcription capacity from promoters of E2-responsive genes, we engineered reporter constructs bearing promoter regions of CatD, pS2, or C3 genes that drive the expression of the luciferase cDNA. We transfected COS-1 and HepG2 cells with a reporter construct together with the expression vector bearing none (pM2 as control) or an ER cDNA (Fig. 8Go). Results revealed that ERß had no effect on luciferase activity driven by the CatD or pS2 promoter whether the cells were treated without or with E2 in transfected COS-1 cells. ERß, on the other hand, augmented luciferase activity driven by the C3 promoter in response to E2. Although the magnitude of response varied with the nature of the promoter, ER{alpha} in the absence or presence of E2 enhanced reporter enzyme activity from all reporter constructs. As both ERs enhance enzyme activity from the C3 promoter in response to E2, we wanted to ensure that this responsiveness to ERß also requires the ERE sequence shown to be critical for the ability of ER{alpha} to enhance transcription (56). We found that the mutation of 5'-GGTGGcccTGACC-3' to a non-ERE sequence, 5'-GTACCTCAGGCAT-3' (Mut), abolished responses to the E2-ER complex, whereas changing it to the consensus ERE, 5'-GGTCActcTGACC-3' (Con), further augmented enzyme activity compared with the WT (C3) promoter. 4-OHT or ICI had no effect on the enzyme activity induced by either ER subtype (data not shown).



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Figure 8. Transcription from Promoters of E2-Responsive Genes by ERß or ER{alpha} in Response to ER Ligands in Transfected Cells

COS-1 or HepG2 cells were transfected with 500 ng CatD, pS2 or C3 promoter constructs that drive the firefly luciferase cDNA as the reporter. Mut and Con indicate mutant C3 promoters in which the upstream 5'-GGTGGcccTGACC-3' ERE sequence, located between residues -235 to -222 from the transcription initiation site, is mutated to a non-ERE sequence, 5'-GTACCTCAGGCAT-3', and to the core consensus ERE, 5'-GGTCActcTGACC-3', respectively. The reporter plasmids were cotransfected with 300 ng expression vector expressing no cDNA (pM2 ), ERß, or ER{alpha} cDNA and 2 ng internal control plasmid bearing CMV promoter that drives the expression of Renilla luciferase cDNA. COS-1 cells were treated for 24 h in the absence (-E2) or presence (+E2) of 10-9 M E2. HepG2 cells were treated without (-Ligand) or with 10-7 M E2, 10-8 M 4-OHT, or 10-6 M ICI. The cell extracts were assayed for luciferases, and the normalized firefly/Renilla luciferase activities are presented as the fold change compared with the control. Shown are the mean ± SEM of three independent experiments performed in duplicate.

 
In HepG2 cells, ERß in the absence or presence of ER ligands had minimal effect on enzyme activity from any of the promoters. Although the E2 or 4-OHT, but not ICI, complexed ER{alpha} augmented enzyme activity from the C3 construct, the receptor had minimal effect on luciferase activity from the CatD or pS2 promoter in the absence or presence of ER ligands.

Thus, it appears that ER{alpha} is the major subtype critical for the regulation of E2-responsive gene in a ligand- and ERE-dependent pathway, independently from the nature of ER ligand, ERE sequence, promoter, or cell context.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We show here that although ER{alpha} and ERß display similar biochemical and intracellular characteristics in transfected cells, the ER subtypes differ in the ability to induce gene transcription. The major findings are: 1) ER{alpha}, but not ERß, mediates transcription synergistically in response to E2 from heterologous promoters bearing tandem ERE sequences. The receptor subtype-specific partial agonistic activity of an antagonist appears to be an additive type of induction. 2) Although a strong AF-1 is critical for the mode of transcription, the subtype-specific AF-2 contributes to the mode and extent of transcription. Differences in the abilities of ERs to integrate both AF-1 and AF-2 are apparently central to the underlying mechanism for the subtype-specific responses in the ligand- and ERE-dependent ER signaling.

ER{alpha}, But Not ERß, Mediates Synergy from Tandem ERE Sequences
We found, as shown previously (3, 4, 5, 6, 7), that the presence of EREs in tandem results in a synergistic response to the E2-ER{alpha} complex in a promoter- and cell context-dependent manner. Moreover, the extent of synergy is determined by the identity of the ERE. Imperfect EREs that deviate from the consensus by one or more nucleotides are less potent transcriptional enhancers than the consensus ERE (3, 4, 5). In contrast, ERß failed to induce trans-activation synergistically, independently from the ERE sequence, ER ligand, promoter, or cell context.

