The Effects of Estrogen-Responsive Element- and Ligand-Induced Structural Changes on the Recruitment of Cofactors and Transcriptional Responses by ER{alpha} and ERß

Ping Yi, Mark D. Driscoll1, Jing Huang, 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, 601 Elmwood Avenue, Box 712, Rochester, New York 14642. E-mail: Mesut_Muyan{at}urmc.rochester.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen signaling is mediated by ER{alpha} and -ß. ERs are converted from an inactive form to a transcriptionally active state through conformational changes induced by ligand and estrogen-responsive element (ERE) sequences. We show here that ER{alpha} and ERß bind to an ERE independently from ER ligands. We found that although the binding affinity of ERß for an ERE is 2-fold lower than that of ER{alpha}, both ERs use the same nucleotides for DNA contacts. We show that both EREs and ligands are independent modulators of ER conformation. Specifically, the ERE primarily determines the receptor-DNA affinity, whereas the structure of the ER ligand dictates the affinity of ER for particular cofactors. We found that the ligand-dependent cofactor transcriptional intermediary factor-2, through a distinct surface, also interacts with ER{alpha} preferentially and independently of ligand. The extent of interaction, however, is dependent upon the ER-ERE affinity. In transfected cells, ER{alpha} is more transcriptionally active than ERß. The ERE sequence, however, determines the potency of gene induction when either ER subtype binds to an agonist. Antagonists prevent ERs from inducing transcription independently from ERE sequences. Thus, ERE- and ligand-induced structural changes are independent determinants for the recruitment of cofactors and transcriptional responses. The ability of ER{alpha} to differentially recruit a cofactor could contribute to ER subtype-specific gene responses.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGEN RESPONSES ARE mediated by ER{alpha} and -ß. ERs are members of a superfamily of nuclear receptors that function as ligand-modulated transcriptional regulators. Although ER{alpha} and -ß share high structural homology in their DNA binding domain (DBD), the amino- and carboxyl-terminal regions of the receptors that possess ligand-independent and -dependent activation functions, AF-1 and AF-2, respectively, are less conserved (1). ER{alpha} and ERß display similar biochemical characteristics (2, 3). However, distinct structural features that account for different transcriptional responses to ligands in a promoter- and cell-dependent manner also indicate functional differences between the receptor subtypes (4, 5, 6, 7, 8, 9).

The elements of estrogen-responsive gene transcription involve a multistep regulation in which ER is converted from an inactive form to a transcriptionally active state. This regulation is initiated by a conformational change in ER upon estrogen binding, dissociation from chaperone proteins, and dimerization (10). The ER then binds to a palindromic DNA motif, estrogen-responsive element (ERE), recruits cofactors, and alters transcription (11). The minimal, or the core consensus ERE, is a 13-bp inverted repeat, 5'-GGTCAnnnTGACC-3' (4, 6). Estrogen-responsive genes, however, contain single or multiple copies of EREs that deviate from the consensus sequence by one or more nucleotides. Although these EREs confer estrogen responsiveness mediated by ER{alpha}, they are less potent enhancers of transcription than the consensus ERE (12, 13, 14, 15, 16). EREs are modulators of the conformation of the DBD of ER (17, 18, 19, 20, 21), as shown for numerous transcription factors and nuclear receptors (22). A single nucleotide change in the consensus ERE, for example, requires the formation of new interconnected hydrogen bonds between the response element and the DBD of ER{alpha}, thereby altering the conformation of the region (18, 19).

In addition to estrogens, ER also binds antiestrogenic compounds that act as agonist and/or antagonist. Although the pure antiestrogens, ICI 182,780 (ICI), are effective antagonists, tamoxifen, or its active metabolite trans-4-hydroxytamoxifen (4-OHT), displays mixed agonist/antagonist properties depending upon promoter and cell context (23, 24, 25). Antiestrogens sterically hinder correct alignment with the interacting surfaces of the carboxyl-terminal ligand binding domain (LBD) of ERs and alter the conformation of the region. Recent studies indicate that the LBDs of ER{alpha} and ERß, despite the poor amino acid homology, display similar tertiary and quaternary architecture (26, 27). Although this accounts for the similar binding affinity of ligands to both receptors (9), differences in the amino acid sequences between the LBDs of ERs are also responsible for subtype-specific alterations in the conformation of LBD induced by antiestrogens (27). Studies also suggest that interactive conformational changes occur among the receptor domains upon ligand binding (21, 28, 29).

In this report, we address how various ligands cause alterations in the conformation of ERß and ER{alpha} that influence the receptor affinity and specificity for binding with ERE sequences, and, in turn, the ability of DNA-bound and ligand-occupied receptors to recruit cofactors. We show here that the extent of AF-2-dependent cofactor recruitment by ER{alpha} or ERß is affected by both ER ligands and ERE sequences. When ER is liganded with E2, the extent of cofactor recruitment is primarily affected by the ERE sequence, which determines the affinity, and thereby the relative amount, of receptor binding. 4-OHT or ICI occupation of the receptors prevents cofactor interactions with the receptors independently of the identity of ERE sequences. The recruitment of both AF-1- and AF-2-dependent cofactors through different domains, exemplified by transcriptional intermediary factor (TIF)-2, occurs in a receptor subtype-specific manner. In transfected mammalian cells, we found that ER{alpha} is a more potent transcription activator in response to an agonist than ERß, regardless of ERE sequence. However, the identity of ERE sequence determines the potency of transcription when ERs bind to E2. This was also the case for 4-OHT or ICI when both compounds display partial agonist activity. When 4-OHT or ICI acts as pure antagonist, it prevents ERs from inducing transcription independently from ERE sequences. These results collectively indicate that ERE- and ligand-induced structural changes are independent determinants for the recruitment of cofactors and transcriptional responses by ERs. Moreover, we suggest that the ability of ER{alpha} to differentially recruit a cofactor through distinct interacting surfaces could contribute to ER subtype-specific gene responses.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ERE Sequences That Bind ERß with High Affinity
The core ERE sequence is a 13-bp palindrome, 5'-GGTCAnnnTGACC-3'. ER{alpha} binds this sequence efficiently and mediates gene transcription in situ in response to estrogen (30). However, only a few of the most highly responsive genes have this palindromic sequence. Most estrogen-regulated gene sequences vary from the core by one or more nucleotides (12, 13). Additionally, many genes have commonly appearing nucleotides that flank the core sequence. Guided by sequences present in natural settings, we previously designed and tested a series of ERE oligomers for binding affinity to ER{alpha} by using EMSA and binding competition assays (12). Results indicated that sequences with variations in the core retain effective binding affinity for ER{alpha} if they have appropriate flanking sequences. We then proposed rules that predict whether an ERE-like sequence confers high-enough ER affinity to be a functional ERE.

Although the DBDs of ERß and ER{alpha} are highly homologous, a recent study suggests that ER{alpha} and ERß display different binding patterns for natural EREs having one or more nucleotide changes from the core sequence (13). This implies that the binding affinity and specificity of ERs to various EREs may not be the same. To examine whether the rules developed for ER{alpha} also serve as a useful guide for the binding of ERß to ERE-like sequences, we measured affinity to a series of EREs varying in sequence (Fig. 1Go). The DNA substrates consist of a test sequence with one, two, or three nucleotide changes (boldfaced) from the core ERE embedded (braces) within a larger oligomer with no ERE features (background oligomer). The binding ability of ERs to EREs (summarized in Fig. 2BGoGo) was assessed by EMSA (Fig. 2AGo).



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Figure 1. Test ERE Sequences

A, The base oligomer sequence that surrounds the ERE test sequence. Braces enclose the region into which the test sequence is inserted. B, 5' to 3' of the upper strand of a test ERE sequence. Underlining indicates the extended bases that surround the 13-base perfect palindromic sequence (p13). Boldfaced type indicates base changes in the p13 sequence. Center nucleotides are shown in lowercase type. C, Numeration of bases in the optimal ERE sequence depicting the vitellogenin A2 gene ERE. The bases are numbered according to their distance from the center nucleotide A (0).