A recent study (60) suggests that in transfected CHO cells, rat ERß, like human ER{alpha}, induces synergistic transcriptional responses. Those investigators observed that ERß, similar to ER{alpha}, induced synergy in response to E2 from the enhancer-less simian virus 40 (SV-40) promoter construct bearing three consensus ERE sequences in tandem with 38-bp center to center distances juxtaposed to the promoter. We, on the other hand, using the same enhancer-less SV-40 promoter reporter construct that bears three consensus EREs in tandem with the same center to center spacing (see Materials and Methods), observed that either ER subtype at various concentrations induces only a minimal response to E2. This was reflected in the negligible transcriptional enhancement [1.4 ± 0.4- and 1.1 ± 0.13-fold (n = 3) for ER{alpha} and ERß, respectively] from three EREs compared with basal transcription. Consistent with our results, a recent report showed that either ER induces transcription minimally from the same enhancer-less SV-40 promoter that bears four consensus ERE sequences (61). Although it is not clear, differences in the experimental conditions, including potential species differences among ERßs, could contribute to the discrepancy between our results and those reported by Tyulmenkov et al. (60).

Cooperative binding to adjacent response elements is thought to be one of the factors that contribute to the steroid hormone receptor-mediated synergy (62). We show here, as shown for ER{alpha} (4, 5), that both ER subtypes bind to adjacent ERE sequences cooperatively. These results indicate that the binding mode to tandem EREs is not responsible for the ERß-specific trans-activation. It appears that the events subsequent to the formation of the agonist-ER-ERE complex are critical for subtype-specific transcriptional responses.

Structural Features Are Critical for ER Subtype-Specific Responses
Structure and function analyses of ER{alpha} led to the identification of two distinct and independent transcriptional ADs, AF-1 and AF-2, localized at the amino and carboxyl termini of ER{alpha}, respectively (63). AF-2 is a hormone-dependent AD. Among the cofactors, the p160 family of coregulators that includes steroid receptor coactivator-1, transcriptional intermediary factor-2 (TIF-2), and amplified in breast cancer-1, as well as other nuclear receptor coregulators, including RIP-140 and TIF-1, interact with the carboxyl terminus of ER in an agonist-dependent manner (64, 65, 66). These cofactor interactions through a signature motif with ERs are required for the activation function of the carboxyl terminus. The AF-1 of ER{alpha} functions independently of the AF-2-containing carboxyl region in yeast and chicken cells, but is ineffective in altering transcription in mammalian cells when isolated from the carboxyl terminus (17, 46, 50, 67). The function of the AF-1 domain of ER{alpha} is dependent upon the structural integrity of the hormone-binding domain and its binding to an agonist (18, 21, 46). Studies further showed that the functional integration of both AF-1 and AF-2 is required for the full potency of receptor function (20, 51, 52). Although the functional characterization of structural domains of ERß is limited, it has been suggested that the absence of a strong AF-1 (22, 23, 24, 25) is responsible for the differences in transcriptional abilities between the receptor subtypes. Preferential interaction of TIF-2, through a distinct receptor-interacting domain and the p68 RNA helicase (21, 26, 52, 68), with the amino terminus of ER{alpha} independent of ligands appears to be one of the reasons for the receptor-specific activity in a ligand- and ERE-dependent pathway of E2 signaling.

In addition to WT ERs, several ER isoforms have been identified in both normal and neoplastic E2 target tissues (for reviews, see Refs. 9 and 69). These isoforms exhibit altered responsiveness to ligand and are thought to be one of the mechanisms that contribute to tumor progression and acquisition of resistance to anti-E2 treatments (69, 70, 71, 72). Primarily detected as single or multiple exon-deleted or truncated transcripts expressed together with the WT transcript, with some cases corresponding proteins, these mutant ERs appear largely to be the products of alternative splicing (69, 73, 74, 75). Moreover, transcripts with variable size nucleotide insertions, exon duplications, and point mutations have been detected and are believed to be generated from a mutated ER allele (69, 73, 74, 75). A functionally distinct form of human ERß has been recently reported (76). This variant ERß contains additional 18 amino acids at the amino terminus and appears to contribute to the AF-1 function of the receptor. Understanding the molecular mechanisms of action of these ER isoforms will be critical for elucidation of their roles in E2 signaling.