 


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Figure 2. Binding of ER to ERE Sequences

A, The binding ability of ERß to various ERE sequences was assessed by EMSA. 32P-end labeled ERE (0.125 nM) was incubated with 0, 2.5, 5, 10, 15, and 20 nM of recombinant human ERß on ice for 1 h and subjected to 8% nondenaturing gel electrophoresis. Gels were dried and exposed to PhosphorImager. Free and Bound indicate the unbound and ER-bound ERE oligomers, respectively. B, Summary of comparative analysis of ER binding to various ERE sequences. Depicted is the quantitative analysis of percent bound ERE. The data are the mean of three independent experiments. For simplicity, the SEM, which was less then 10% of the mean is not shown. C, Displacement of radiolabeled p17 ERE bound to ER by unlabeled p17, p17d1, or p17d2 ERE. 32P-End-labeled ERE (0.125 nM) was incubated with 15 nM ER on ice for 30 min. Reactions were then incubated with increasing concentrations of p17 ERE (0, 1.875, 3.75, 5.625, 11.25, 15 nM), p17d1 ERE (0, 2.5, 5, 10, 15, 20 nM), or p17d2 ERE (0, 25, 50, 75, 100, 125 nM). Bound and free fractions quantified by PhosphorImager were used in the estimation of binding affinity (Kd). A representative phosphor image of three independent experiments is shown.

 


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Figure 2A. Continued.

 
We first examined how one or more nucleotide substitutions in the core affect ER binding. ERß interacts in a concentration-dependent manner with the p13 that contains the Vitellogenin A2 core (Fig. 2AGo). Increasing formation of ERß-ERE complex (Bound) was correlated with a decrease in the labeled ERE that remained unbound (Free). A single change in the core (13d1) reduced ERß binding about 10-fold (Fig. 2AGo). Binding was undetectable when two nucleotide changes were introduced (13d2). The decreased binding of ERß to p13d1 was independent from the identity of the nucleotide or the position of substitution (data not shown).

We also tested whether specific flanking nucleotides could increase or restore receptor binding. As a first approximation, we used the flanking nucleotides of the Vitellogenin A2 gene ERE. Placement of an A residue at position -7 and a T at position +7 without (p15) or with one nucleotide substitution (p15d1) increased the ERß binding compared with that with p13 and p13d1 2- and 10-fold, respectively. The same flanking substitutions also restored binding by compensating for two substitutions within the core (p15d2). These results indicate that the flanking sequences are important for binding (Fig. 2AGo). Binding of the receptors to p15 with three nucleotide changes in the core (p15d3) was negligible (data not shown). A G residue at the 5'-end with a C placement at the 3'-end of the core (15GC) also enhanced the ERß binding. A C residue (15CG) or a T (15TA) was not as effective as the A or G (Fig. 2BGo). Similar results were obtained for ER{alpha} (data not shown). These results indicate that the identity of the nucleotides immediately flanking the core can be critical for effective binding.

We also examined whether nucleotides that are two residues distant from the core sequence further affect receptor binding to p15. We placed a 5'-C two nucleotides away from the core, i.e. at position -8, and a 3'-G at position +8, reflecting the Vitellogenin A2 gene ERE, CAGGTCAnnnTGACCTG-3'. Results revealed that the binding of ERß to p17 with one (p17d1, data not shown) or two (p17d2, data not shown) nucleotide substitutions displayed a binding specificity and affinity (see below) similar to those for p15, p15d1, and p15d2. However, we also found that ERß binds to p15d1TA with lower efficiency than to the identical sequence containing an additional 5'-C and a 3'-G residues at position -8 and +8, respectively, (p17d1TA; data not shown). Thus, bases that are two residues away from the core sequence can have an impact for binding of ERß if the core ERE has a variation from the consensus. Overall, we found that ERß binds similarly to the same range of ERE sequences, as does ER{alpha}. ERß, as ER{alpha}, binds to 5'-C(A/G)GGTCAnnnTGACC(T/C)G-3' sequence with an optimal efficiency.

We also assessed the relative affinity of ERß to various ERE sequences in comparison with ER{alpha} by competitive displacement assays (Fig. 2CGo). EREs were tested for the ability to compete with the optimal ERE sequence (p17) for ER{alpha} and ERß binding. Increasing concentration of competitor (unlabeled) ERE resulted in a gradual decline in the bound fraction that represents the ER-radiolabeled ERE complex and an increase in the free fraction that represents the displaced radiolabeled ERE. Quantitative analysis revealed that while the binding affinity (Kd) of ERß for p17 was 5.6 ± 0.4 nM, one (p17d1) or two nucleotide substitutions (p17d2) reduced the Kd values of the receptor to 11.3 ± 3.4 and 101.2 ± 5.3 nM, respectively. Although both ERs bound to the ERE sequences with indistinguishable specificity, the affinity of ER{alpha} to EREs was 2-fold higher than that of ERß. Kd values of ER{alpha} for p17, p17d1, and p17d2 ERE sequences were 2.9 ± 0.7, 6.3 ± 1.5, and 53.5 ± 4.2 nM, respectively.

In addition to the natural hormone E2, ERs also bind to compounds that act as partial agonists (4-OHT), or antagonists (ICI 182,780). Because the nature of the ligand is critical for the extent of transcriptional responses from ERE-driven promoters (31), we also addressed whether ligands alter the affinity and specificity of ER{alpha} and ERß binding to various ERE sequences. We preincubated the receptors with a saturating concentration (10-6 M) of E2, 4-OHT, or ICI. We found that ligands had minimal, if any, effects on the binding pattern or affinity of ERs to EREs (data not shown). These results, as shown previously for ER{alpha} (23, 32), demonstrate that ligands do not have an impact on the ability of the receptors to bind to an ERE.

Although the affinity of ERß for EREs is lower than that of ER{alpha} independent of ligands, the similar DNA binding specificity suggests that the same range of sequences binds to both receptors. However, because the binding affinity of ER{alpha} to an ERE correlates with the extent of transcription (14, 15, 33, 34, 35), a lower binding of ERß could be one of the underlying mechanisms for the differences in the transcriptional strength of the receptors (1, 4).

ER{alpha} and ERß Utilize the Same Nucleotides for Binding to an ERE
Previous studies indicated that ER{alpha} makes contacts with one face of the palindromic sequence in adjacent major grooves of DNA (19, 36). The interactions are mediated by the binding of the first zinc-finger motif of each DBD that makes base-specific contacts within the major groove of the DNA helix, while the second zinc-finger motif forms a dimer interface between the two DBDs. These interactions determine the specificity of the response element recognition. Highly conserved amino acid sequence identity of the two DBDs, together with a similar binding specificity to EREs, as we showed here, suggests that the ER{alpha} and ERß employ similar contacts with EREs. However, our results also indicate that ERß binds to an ERE with a lower affinity than ER{alpha}. Because reduced binding affinity to nonconsensus EREs results from the formation of alternative patterns of intermolecular contacts between DNA and ER (19), it is also possible that ERß makes different nucleotide contacts when binding to an ERE compared with ER{alpha}. To address this issue, we used missing nucleoside hydroxyl radical assay (HRA) to detect differences in binding contacts between the two receptors. Because the affinities of both ERs to p17d2 are substantially lower than that for p17, we also examined whether contact sites are altered when ERs bind to a nonconsensus ERE.

HRA assesses the contribution to protein binding of each member of a base pair independently of all other nucleotides in a linear double-stranded DNA molecule (37). This method is based on the expectation that if a base important for binding were missing in a particular DNA molecule, the protein binding affinity would be adversely affected. For this assay, hydroxyl radical treatment is used to remove a single nucleoside from DNA. This generates DNA fragments containing fewer than one randomly placed one-nucleoside gap per fragment, allowing the analysis of DNA-protein interaction at single-nucleotide resolution.