Mechanism(s) of Transcriptional Synergy
The mechanism of synergistic transcription induction is yet unknown. Nuclear receptor-mediated gene expression is a dynamic event in which transcription of target genes is modulated by sequential cycles of association and dissociation of cofactor complexes with receptors through acetylation/deacetylation processes (77, 78, 79). It is possible that the ability of ER{alpha} bound to tandem EREs to interact with a variety of cofactors through both the amino and carboxyl termini is a mechanism for synergy that operates by increasing the local concentrations of cofactors. This heterogeneous population of cofactors could lead to extensive modifications of local chromatin architecture, extended transcriptional initiation and reinitiation events, and subsequent synergy, the extent of which is determined by cofactor concentrations that vary in a cell type-dependent manner (8). We observed here that both the ßN{alpha}C and the {alpha}NßC chimeras induce synergy, but neither is capable of inducing transcription with a magnitude similar to that observed with ER{alpha}. Furthermore, the amino-terminal region of ERß functionally interacted with the carboxyl-terminal region of ER{alpha} to induce, albeit weakly, transcription in the two-hybrid system. In contrast, the amino-terminal region of ERß failed to interact with its own carboxyl terminal to induce transcription. This together with potential differences in the affinities of ERs to ligand-dependent cofactors suggest that despite the structural similarity, the carboxyl-terminal activation functions of ERs also differ. Thus, it appears that the extent of functional interactions between the termini correlates with the transcriptional strength of the receptor. This, in turn, implies that although a strong AF-1 is critical for synergy, the integrated effects of AF-1 and AF-2 are required for the receptor subtype-specific transcriptional responses.

We observed, as reported previously (52), that there is no physical interaction between the amino and the carboxyl termini of ER{alpha} as assessed by immunoprecipitation and EMSA (data not shown). These observations suggest that a factor capable of bringing two domains together is involved in the integration of both ADs of ER{alpha}. We (21, 26) and others (52, 68) have recently shown that AF2-dependent TIF-2, through a distinct receptor-interacting domain, is also recruited preferentially by the amino-terminal region of ER{alpha}. Indeed, it appears that TIF-2 can bridge the amino and carboxyl termini of ER{alpha} in vitro and in situ (52). TIF-2-mediated functional interactions between amino and carboxyl termini of the androgen receptor also appear mandatory for the optimal activation of androgen- induced transcription (80, 81, 82). The ability of a cofactor, as exemplified by TIF-2, to concurrently interact with both termini of ER{alpha} could allow the receptor to form a scaffold necessary for an intramolecular interaction within an ER{alpha} dimer or an intermolecular interaction between ER{alpha} dimers bound to tandem EREs. This scaffold could stabilize the E2-ER{alpha}-ERE-cofactor complex interaction, which, in turn, provides the necessary architecture to form a more stable interaction with other cofactors, cofactor-interacting proteins (83), or transcription complexes (84, 85). This would result in high transcriptional responses.

As the mode of transcription primarily describes the magnitude of a response, it is also likely that underlying mechanisms for synergy from tandem ERE sequences are critical for E2-responsive genes wherein a single ERE or multiple EREs are clustered among binding sites for other transcription factors. The ability of the E2-ER-ERE complex to share cofactor or integrator proteins with the other transacting factors specific to each gene could generate an integrated transcriptional complex that will ultimately determine the direction and magnitude of gene expression.

How do antagonists mediate ER subtype-specific responses as partial agonists? 4-OHT and ICI as antagonists exert their effects by impeding the interaction of both ER{alpha} and ERß with AF-2-dependent cofactors by inducing distinct conformation in the ligand-binding domain and recruitment of corepressor proteins (26, 86). We observed here that 4-OHT and ICI prevent transcription induction by hindering the interaction between the amino and carboxyl termini of ER{alpha}. Disruption of the functional interaction between the ADs through prevention of the recruitment of bridging cofactors, as with TIF-2, lends credence to the concept that the integrated effects of both AF-1 and AF-2 are critical for defining the receptor subtype.