The receptors were incubated with 5'- or 3'- (data not shown) end labeled and gapped test oligomers. The ER-bound EREs were separated from unbound EREs by nondenaturing PAGE. Radioactive bands containing the bound and free ERE were excised from the gel and eluted and were then subjected to denaturing PAGE analysis (Fig. 3Go). A low-intensity, or missing, band in the lane containing ERE-bound ERs (lanes 5 and 6) or, conversely, a high-intensity band in the lane containing free ERE (lanes 4 and 7) identifies a nucleoside important for the formation of the ER-ERE complex. The intensities of individual DNA bands were quantified by PhosphorImager. Shown in Fig. 3Go is the TGACC half-arm of the bottom strand. A high ratio of free-to-bound ERE is represented as long horizontal bars and approximates the strength of contacts with ERs. The crystal structure of the DBD of ER{alpha} indicates that the G at position +3, and the T and A at position +2 and +4, respectively, make multiple contacts with the region (19, 36). We also observed that both ER{alpha} and ERß have a strong interaction with the G at position -3 and the T and A at position -2 and -4, respectively, of the TGACC half-arm of the bottom strand of the consensus ERE. Similarly, the G at position +3 and surrounding T and A contacted the receptors in the TGACC half-arm of the upper strand (data not shown). The G residues at positions -5 and -6 on the GGTCA half-arm of the upper strand, together with flanking bases A and C at position -7 and -8, also showed contacts with both receptors (data not shown). These results demonstrate that both ER{alpha} and ERß make contacts with the same nucleosides in the consensus ERE.



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Figure 3. Identification of Critical Nucleosides of ERE Sequences for Interaction with ER

The missing nucleoside HRA was used to identify the critical contact sites in ERE sequences that are used by ER{alpha} and ERß. The receptors were incubated with the 5'-end-labeled and gapped optimal ERE without (p17; upper panel) or with two nucleotide substitutions (p17d2; lower panel) that are circled. The ER-bound EREs were separated from the free ERE by nondenaturing PAGE. Radioactive bands containing the bound and free ERE were excised from the gel and eluted. DNA was then subjected to denaturing PAGE analysis. A low-intensity, or missing, band in the lane containing ER{alpha} (lane 5) or ERß (lanes 6), or conversely a high-intensity band in the lane containing free ERE (lanes 4 and 7), identifies a nucleoside important for the formation of the ER-ERE complex. This was assessed by the Maxam-Gilbert G-specific sequencing reaction (lanes 3 and 8). Lanes 1 and 2 represent the uncut DNA and DNA subjected to hydroxyl radical treatment in the absence of any protein, respectively. The intensities of individual DNA bands were quantified with PhosphorImager. The ratio of free to bound DNA at each base was plotted for the half-arm of the CCAGT sequence or the nonconsensus CCCGA sequence (changes are in bold) in the bottom strand of ER{alpha} (left column) or ERß (right column). A high ratio, represented as long horizontal bars, approximates the strength of nucleoside contact with the receptors. Arrows indicate A residues deciphered by the Maxam-Gilbert G-specific sequencing reaction. A representative autoradiogram of three independent experiments is shown. Bars are the mean of three independent experiments. The SEM, which was less than 15% of the mean, is not shown for simplicity.

 
When two nucleotide substitutions were introduced into the consensus sequence (p17d2), the contact sites were altered only in the TGACC half-arm of the bottom strand. Changing the T to an A at position -2 had no significant effects on contacts. The G at position -3 remained critical for interaction with both ER{alpha} and ERß. Strong interaction was lost when the A at position -4 was substituted with a C. The C at position -5 and the flanking C and T then became the critical bases for contacts, further emphasizing the role of flanking sequences in the binding of receptors to nonconsensus EREs.

Thus, despite differences in affinities, ER{alpha} and ERß utilize the same nucleotides for binding to an ERE. Base contact sites are altered when the receptors interact with a nonconsensus ERE. These results reaffirm our conclusion that the ER subtypes interact with EREs similarly. Furthermore, they suggest that the decreased DNA affinity of ERs for nonconsensus EREs result from the formation of alternative patterns of intermolecular contacts.

ER Ligand-Induced Conformational Changes in ER
Ligand binding results in substantial alterations in the conformation of ER{alpha} manifested as changes in receptor hydrophobicity, epitope exposure, and protease sensitivity (38, 39, 40, 41, 42). Limited proteolysis has been a useful structural probe for investigating the effects of ligand on ER conformation (41, 42, 43). To examine how different ligands alter protease accessibility of ER{alpha} and ERß, we preincubated equal molar concentrations of [35S]-methionine-labeled receptors synthesized in vitro in the absence (control, 0.01% ethanol) or presence of 10-6 M E2, 4-OHT, and ICI. They were then subjected to partial proteolysis with varying concentrations of chymotrypsin or trypsin. Reactions were resolved on an 8–16% gradient SDS-PAGE.

As shown for ER{alpha} (42, 43), and recently for ERß (43), proteolysis of ERß with increasing concentrations of chymotrypsin (Fig. 4Go) resulted in proteolytic fragments with distinct electrophoretic migration. In the absence of ligand, proteolysis of the full-length ERß (molecular mass of 60 kDa, lane 1) produced four distinct fragments that migrate with molecular mass of 35, 33, 31, and 27 kDa, corresponding to ßC1, ßC2, ßC3, and ßC4, respectively (lanes 2–6). When the receptor was occupied with E2 (lanes 7–12), the ßC1 and ßC2 (lanes 11 and 12) were more resistant to the same concentration of protease than in the absence of ligand (lanes 5 and 6). This was reflected in a slower disappearance of ßC1 and a delayed appearance of ßC4. The electrophoretic migration patterns of proteolytic fragments of ERß complexed with 4-OHT (lanes 13–18) were similar to those of ERß bound to ICI (19, 20, 21, 22, 23, 24). However, the degree of enzyme sensitivity of these fragments differed from those observed for ERß both in the absence or presence of E2. Quantitative appearance of ßC3 and subsequent further proteolysis to ßC4 occurred at lower concentrations of protease. Moreover, we also observed that the appearance of ßC3 occurs at a lower enzyme concentration when ERß was bound to 4-OHT (lane 16) in contrast to ICI (lane 22). Results were similar with exposure to trypsin (data not shown). These results indicate that each ligand is capable of inducing a distinct conformation in the receptor.



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Figure 4. The Effects of ER Ligands on the Pattern of Partial Proteolysis of Radiolabeled ERs

[35S]-Methionine-labeled equal molar concentrations of ERß and ER{alpha} synthesized in vitro were incubated without (0.01% ethanol) or with 10-6 M E2, 4-OHT, or ICI at room temperature for 10 min. Reactions were then subjected to 1, 2.5, 5, 10, and 25 µg/reaction chymotrypsin for 10 min at room temperature. Reactions were analyzed by 8–16% gradient SDS-PAGE and visualized by PhosphorImager. The positions of molecular mass markers in kilodaltons are shown on the left. The estimated molecular mass of the full-length ERß is 60 kDa, and the proteolytic fragments that migrate with molecular masses of 35, 33, 31, and 27 kDa correspond to ßC1, ßC2, ßC3, ßC4, respectively, are shown on the right. The full-length ER{alpha} migrates with a molecular mass of 65 kDa. The proteolytic fragments of {alpha}C1, {alpha}C2, and {alpha}C3 migrate with molecular masses of 35, 33, and 30 kDa, respectively. Shown is a representative experiment from three to five independent determinations.

 
Digestion of the full-length ER{alpha} (molecular mass of 65 kDa, lower panel, lane 1) produced three distinct fragments, {alpha}C1, {alpha}C2, and {alpha}C3, that migrated at 35, 33, and 30 kDa, respectively, in the absence of ligand (lanes 2–6). When the receptor was occupied with E2 (lanes 7–12), the {alpha}C1 and {alpha}C2 (lanes 11 and 12) showed more resistance to the same concentrations of protease. Similar resistance of {alpha}C1 and {alpha}C2 was also observed at the penultimate concentration of protease when ER{alpha} was bound to 4-OHT (lane 17) or ICI (lane 23) compared with the receptor in the absence of ligand (lane 5). In contrast to E2-bound receptor (lane 12), the highest concentration of chymotrypsin produced identical proteolytic fragments in the absence (lane 6) and the presence of 4-OHT (lane 18) and ICI (lane 24). Consistent with previous observations (41, 42, 43), these results indicate that ER ligands also induce distinct conformational changes in ER{alpha}.