As shown here and previously (24, 29, 46, 87, 88, 89), 4-OHT or ICI displays a partial agonistic effect in an ER subtype-, promoter-, and cell context-dependent manner. The partial agonist activity is manifested as lower transcriptional responses compared with the E2-ER{alpha} complex. As 4-OHT or ICI does not affect the recruitment of cofactors to the amino terminus (26, 68), it is likely that the ability of the antagonist-ER{alpha} complex to recruit AF-1-dependent cofactors is responsible for the partial activity (24, 46). However, as 4-OHT or ICI induces an additive response, in contrast to E2, in a cell-context-dependent manner, as we show here, a balance between cell-specific coactivators and corepressors recruited by the antagonist-ER{alpha} complex may be critical for transcription. These findings together with observations that ICI (30, 31, 32, 33, 34, 35, 36) displays an agonistic effect depending upon the promoter and cell context reinforce the need for the development of pure antiestrogenic compounds for the treatment of E2-dependent cancers.

Although it is appears that ER{alpha} is the critical subtype for the regulation of E2-responsive genes in a ligand- and ERE-dependent manner, convergent as well as divergent regulation of these genes through ligand- and/or ERE-independent pathways is also important to ER signaling. In a ligand-independent, but ERE-dependent, pathway, the phosphorylation state of both amino and carboxyl termini of ER{alpha} by different signaling pathways correlates with an altered activity of the receptor in the absence of ligands (9). Although SRC-1 interacts with the carboxyl termini of both ER{alpha} and ERß, recent studies indicate that the ligand-independent recruitment of SRC-1 preferentially by the amino terminus of the murine ERß through phosphorylation by kinase signaling pathways enhances the ability of the receptor to induce transcription (90). Thus, it is also likely that differential post-translational processing of ERs by different signaling pathways could provide ERs with divergent regulatory potential for the modulation of E2-responsive genes. Moreover, studies showed that ER{alpha} and ERß act in opposite ways to modulate gene expression in a ligand-dependent, but ERE-independent, manner (91). Although the E2-ER{alpha} complex activates the gene transcription from the activator protein-1 site, ERß bound to E2 inhibits transcription. Conversely, the antagonist-occupied ERß is a potent transcription activator. The divergent activation of E2-responsive genes is shown to occur also at a GC box site (92). The E2-ER{alpha} complex activates transcription through an interaction with the DNA-bound GC box-binding protein (Sp-1), whereas ERß fails to induce transcription at this site.

It is evident that the integrated effects of a complex array of pathways achieve the transcription of an E2-responsive gene in a spatio-temporal manner. Delineation of the molecular mechanism of action of ERs and their isoforms in these pathways will be crucial for the understanding the physiology and pathophysiology of ER signaling.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
The human WT and mutant ER{alpha} and ERß cDNAs without or with the Flag epitope was described previously (21, 26, 48). The WT ERß cDNA encodes a 530-amino acid-long protein. All ER truncation mutants were generated by PCR. In all constructs, the first methionine and the stop codon, which are underlined, are within the context of the Kozak sequence (CGCCATG) and a Poly A (TAATAAA) signal, respectively. The cDNA for ER{alpha}-AF2 mutant contains a three-amino acid replacement (D538A, E542A, and D545A) that destroys the AF-2 function of ER{alpha} (18, 48). The same amino acid substitutions at corresponding positions (D489A, E493A, and N496A) were also created in ERß to generate the AF2 mutant using an overlapping PCR. All cDNAs were inserted into the expression vector pM2 as previously described (48).

To examine the roles of the ADs, we constructed chimeric ER{alpha} and ERß receptors. The ERß cDNA contains an EcoRV restriction enzyme site at sequences surrounding codon 158 [based on the report by Ogawa et al. (93)]. The ER{alpha} and ERß cDNAs share high sequence homology in regions corresponding to the DNA-binding domains of both receptors (40, 93). This conservation allowed us to create an EcoRV site (24) at the same region of ER{alpha} that flanks codon 194 [based on the report by Green et al. (94)] by PCR-directed mutagenesis. We used primers containing nucleotide sequences that introduce silent changes without altering the amino acid sequences of the receptor. The mutant ER{alpha} cDNA was then sequenced. We then exchanged the SalI-EcoRV fragment, which contains the entire A/B region, of ERß with that of ER{alpha} and vice versa. These exchanges generated the chimeric ßN{alpha}C and {alpha}NßC receptors.

The simple TATA box promoter reporter construct contains a TATA box. Double-stranded oligomers containing one or two ERE sequences were inserted into this construct at various distances (2–14 helical turns; 10.5 bp/turn) from the promoter. The center to center distance between the two EREs were 38 bp, which is an optimal distance between EREs for synergistic transcription (5, 6, 7). In generating reporter plasmids bearing complex promoters, we used promoters with or without their enhancer regions. We reasoned that induction of transcription of the reporter enzyme from a promoter whose enhancer region was replaced by an ERE sequence(s) could be more readily observable in response to ERs and ER ligands. For construction of the moderately strong reporter plasmid bearing the TK promoter/enhancer of the herpes simplex virus, the entire TK promoter/enhancer (760 bp) was excised from the pRL-TK plasmid (Promega Corp., Madison, WI) and inserted into pGL3-Basic (Promega Corp.) linearized with the same restriction enzymes. The enhancer-less TK promoter was generated by PCR using PRL-TK as the template. The 236-bp-long fragment generated corresponding to the TK promoter was inserted into pGL3-Basic. For construction of the SV-40 promoter/enhancer plasmid, the excised fragment containing the entire SV-40 early promoter/enhancer from the plasmid pRL-SV40 (Promega Corp.) was inserted into the pGL3-Basic plasmid, which was linearized with the same restriction enzymes. The pGL3-Promoter plasmid that bears the enhancer-less SV-40 promoter was used in generating the SV40 enhancer-less reporter plasmid. Double-stranded oligomers containing one or two ERE sequences in tandem as described above were then inserted immediately upstream of a given promoter linearized with appropriate restriction enzymes. All constructs were sequenced. In all reporter constructs, promoters drive the firefly luciferase enzyme cDNA as the reporter.

In the construction of reporter plasmids bearing promoters from E2-responsive genes, we used CatD, pS2, and C3 promoters. The CatD and C3 promoters were generated by PCR using pCD3542 (58) and pC3T1 (18, 56) reporter plasmids as templates. A fragment of 730 bp [-750 to -22, +1 is referred to the first ATG (58)] of the promoter region of CatD gene or 580 bp (-516 to +61, +1 as the transcription start site) of the C3 gene (95) was generated. A 750-bp (-703 to +34, +1 as the transcription start site) fragment upstream of the initiation codon (ATG) of the pS2 gene was obtained by PCR from MCF-7 cell genomic DNA using oligonucleotides based on published sequences (96). PCR fragments were then inserted into the reporter plasmid pGL3-Basic linearized with appropriate restriction enzyme sites. We used an overlapping PCR approach in the generation of ERE mutant C3 promoters by using oligomers containing no or the consensus ERE sequence. Mut and Con indicate mutant C3 promoters in which the upstream 5'-GGTGGcccTGACC-3' ERE sequence, -235 to -222 from the transcription initiation site (95), is mutated to a non-ERE sequence, 5'-GTACCTCAGGCAT-3', and to the core consensus ERE, 5'-GGTCActcTGACC-3', respectively. All constructs were sequenced.

Cell Culture
Culturing and transfection of mammalian cells were described previously (21, 48), except that a reporter plasmid bearing the Renilla luciferase cDNA (Promega Corp.; 2 ng/well) was used to monitor transfection efficiency. Three hours after transfection, cells were washed once with PBS and incubated with fresh medium containing no (0.01% ethanol) or various concentrations (10-11–10-6 M) of E2 (Sigma- Aldrich Corp., St. Louis, MO), 4-OHT (Sigma-Aldrich Corp.), or ICI (Tocris, Inc., Ballwin, MO) for 24 h. Luciferase assays were performed with a dual luciferase assay kit (Promega Corp.).

We found that transcription response from a single ERE to either ER in the absence or presence of ligands was minimal in transfected mammalian cells independent of the nature of promoter and the distance of ERE to the promoter. For example, when the optimal ERE was placed 2, 3, 4, or 5 helical turns (10.5 bp/turn) away from the TATA box, there was no change in the level of enzyme activity in response to ER{alpha} regardless of whether E2 was present in transfected cells. The response to ER increased gradually with additional helical turns and maximized at 8 turns (5.3 ± 0.8-fold; n = 3) when cells were treated with 10-9 M E2 compared with the basal transcription. The activity remained the same at a distance of 14 helical turns. A similar response pattern, but not the magnitude, which was 2.1 ± 0.3-fold (n = 3) compared with the TATA box alone, was observed for ERß.