ERE-Induced Conformational Changes in Ligand-Occupied ER
Previous studies showed that the ligand-ER{alpha} complex bound to the consensus ERE displays different electrophoretic migration patterns depending upon the identity of ligand. Although E2-coupled ER{alpha} migrates faster in EMSA compared with the unliganded or ICI-bound receptor, the tamoxifen-ER{alpha} complex displays the slowest electrophoretic migration (44, 45). This suggested a role of ligand in ER{alpha} conformation when the receptor is bound to the consensus ERE. Consistent with these results, we also observed that the ERE-ERß complex, just as the ERE-ER{alpha} complex (data not shown), displays differences in electrophoretic migration depending upon the nature of ER ligand (Fig. 5AGo). However, the migration pattern was the same whether or not either ER interacted with p17, p17d1, or p17d2. This implies that the ligand is responsible for the observed differences in migration. However, studies also showed that, in addition to ER ligands, EREs also allosterically modulate receptor conformation upon binding (17, 18, 19, 20, 21), as shown for numerous transcription factors and nuclear receptors (22). Reports have indicated that a single nucleotide change in the consensus ERE causes the formation of new interconnected hydrogen bonds between the response element and the DBD of ER{alpha}, thereby altering the conformation of the region (18, 19). We therefore wanted to address whether variant ERE sequences further alter the conformation of ERs induced by various ligands using a gel shift-proteolysis assay (21). The assay combines EMSA as a detection approach for functional ER-ERE interaction and partial proteolysis as an approach for the analysis of the conformation of the ERE-bound ER complexed with an ER ligand.



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Figure 5. The Effects of ERE Sequences and ER Ligands on the Pattern of Partial Proteolysis ERs by Proteases

A, Electrophoretic migrations of ligand-ERß-ERE complexes. ERß was incubated without (control) or with 10-6 M ER ligands for 15 min followed by incubation with radiolabeled p17, p17d1, or p17d2 ERE for an additional 15 min. Reactions were resolved on 8% nondenaturing PAGE. B, Recombinant ER{alpha} (left panels) and ERß (right panels) were incubated without (ethanol 0.01%; Control, lanes 1–6) or with 10-6 M E2 (lanes 7–12), 4-OHT (lanes 13–18), or ICI (19 20 21 22 23 24 ) for 15 min. Equal molar concentrations of [32P] end-labeled p17 (upper panel), p17d1 (middle panel) or p17d2 (lower panel) oligonucleotides were then added into reaction mixtures and incubated for an additional 15 min. This was followed by exposure of the ligand-ER-ERE complexes to 0, 1, 2.5, 5, 10, and 25 µg/reaction chymotrypsin. After termination, the reactions were immediately analyzed by 8% nondenaturing PAGE. Note that due to lower affinity of both receptors to p17d2, images of p17d2-bound ERs were enhanced severalfold for comparable intensities. Shown is a representative experiment from at least three independent determinations.

 
End-labeled p17, p17d1, or p17d2 was incubated with equal amounts of ERs. This was followed by exposure of the ER-ERE complexes to various concentrations of chymotrypsin. Reaction mixtures were then subjected to 8% nondenaturing PAGE (Fig. 5BGo). Chymotrypsin treatment of the ERE-ER{alpha} or -ERß complex (lanes 1–6) generated proteolytic fragments having faster electrophoretic migration with increasing concentrations of the enzyme. The impact of ERE sequence on the extent of proteolytic fragmentation is evident. ER{alpha} or ERß bound to the consensus ERE (p17) showed varying resistance to the protease compared with ERs bound to ERE sequences containing one (p17d1) or two (p17d2) nucleotide substitutions. This was particularly reflected in the pronounced (p17d1 and p17d2) and early (p17d2) appearance of C2 and C3 for ER{alpha} and C3 and C4 for ERß. These data suggest that the ERE induces conformational changes in ERs. We next examined whether ligands further influence the ERE-induced conformational changes. If a ligand induces a distinct fragmentation pattern in ERs that are bound to different EREs compared with unliganded receptors, this would suggest that ERE and ligand alter the conformation of ERs in an integrated, interactive manner. We observed that exposure of ERE-bound ER{alpha} or ERß in the presence of 10-6 M E2, (lanes 7–12), 4-OHT (lanes 13–18), or ICI (lanes 19–24) to varying concentrations of chymotrypsin produced proteolytic patterns that were similar to those of the unliganded receptors (lanes 1–6). Thus, it appears that ligands do not produce altered protease accessibility in the ER-ERE complex compared with the unliganded ER.

To further confirm that the observed ERE effects on ER conformation in the absence or presence of ligand were not due to the protease used, we subjected both ERs to varying concentrations of Endoproteinase Glu-C. Although the exposure of ERs to the protease produced a fragmentation pattern distinct from that observed with chymotrypsin, the proteolysis pattern of the ER species was similar whether ERs were bound to p17, p17d1, or p17d2 in the absence or presence of ER ligands (data not shown).

These results demonstrate, as shown for other DNA-binding proteins including ER{alpha} (17, 18, 19, 20, 21, 22), that EREs are potent modulators of ER conformation. Because ligands do not affect either the affinity of ER binding to EREs or the protease accessibility of an ER-ERE complex, these data imply that conformational changes in DBDs and LBDs are independent.

The Recruitment of Cofactors by Ligand-ER-ERE Complexes
If, as our data suggest, the DBD and the LBD of ER do not interact functionally to provide the receptors with novel features, the nature of cofactor interaction with the receptors in response to ligands should not be affected by the ERE sequence. Studies have indicated that ERs interact with a complex array of coregulator proteins that mediate the interactions between receptors and the basal transcription apparatus and the remodeling of chromatin structure (46, 47, 48, 49). Among the coregulators, steroid receptor coactivator 1 (SRC-1), TIF-1, TIF-2, and amplified in breast cancer-1 (AIB-1) have been shown to interact with the LBD in an agonist-dependent manner by glutathione-S-transferase (GST)-pull down and by yeast and mammalian two-hybrid analysis (43, 50, 51, 52, 53, 54, 55, 56, 57, 58). To examine the effects of ERE and ligand on cofactor interaction with the receptors, we used EMSA. Because it appears that ER and cofactor interactions are sensitive to ligand concentrations (43, 56), we initially addressed how various concentrations of ligands affect the recruitment of cofactors by ER and whether or not different ERE sequences have an impact on these interactions. Equal molar concentrations of ER{alpha} and ERß were preincubated without or with E2, 4-OHT, and ICI ranging from 10-9 to 10-6 M. This was followed by incubation of the reaction mixtures with 32P-end-labeled ERE. The polypeptides of cofactors, as GST fusion proteins, were then added into reaction mixtures in the amount of 1 µg/reaction. After the reaction, samples were subjected to nondenaturing gel electrophoresis. Because of the substantially lower affinity of ERs to p17d2 compared with other test EREs (see above), assessing the effects of ER ligands on the interaction of cofactors with ERs was difficult when we used an ERE sequence of which a single strand was end labeled with [32P]. To overcome this difficulty, both strands of ERE were end labeled with a high specific activity (6,000 Ci/mmol) [32P] isotope. As shown in Figs. 6AGo and 7AGo, the ERE-bound ER{alpha} or ERß (lane 2) was quantitatively supershifted by the TIF-2623-986 (TIF-2) in the absence of ligand (lane 3). This interaction was independent of the identity of ERE sequence in that the pattern of interaction was the same whether the receptors bound to p17, p17d1, or p17d2 (upper, middle, and lower panels, respectively). However, the extent of TIF-2 interaction with both receptors was correlated with the affinity, hence, the relative amount, of receptor binding to EREs. Binding of ER{alpha} to E2 over a range of hormone concentration increased the proportion of receptor that bound TIF-2 (lanes 4–7). The cofactor retarded the migration of the entire population of ER-ERE complexes in the presence of 10-8 M E2 (lane 5). This retardation was specific to TIF-2 as GST alone did not alter the migration of the receptors (data not shown). Significantly, binding of the receptors to increasing concentrations of either 4-OHT (lanes 8–11) or ICI (lanes 12–15) abolished TIF-2 interaction with the receptors bound to p17, p17d1, or p17d2.



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Figure 6. The Effects of ER Ligands and ERE Sequences on the Recruitment of AF-2-Dependent TIF-2 by ER{alpha}

Recombinant ER{alpha} was incubated on ice without (ethanol 0.01%; Control, lanes 2 and 3) or with 10-9, 10-8, 10-7, and 10-6 M E2 (lanes 4–7), 4-OHT (lanes 8–11), or ICI (12 13 14 15 ) for 15 min. Equal molar concentrations of p17 (upper panel), p17d1 (middle panel) or p17d2 (lower panel) oligonucleotide of which both strands were [32P] end labeled were then added into reaction mixtures and incubated on ice for an additional 15 min. This was followed by the addition of 1 µg/reaction (A) or 0.125 µg/reaction (B) TIF-2623-986 (TIF-2) fragment produced as GST fusion protein into the reactions. Samples were subjected to 6% nondenaturing gel electrophoresis. Gels were dried and phosphoimaged. Free DNA is not shown. Lane 1 represents DNA in the absence of any protein. C, ER{alpha} were preincubated in the absence (Control, ethanol 0.01%) or the presence of 10-7 M E2 for 15 min followed by the additional incubation with [32P]-end-labeled p17 on ice for 15 min. GST fusion TIF-2623-986 (TIF-2) was added in the amount of 0 (lanes 1 and 6), 0.125, 0.25, 0.5, and 1 µg (lanes 2, 3, 4, and 5 and 7, 8, 9, and 10, respectively). The reaction was further incubated for 30 min. Samples were resolved on 6% nondenaturing gels. Shown is a representative image from three independent experiments. Free DNA is not shown. ER and ER-CF represent ER-ERE complexes in the absence or presence of cofactors (CF), respectively. D, ER{alpha} was preincubated with a physiological level (10-9 M) of E2. The E2-ER complex was then incubated with p17 (upper panel), p17d1 (middle panel), or p17d2 (lower panel) oligonucleotide of which both strands were [32P] end labeled. This was followed by the incubation of the complex with TIF-2 at the concentration of 0.125 µg. The complex was then treated with increasing concentrations (10-9 to 10-6 M) of 4-OHT or ICI. Samples were resolved on 6% nondenaturing gels. Shown is a representative image from three independent experiments. Free DNA is not shown.

 


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Figure 7. The Effects of ER Ligands and ERE Sequences on the Recruitment of AF-2-Dependent TIF-2 by ERß

The effects of ERE sequences and ligands on the ability of ERß to recruit cofactors were examined as described in legend of Fig. 6Go. Shown is a representative image from three independent experiments. Free DNA is not shown.

 
The quantitative binding of TIF-2 with the unliganded ER{alpha} and ERß, irrespective of the identity of ERE sequences, is surprising, given the fact that recruitment of TIF-2 by ER has been reported to be ligand dependent (43, 50, 51, 52, 53, 54, 55, 56, 57, 58). To address whether the amount of TIF-2 used contributes to the extent of interaction with the unliganded ER, concentrations of TIF-2 ranging from 0.125 to 1 µg/reaction were incubated with ER-ERE complexes in the absence or presence of 10-7 M E2 (Figs. 6CGo and 7CGo). While the unliganded ER interacts with TIF-2 when the cofactor is present at high concentrations (lane 5), E2 increased the affinity of ER dramatically to TIF-2 such that the cofactor bound even at the lowest concentration tested (lane 7). This resulted in the complete retardation of the ER-ERE complex in the presence of E2 independent of the identity of ERE sequence.

To ensure that high concentration (1 µg) of TIF-2 did not mask the effects of ER ligands on coactivator interactions with ERs bound to various EREs, we also used a low concentration of the cofactor. TIF-2 at 0.125 µg concentration (Figs. 6BGo and 7BGo) had no effect on the electrophoretic mobility of the ER-ERE complex in the absence of ligand (lane 3). The preincubation of ERs with E2 (10-9 M to 10-6 M) led to a quantitative interaction with p160 proteins with the E2-ER-ERE complex (lanes 4–7). This was reflected as a gradual retardation in the migration of the complex. In contrast, 4-OHT or ICI at any concentration tested (10-9 to 10-6 M; lanes 8–11 and 12–15, respectively) had no effect on the ER-ERE complex, whether the ERE sequence was p17, p17d1, or p17d2.

Because 4-OHT and ICI act as antiestrogens, we further examined the effects of these compounds on the interactions of cofactors with the E2-ER complex bound to the test EREs (Figs. 6DGo and 7DGo). We preincubated ERs with a physiological level (10-9 M) of E2. The E2-ER complex was then incubated with an ERE. This was followed by the incubation of the complex with TIF-2 at the concentration of 0.125 µg. The complex was then treated with increasing concentrations (10-9 to 10-6 M) of 4-OHT or ICI. The results revealed that increasing concentrations of 4-OHT (lanes 6–9) or ICI (lanes 10–13) decreased the interaction of TIF-2 with the E2-ER-ERE complex (ER-CF). This decrease was inversely correlated with an increase in the E2-ER-ERE complex (ER) regardless of the identity of ERE sequence.

As with TIF-2, TIF-1638-851 (TIF-1), AIB-1522-827 (AIB-1), and SRC-1219-399 (SRC-1) at high (1 µg) concentration were also recruited by the unliganded ER{alpha} and ERß bound to p17 (Fig. 8, A and BGo; lane 3), p17d1, or p17d2 (data not shown). Increasing concentrations of E2 enhanced the ability of the receptors to interact with the cofactors. This was reflected in the complete mobility shift of the E2-ER-ERE-cofactor complex at 10-8 M E2 (lane 5).



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Figure 8. The Effects of ER Ligands and ERE Sequences on the Recruitment of AF-2-Dependent Cofactor by ER{alpha} and ERß

Shown is the interaction of p17 bound ER{alpha} (panel A) and ERß (panel B) in the absence (lanes 2 and 3) or the presence of 10-9, 10-8, 10-7, and 10-6 M E2 (lanes 4–7), 4-OHT (lanes 8–11), or ICI (12 13 14 15 ) with 1 µg/reaction of TIF-1638-851 (TIF1), AIB-1522-827 (AIB-1), and SRC-1219-399 (SRC-1) produced as GST fusion proteins. Panels C and D depict the interaction of 0 (dashed, lanes 1 and 6), 0.125, 0.25, 0.5, and 1 µg/reaction (lanes 2, 3, 4, and 5 and 7, 8, 9, and 10, respectively) GST fusion TIF-1, AIB-1, and SRC-1 with p17 ERE-bound ER{alpha} (panel C) or ERß (panel D) in the absence (Control, ethanol 0.01%, lanes 1–5) or presence of 10-7 M E2 (lanes 6–10). Incubation, processing, and resolving of samples were identical with those described in legend of Fig. 6Go. Shown is a representative image from three independent experiments. Free DNA is not shown. ER and ER-CF represent ER-ERE complexes in the absence or presence of cofactors (CF), respectively.

 
All cofactors in a concentration-dependent manner interacted also with the ERE-ER complexes in the absence of ligands when we used 1 µg cofactor (Fig. 8, C and DGo; lanes 1–5). The presence of 10-7 M E2 increased the interaction of cofactors with both ER{alpha} and ERß by augmenting the affinity of receptors for cofactors (lanes 6–10). Although the extent of interactions of ER{alpha} and ERß with the cofactors was similar, discernable differences were also noted in the binding patterns of E2-ER-ERE-cofactor complexes. Although the TIF-1 (Fig. 8Go) binding to E2-ER{alpha}-ERE complexes showed a gradual decrease in the electrophoretic mobility of the complex, the interaction of equal molar concentration of ERß with TIF-1 under the identical conditions was essentially "all-or-none" as assessed by mobility. Although the underlying reason is not clear, one likely explanation is that the affinities of these cofactors could be different for ERß than for ER{alpha}. Nevertheless, these results suggest that the mode of interaction among cofactors could differ depending upon the ER subtypes. As observed for TIF-2, the effects of ER ligands on the interaction of TIF-1, AIB-I, or SRC-1 at low concentration (0.125 µg) with ERs were independent of the ERE sequence (data not shown).

Thus, these results indicate that the AF-2-dependent cofactor interactions with both ER{alpha} and ERß are similarly affected by ligands regardless of the identity of ERE sequence. The data suggest that the integrated influences of ERE and ligand on conformational states of ER{alpha} and ERß do not provide a means by which either receptor could present distinct functional surfaces to different AF-2-dependent coregulators that could lead to a selective gene activation. Moreover, we observed that the unliganded ER-ERE complexes can recruit cofactors depending upon the amount of cofactor present. The high degree of regional flexibility, e.g. the relative positioning of helix 12 (59), could allow the receptors to interact with cofactors in the absence of ligand, albeit at a much lower efficiency. Although primarily attributed to traces of estrogenic compounds in culture media (60, 61), true hormone-independent cofactor binding could account also for the basal transcriptional responses to ER, the magnitude of which vary in a cell-specific manner (62, 63, 64).