Although transcriptional response from a single ERE to either ER was low, the extent of transcriptional responses from reporter plasmids induced by ERs differed in the presence of a second, adjacent ERE. In transiently transfected mammalian cells, the extent of transcriptional response from two ERE sequences was correlated with the positioning of the first ERE with respect to the TATA box. We found, for example, that ER{alpha} augmented luciferase activity about 20-fold from 2 consensus EREs when the first ERE sequence was placed at 5 helical turns away from the TATA box compared with the promoter construct bearing no ERE sequence in transiently transfected COS-1 cells. However, the response increased to 90-fold when the first ERE was 10 helical turns (106 bp) away from the promoter. This distance was optimal, as there was no further augmentation of the extent of transcription when the first ERE was placed further away from the promoter. We therefore used reporter constructs bearing a single ERE or ERE sequences in tandem with 38 bp center to center spacing in subsequent experiments. In all TATA box constructs, the distance between the center nucleotide in the 13-base core of the first ERE and the TATA box is 106 nucleotides. In the construction of the enhancer-less TK promoters bearing a single ERE or tandem EREs, the ERE sequence was juxtaposed to the end of the promoter, and the center to center spacing between the 2 EREs was 38 nucleotides.

We also found that the magnitude of transcriptional response from reporter constructs bearing ERE sequences in tandem was correlated with promoter strength, as recently reported (61). We observed that the induction of a transcriptional response from two ERE sequences in tandem was the highest with the TATA box promoter, whereas the enhancer-less TK promoter showed a moderate augmentation to ERs (see Figs. 1Go and 2Go). On the other hand, transcription from the TK promoter with its enhancer or the SV40 promoter without or with its enhancer region was minimal in all cell lines examined (data not shown). We therefore used the simple TATA box promoter or the enhancer-less TK promoter constructs that bear zero, one, or two ERE sequences in tandem.

Western Blotting
Western blotting was performed as previously described (48). Proteins were probed with the ER{alpha}-specific polyclonal HC-20 antibody (Santa Cruz, Biotechnology, Inc., Santa Cruz, CA), ERß-specific polyclonal D7N (Zymed Laboratories, Inc., San Francisco, CA), or the monoclonal Flag antibody (M2, Sigma-Aldrich Corp.).

Immunocytochemistry
The preparation and transfection of cells for immunocytochemistry were performed as described previously (48). Cells were incubated in medium in the absence or presence of various concentrations of E2, 4-OHT, or ICI for 24, 3, or 1 h before the termination of an experiment. The primary antibodies were HC-20, PA1-313 (a polyclonal antibody specific for ERß; Affinity BioReagents, Inc., Golden, CO), and M2. Proteins were visualized with fluorescein-conjugated (Santa Cruz Biotechnology, Inc.) secondary antibody. The cover glasses were mounted on a glass slide (VWR Scientific, Bridgeport, NJ) using a mounting medium containing 4',6-diamido-2-phenylindole hydrochloride (Vectashield, Vector Laboratories, Inc., Burlingame, CA) for nucleus staining and were examined by fluorescence microscopy.

Subcellular localizations of the ERs in transfected mammalian cells were independent of the expression vector (pM2 vs. pcDNA3.1; Invitrogen, Carlsbad, CA) bearing the receptor cDNA used, the cell-staining procedure (immunofluorescence vs. immunoperoxidase) employed, or the dilution of the first antibody (1:50 vs. 1:100) used. All experiments were repeated at least two different times in duplicate.

EMSA
Oligomers bearing single or two ERE sequences in tandem were annealed and 32P end-labeled as previously described (12, 48). Labeled oligomers were incubated in the absence or presence of equal amounts of nuclear extract (97) of cells transfected with expression vectors bearing no cDNA (control) or ER{alpha} or ERß cDNA. Reactions were further incubated with or without the antibodies, unlabeled ERE, 10-6 M E2, 4-OHT, or ICI in a total volume of 25 µl at 4 C for 1 h. Reactions were subjected to electrophoresis on 4% nondenaturing polyacrylamide gel. In assessing the cooperative binding of ERs, we used annealed oligomers bearing the consensus (17-17) or nonconsensus (17d2-17d2) EREs in tandem with 38-bp center to center spacing, which are identical with those in reporter constructs used in transfection assays. 32P-end-labeled oligomer (0.125 nM) was then incubated with various concentrations (0–18.75 nM for 17-17; 0–75 nM for 17d2-17d2) of ER{alpha} or ERß for 1 h at 4 C. Reactions were resolved on 4% nondenaturing polyacrylamide gel.