Studies based on alterations in epitope accessibility of ER{alpha} upon binding to an ERE sequence suggest that ERE-induced conformational changes are transmitted to the amino-terminal region (21). Moreover, the partial agonistic effect of tamoxifen-coupled ER{alpha}, but not ERß, from consensus ERE-driven promoters is modulated through the amino-terminal AF-1 (60, 65, 66). These observations raise the possibility that the integrated influences of ERE and ligand could affect the interaction of AF-1-dependent cofactor recruitment. Among the cofactors, GR-interacting protein 1 (GRIP-1) (67) and the human homolog TIF-2 (68), through distinct interacting surfaces other than signature nuclear receptor interacting domains, are shown to interact with the amino-terminal ER{alpha} independently from ligands in GST pull-down assays. ERß, on the other hand, fails to interact quantitatively with GRIP-1 (67).

To examine whether ERE and ligand affect the nature of AF-1-dependent cofactor interaction with ERs, the polypeptide of the glutamine-rich (Q) region of TIF-2 containing residues 1,125–1,325 (TIF2-Q) was produced as a GST fusion protein. This region has been shown previously to interact with the AF-1 domain of ER{alpha} in a ligand-independent manner in GST pull-down assays (67). The recruitment of TIF2-Q by ERs occurred independently from ligands; the extent of interaction, however, was preferential for ER{alpha}. ER{alpha} bound to p17 (Fig. 9AGo, upper panel) effectively and quantitatively recruited TIF2-Q in the absence or presence of various concentrations (10-9 to 10-6 M) of E2 (lanes 4–7), 4-OHT (lanes 8–11), or ICI (lanes 12–15), whereas ERß interacted with the cofactor minimally (Fig. 9AGo, lower panel). Increasing concentrations of the cofactor led to a gradual retardation and the eventual complete shift of the ER{alpha}-p17 complex (Fig. 9BGo, upper panel) in the absence (lanes 1–5) or presence of 10-7 M E2 (lanes 6–10), 4-OHT, or ICI (data not shown). However, TIF2-Q supershifted only about 10% of the ERß bound to p17 at the highest concentration tested whether or not E2 was present (Fig. 9BGo, lower panel). The extent of interaction of the cofactor was correlated with the amount of ERs bound to ERE with one (p17d1) or two nucleotide (p17d2) substitutions in the core (data not shown).



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Figure 9. The Effects of ER Ligands and ERE Sequences on the Recruitment of the Glutamine-Rich Region of TIF-2 by ER{alpha} and ERß

The interaction of TIF21125-1325 (TIF2-Q)-GST fusion protein with p17 bound ER{alpha} or ERß (panel A) in the absence (Control, lane 3) or presence of 10-9 to 10-6 M E2 (lanes 4–7), 4-OHT (lanes 8–11), or ICI (lanes 12–15). Interaction of various concentrations of TIF2-Q with ER{alpha} and ERß (panel B) in the absence (Control, lanes 1–5) or presence of 10-7 M E2 (lanes 6–10). Lanes 1 and 5 represent the ER-ERE complex in the absence of TIF2-Q. Incubation, processing, and resolving of samples were the same as those described in the legend of Fig. 6Go. Free DNA is not shown. ER and ER-CF represent ER-ERE complexes in the absence or presence of cofactors (CF), respectively.

 
Thus, these results indicate that although ERE and ER ligand induce conformational changes, these changes are not interrelated to provide the receptor with novel functional properties.

The Effects of ERE Sequences and ER Ligands on ER-Mediated Transcriptional Responses
If, indeed, cofactor recruitment by both ER{alpha} and ERß is affected by ERE sequences and ER ligands acting independently, the extent of E2-induced transcriptional responses mediated by either ER subtype should primarily be dependent upon the ERE sequence. To test this prediction, we transfected mammalian cells with an expression vector bearing the ER{alpha} or ERß cDNA together with a reporter plasmid containing none or two EREs in tandem upstream of a minimal TATA box or the complex thymidine kinase (TK) promoter. Both promoters drive the firefly luciferase cDNA as the reporter. The transfection efficiency was normalized with the ß-galactosidase activity from a cotransfected expression vector bearing the enzyme cDNA. E2 at 10-9 M concentration dramatically augmented the luciferase activity induced by ER{alpha} in the absence of the hormone from the reporter plasmid bearing two optimal EREs (2x17) compared with the reporter plasmid bearing no ERE sequence (TATA) in transiently transfected COS-1 (Fig. 10AGo), Chinese hamster ovary (CHO), or HeLa (data not shown) cells. Although ER{alpha} augmented the reporter enzyme activity from two variant EREs (2x17d2) in the absence or presence of E2, the extent of transcription was substantially lower than that from the consensus EREs. Similar results were observed also for ERß, which displayed significantly lower transcriptional activity compared with ER{alpha} in COS-1 (Fig. 10AGo), CHO, or HeLa cells (data not shown), as shown previously (4, 5, 6, 7, 8, 9). Although the magnitude of transcriptional responses was lower compared with the responses from the TATA box constructs, the results were similar when we used the TK promoter (data not shown).



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Figure 10. The Effects of ERE Sequences and ER Ligands on Transcriptional Responses Induced by ERs

A, COS-1 cells were transiently transfected with 300 ng expression vector bearing cDNA for ER{alpha} or ERß, together with 500 ng reporter plasmid. The reporter plasmid contained either no EREs (TATA) or two copies of the optimal ERE (2x17) or of a variant ERE containing two nucleotide substitutions (2x17d2) in tandem located upstream of a simple promoter, TATA box, driving the luciferase gene. Transfection efficiency was monitored by coexpression of 200 ng reporter plasmid bearing the ß-galactosidase gene. Cells were treated without (no ligand) or with 10-9 M E2 (E2) for 24 h. B, Shown are the effects of 10-7 M E2, 10-8 M 4-OHT, or 10-6 M ICI on ER{alpha}-induced transcriptional responses from the TATA, 2x17, or 2x17d2 reporter plasmid in transiently transfected HepG2 cells. The data represent the mean ± SEM of three independent experiments in duplicate.

 
To examine whether 4-OHT and ICI can alter the pattern of transcriptional responses from the EREs in a promoter- and cell-context dependent manner, we tested their effects on ER-induced transcriptional responses in transiently transfected COS-1, CHO, and HeLa cells. We found that neither ER affected the luciferase activity from either promoter construct when cells were treated with various concentrations (10-11 to 10-5 M) of 4-OHT or ICI. Both compounds, as expected, effectively antagonized the E2-induced enzyme activity in response to either ER (data not shown).

HepG2 cells have been used as a model system in examining the partial agonist activities of ER ligands (69). In these cells, similar to E2, 4-OHT or ICI, in a concentration-dependent manner (Fig. 10BGo; shown is 10-7, 10-8, and 10-6 M for E2, 4-OHT, and ICI, respectively), augmented ER{alpha}-induced luciferase activity from the TATA box or TK promoter (data not shown) bearing EREs in tandem. As observed for E2-mediated induction, the extent of luciferase activity induced by 4-OHT or ICI was also correlated with the affinity of ER{alpha} to the ERE sequence. Although ERß-induced enzyme activity was minimal in response to all three compounds in this cell line, the transcriptional induction from different ERE sequences showed a pattern similar to that observed for ER{alpha} (data not shown).

Thus, these results collectively indicate that the integrated influences of ERE and ER ligand do not provide ERs with altered functional features responsible for a differential regulation of estrogen-responsive genes in an ERE-, ER ligand-, promoter-, and cell context-dependent manner. These data corroborate our conclusion that the nature of ERE sequence determines the potency of transcriptional responses to either ER when bound to E2 or an E2 agonist. This is also consistent with numerous observations that nonconsensus ERE sequences are less potent transcriptional enhancers than the consensus ERE in response to E2 (14, 15, 16). Moreover, because the interaction of the AF-2-dependent cofactors with the AF-1 domain (67) occurs in a receptor-specific manner, our results further suggest that structural differences between the AF-1 domains of ERs reflect the functional differences between ER subtypes. These are manifested here as differences in the extent of transcriptional responses.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this report, we addressed how various ligands cause alterations in the conformation of ERß and ER{alpha} that influence the receptor affinity and specificity for binding with ERE sequences, and, in turn, the ability of DNA-bound and ligand-occupied receptors to recruit cofactors and induce transcription.

ER Subtypes Utilize the Same Nucleotides to Interact with an ERE Independently from ER Ligands
The recognition of an ERE sequence by the ERs is central to transcription of estrogen-responsive genes. Although the consensus ERE is a palindrome, most of the responsive genes contain sequences that vary from the consensus by one or more nucleotides. Our results indicate that the characteristics of nonconsensus EREs that effectively predict the relative binding affinity of ER{alpha} (12, 13) are equivalently relevant for ERß. These data indicate that ERE sequences in responsive genes are not a predictor for receptor specificity.

Although both receptors 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 for an ERE sequence to both receptors was correlated with the number of nucleotide variations from the consensus. We determined that a single nucleotide substitution within the core reduced both ER{alpha} and ERß binding, whereas two nucleotide changes abolished the interaction. Furthermore, substitution of certain nucleotides immediately flanking the consensus or variant core could substantially improve receptor binding. The nature of nucleotides positioned two bases away from a variant core sequence can also have an impact on the binding of the receptors. For example, both ER{alpha} and ERß bind to 5'-C(A/G)GGTCAnnnTGACC(T/C)G-3' with an optimal efficiency, as we proposed previously for ER{alpha} (12). One or two nucleotide substitutions within this ERE, however, reduced the binding affinities of the receptors 2- and 20-fold, respectively. These observations allow reliable predictions of ER binding affinity to EREs and effectiveness of an ERE in vivo [see Driscoll et al. (12) for further discussion].

We used HRA to examine the DNA contact sites of ERs. HRA assesses the independent contribution of a base pair to protein binding that allows the analysis of DNA-protein interaction at a single-nucleotide resolution (37). One of our major findings is that both ER{alpha} and ERß utilize the same nucleotides to bind to ERE sequences, despite the differences in the binding affinities to an ERE. We also found that introduction of two nucleotide substitutions into the consensus ERE leads to asymmetrical changes in the contact sites. Rearrangement of contact sites in response to substitutions includes bases that flank the core repeat, further emphasizing the importance of flanking sequences in the binding of the receptors to a nonconsensus ERE.

ER Ligand- and ERE-Induced Conformational Changes in ER Are Not Interrelated
Protease sensitivity assays, which rely on access to cleavage sites that possess conformational flexibility between the tightly folded domains (70), have been widely used to characterize the ligand-induced conformational changes in ERs (22, 42, 43, 69, 71, 72, 73). Using similar approaches, we show here also that each ligand or ERE induces different conformations in ERs. It has been reported that DNA-induced structural changes are transmitted to both the amino and the carboxyl termini of ER{alpha} as assessed by the alterations in epitope accessibility (21, 28, 29). Does this mean that ERE- and ER ligand-induced conformational changes influence each other? Recent fluorescence anisotropy studies in concordance with previous reports (32, 74, 75) indicate that E2, 4-OHT, or ICI have no effect on the binding affinity of ER{alpha} to the consensus ERE (76). Similarly, we observe here that ligands do not affect the binding specificity or affinity of ERs to EREs. Moreover, the binding of ligands does not alter the fragmentation patterns of ERs bound to EREs. These results collectively suggest that ERE- and ER ligand-induced conformational changes are not integrated to provide ERs with distinct functional features. Indeed, our results that the extent of cofactor interaction with the receptors is primarily determined by the binding affinity to an ERE, while ER ligand effects the affinity of ER to a cofactor, support this conclusion.

The Structure of the Ligand Dictates the Affinity of ER for Cofactors Independently from ERE Sequence
SRC-1, TIF-1, TIF-2, and AIB-1 interact with the AF-2 domain of ERs in an agonist-dependent manner through receptor interacting domains in GST pull-down assays and yeast and mammalian two-hybrid systems (43, 50, 51, 52, 53, 54, 55, 56, 57, 58). Consistent with those, our results further indicate that the effects of E2 on conformational changes of the LBD are critical for the efficient recruitment of cofactors. This was manifested as a dramatic increase in the receptor affinity for cofactors upon binding of E2. This contrasts to 4-OHT and ICI, which impede the interactions of both ER{alpha} and ERß with cofactors by inducing conformations in the LBD that are distinct from those of the unliganded receptor or of the receptor bound to E2.

We show here that the effects of E2, 4-OHT, and ICI on the pattern of AF-2-dependent cofactor interactions with both receptors are independent from the identity of ERE sequences. Moreover, distinct surfaces of a cofactor, as observed here and previously for TIF-2 (68) and GRIP-1 (67), can preferentially interact also with the ER{alpha} in a ligand-independent manner. Because the ERE sequence is critical for the affinity (and therefore the relative amount of receptor binding), these observations imply that the amount of cofactor recruitment is ultimately determined by the amount of receptor bound to an ERE, which is correlated with transcription, as shown here and previously (14, 15, 16). Distinct transcription activation domains, AF-1 and AF-2, of ER{alpha} have been shown to act independently and in concert to regulate estrogen-responsive gene transcription in a ligand-, promoter-, and cell-specific manner (63, 77). Because AF-1 is a critical domain for the transcriptional strength of ER{alpha} and ERß (78, 79) and because AF-2-dependent TIF-2 can also be recruited by ER subtype preferentially and independently from ligands, the ability of ER{alpha} to interact with distinct surfaces of a cofactor could be critical for an intramolecular interaction within ER{alpha} and an intermolecular interaction between ER{alpha} molecules when bound to tandem EREs (67). This mechanism could allow the receptor to effectively regulate natural estrogen-responsive genes in which ERE sequences are dispersed among other hormonal or nonhormonal response elements.

Recently, Loven et al. (80) and Wood et al. (81) reported that ERs bind to the consensus ERE of vitellogenin A2 gene with a 2-fold higher affinity than the nonconsensus EREs with one nucleotide change (underlined) of the Xenopus laevis B1 (AGTCAnnnTGACC), the human pS2 (GGTCAnnnTGGCC), or the human oxytocin (GGTGAnnnTGACC) gene. Wood et al. (81) reported, as we show here, that ERE-induced changes in the receptor conformation do not alter the interaction of the E2-ER{alpha} complex. This was based on the observations that RIDs of SRC-1 and GRIP-1 as GST fusion proteins are recruited by the E2-ER{alpha} complex whether or not the receptor is bound to the A2, B1, pS2, or oxytocin ERE in EMSA. In contrast, however, they found that the E2-ER{alpha} complex bound to B1, but not to A2, pS2, or oxytocin, ERE recruits lower amounts of TIF-2, but not AIB-1, from HeLa cell extracts, as assessed by Western blotting, in a DNA pull-down assay with biotinylated oligomers. It was concluded that the identity of the B1 ERE affects the ability of ER{alpha} to interact with cofactors differentially. It is not clear, however, why B1 but not the pS2 or oxytocin EREs, all of which show similar binding affinities to ER{alpha} (80), is capable of affecting the recruitment of TIF-2 but not AIB-1. It was also reported that ERß undergoes ERE-dependent changes in conformation resulting in differential recruitment of cofactors from HeLa cell extracts in a DNA pull-down assay with biotinylated oligomers (82). Although the interaction of TIF-2 with ERß is affected when bound to pS2 or B1 ERE but not A2 or oxytocin ERE, the recruitment of AIB-1 by ERß is altered when bound to B1, pS2, or oxytocin ERE but not to A1 ERE. Cell extracts contain a variety of proteins including varying amounts of ligand-dependent cofactors that interact with the AF-2 domain of ER{alpha} competitively (58). Because TIF-2 can bind to both the amino and carboxyl termini of ER{alpha}, but not of ERß, through distinct interacting domains, it is possible that while TIF-2 interacts with a particular ER{alpha} domain, other cofactor(s), including AIB-1, can interact simultaneously with the remaining region. On the other hand, competition of cofactors for binding to the carboxyl terminus of ERß, together with differences in the affinities of cofactors to the ER subtypes, could have led to the differences in the pattern of recruitment of cofactors, depending upon the ERE sequence. The combinatorial effects of this heterogenous population of cofactors, rather than a sole cofactor, associated with ER{alpha} could indeed magnify small differences in ERE-induced alteration in ER conformation.