E2 Binding Assay
E2 binding was performed with equal amounts of COS-1 or CHO nuclear extracts, transfected with the expression vector bearing none (as control), ER{alpha}, or ERß in the absence of exogenously added hormone. Extracts were incubated with 10-9 M 16{alpha}-[125I]-iodo-3,17ß-estradiol (2200 Ci/mmol; NEN Life Science Products, Boston, MA) in the absence or presence of 10-6 M unlabeled E2 as described previously (48, 98). Bound radioligand was separated from unbound by column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA) and counted.

Interference Assay
The interference assay is based on the physical interference of transcription through the binding of ER to two consensus ERE sequences placed between the TATA box of the strong CMV promoter and the start site of transcription of CAT cDNA (45). The reporter plasmid (100 ng) pCMV(ERE)2CAT (provided by Dr. Benita S. Katzenellenbogen) or pCMV-CAT control plasmid that does not bear ERE sequences was transfected together with expression vectors bearing WT or mutant ER cDNA (0–2000 ng/well) into mammalian cells as described above. A reporter plasmid bearing ß-galactosidase cDNA (200 ng/well) was also cotransfected for monitoring transfection efficiency. Cells were then incubated in the absence or presence of ER ligands for 24 h, and cell lysates were assayed for the expression of CAT as previously described (99). CAT values were normalized with ß-galactosidase expression.

Two-Hybrid Assay
In generating the fusion proteins for the two-hybrid system, we used the Mammalian Matchmaker Two-Hybrid Assay Kit from CLONTECH Laboratories, Inc. (Palo Alto, CA). DNA fragments bearing the amino terminus of ER{alpha} (amino acids 1–180) and that of ERß (amino acids 1–148) and the carboxyl terminus of ER{alpha} (amino acids 301–595) and that of ERß (amino acids 287–530) were generated by PCR using the WT ERs as templates. The amino-terminal region of either ER was inserted into the pVP16 activation domain vector with appropriate restriction enzyme sites, whereas the carboxyl termini were inserted into the pM Gal4 DBD cloning vector. Constructs (300 ng) with appropriate combinations were transfected into COS-1 cells together with a reporter plasmid (500 ng) with five Gal4 response elements juxtaposed to a simple TATA box promoter that drives the expression of the firefly luciferase enzyme cDNA (Stratagene, La Jolla, CA) as described above. Transfection efficiency was monitored by coexpression of a reporter plasmid bearing SV40 promoter that drives the expression of Renilla luciferase cDNA (2 ng). Maximal enhancement of luciferase activity was observed at 48 h after transfection and only in the presence of 10-8 M E2, the minimal concentration that maximally stimulated luciferase activity in response to receptor constructs. We therefore incubated transfected cells without or with 10-8 M E2, 10-6 M 4-OHT, or ICI for 48 h. Cell extracts were processed for the dual luciferase assay.


    ACKNOWLEDGMENTS
 
We thank Drs. Patrick Augereau, Benita Katzenellenbogen, and Donald P. McDonnell for providing the pCD3542, pCMV(ERE)2 and pC3T1 plasmids. We thank Drs. Patricia M. Hinkle, Mark E. Dumont, and Scott Gibson for critical reading of the manuscript.


    FOOTNOTES
 
This work was supported by NIH Grant HD-24459 (to R.H., R.A.B., and M.M.).

P.Y. is the Recipient of a predoctoral fellowship from the Department of Defense Breast Cancer Research Program (DAMD 179717227).

Abbreviations: AD, Activation domain; AF-1, activation function-1; C3, third component of complement; CAT, chloramphenicol acetyltransferase; CatD, cathepsin D; CMV, cytomegalovirus; DBD, DNA-binding domain; E2, 17ß-estradiol; EMSA, electrophoretic mobility shift assay; ER, estrogen receptor; ERE, estrogen response element; ICI, ICI 182,780; 4-OHT, 4-hydroxytamoxifen; SV-40, simian virus 40; TIF-2, transcriptional intermediary factor-2; TK, thymidine kinase; WT, wild-type.

Received for publication November 29, 2001. Accepted for publication April 22, 2002.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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