In summary, our results indicate that the extent of ligand-, or AF-2-dependent cofactor recruitment by both ER{alpha} and ERß is altered by two factors acting independently: ER ligands and ERE sequences. After agonist binding, interaction of cofactors with each of the receptor subtypes is indirectly affected by the ERE sequence, which determines the binding affinity, and therefore the relative amount, of ERs that use the same contacts in DNA. The interference of cofactor interaction with both ERs by an antagonist, on the other hand, occurs independently of the ERE sequences. Because ERE sequence influences the amount of receptor binding to DNA, it also indirectly influences the amount of coactivator that is recruited to ER bound to an ERE sequence. The ability of ER{alpha} to differentially recruit a cofactor could contribute to ER subtype-specific gene responses.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
The human wild-type ER{alpha} cDNA, provided by the late Dr. Angelo Notides, was inserted into pBluescript II KS (+) (pBS-KS). An expression vector bearing human ERß cDNA was a gift from Dr. Simak Ali. This ERß cDNA encodes a 477-amino acid ERß. The extended sequence encoding the additional amino-terminal 53 amino acids was generated using PCR from human placental DNA (Sigma-Aldrich Corp., St. Louis, MO) with primers based on the published sequence of the full-length ERß (83) and inserted in frame into the pBS-KS bearing the parent short ERß cDNA. The resultant long ERß, referred to here as the wild-type ERß, was then sequenced. Receptor cDNA was excised from pBS-KS and inserted into a mammalian expression vector [pM2-AH] as described previously (84).

Fragments of AIB-1 containing nuclear interacting signature motifs within the region encompassing residues 522–827 (50), residues 219–399 of SRC-1 (52), and 623–986 of TIF-2 (51) were obtained by PCR and inserted into pGEX-2TK (Amersham Pharmacia Biotech, Piscataway, NJ). The residues 1,125–1,325 of TIF-2 containing a glutamine-rich region that is shown to interact with ER (68) was also obtained with PCR. A fragment of TIF-1 containing residues 638–851 was amplified with PCR from a human testis cDNA library (CLONTECH Laboratories, Inc., Palo Alto, CA) with primers based on the published sequence (53, 85).

GST-cofactor fusion proteins were expressed in Escherichia coli BL21 (DE3) cells and purified using GST purification modules as recommended by the manufacturer (Amersham Pharmacia Biotech). Protein contents in eluates were estimated using the Protein Assay Kit (Bio-Rad Laboratories, Inc., Hercules, CA). Equal aliquots of GST-fusion proteins were resolved by SDS-PAGE and visualized by Coomassie staining of the gel.

EMSA
Both strands of oligomers (Sigma-Genosys, The Woodlands, TX, or Integrated DNA Technologies, Inc., Coralville, IA) were annealed and [32P]-end labeled with a 3,000 Ci/mmol specific activity isotope, as described previously (12). The end-labeled DNA (0.125 nM) was incubated with 0, 2.5, 5, 10, 15, and 20 nM of human recombinant ER{alpha} or ERß expressed in baculovirus-infected insect cells (Panvera, Madison, WI) in a binding buffer in a total volume of 10 µl. The concentrations of recombinant ERs provided by the supplier were based on the quantitation of [3H]-E2-receptor complexes using a hydroxylapatite assay. The binding buffer contains final concentrations of 40 mM Tris-HCl, pH 7.5, 100 mM KCl, 1 mM EDTA, 0.1% Nonidet P-40, 5% glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/µl BSA, 0.1 µg/µl poly (dI-dC) (Midland Certified Reagents, Midland, TX). Reactions were incubated on ice for 1 h and loaded onto an 8% native PAGE. The dried gel was visualized and quantified by PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). In assessing the effects of ligands on the binding specificity and affinity of the receptors, varying concentrations of receptors were preincubated without (0.01% ethanol) or with a saturating concentration (10-6 M) of 17ß-E2 (Sigma-Aldrich Corp.), 4-OHT (Sigma-Aldrich Corp.), or ICI 182,780 (ICI; Tocris, Ballwin, MO) for 15 min on ice. This was followed by incubation with 0.125 nM end-labeled ERE for 30 min. Reactions were resolved as described above.

The affinity of ER for various EREs was determined by competition assays. The concentration of unlabeled oligonucleotides that contain various ERE sequences required to reduce ER complex formation with the labeled optimal ERE (p17) by 50% (IC50) is proportional to the affinity of ER for the unlabeled oligonucleotide by the equation (86) Ki = IC50/(1 + F/Kd), where Ki is the equilibrium constant for the unlabeled oligonucleotide and Kd is the equilibrium dissociation constant for the labeled p17. F is the concentration of labeled p17 in the assay. When the competition was performed with unlabeled p17, Ki = Kd.

The affinity of ERs to various ERE sequences was similar whether or not the ERs were from different preparations or were obtained from different sources (Panvera vs. Affinity BioReagents, Inc., Golden, CO).

For the recruitment of cofactors, 15 nM of the receptors were preincubated in the absence (0.01% ethanol) or presence of varying concentrations (10-9 to 10-6 M) of E2, 4-OHT, or ICI for 15 min on ice. The end-labeled consensus ERE was then added and reactions were incubated for an additional 30 min. This was followed by the addition of cofactors in the amount of 0.125 or 1 µg/reaction for 30 min. Reactions were then resolved on 6% nondenaturing PAGE. Due to lower affinities of ERs to p17d2 compared with other test EREs, assessing the effects of ER ligands on the interaction of cofactors with ERs was difficult when we used an ERE sequence of which a single strand was 32P-end labeled. To overcome this difficulty, both strands of ERE were end labeled with a high specific activity (6,000 Ci/mmol) [32P] isotope.

Missing Nucleoside HRA
Single-stranded oligonucleotides were labeled at the 5'-end with [{gamma}-32P] by polynucleotide kinase, gel purified, and annealed with unlabeled complementary strand. Double-stranded DNA was 3'-labeled using DNA polymerase I Klenow fragment by incorporation of [{alpha}-32P] dGTP. The labeled DNA, randomly cleaved by hydroxyl radicals as described previously (37), was incubated with ER on ice for 30 min. The amount of ER used was adjusted to bind the majority of the end-labeled ERE oligomers. ER-bound EREs were separated from free EREs by 5% native PAGE. Radioactive bands containing bound and free ERE were excised from the gel. The EREs were eluted, precipitated, and dissolved. Equal concentrations of bound and free EREs were subjected to 18% sequencing gel electrophoresis. Maxam-Gilbert G-specific sequencing reactions were performed simultaneously. It should be noted that identification of critical residues at the 5'-end of both strands when the oligomers were 5'-end labeled was difficult. This was also the case for the 3'-end resolution of oligomers that were 3'-end labeled. The difficulty most likely arose from the length of the oligomers used in HRA (87). Using the same ERE oligomers labeled at either end, however, allowed us to obtain a corroborative assessment of critical residues.

Preparation of Radiolabeled ER{alpha} and ERß
Supercoiled pBS-KS bearing none, as control, or receptor cDNA was transcribed/translated using a rabbit reticulocyte translation system with 2 µl L-[35S]methionine (1,175 Ci/mmol; NEN Life Science Products, Boston, MA) as directed (Promega Corp., Madison, WI). Equal aliquots of reaction mixtures (5 µl of 50 µl reactions) were subjected to electrophoresis under reducing conditions on 10% SDS-PAGE. Bands corresponding to ER{alpha} and ERß were excised from the gel and counted to estimate receptor concentrations.

Partial Proteolysis of ER{alpha} and ERß
To assess the conformational changes induced by ER ligands, equal molar concentrations of receptors were incubated without (0.01% ethanol) or with 10-6 M E2, 4-OHT, or ICI for 10 min at room temperature. The reactions were then subjected to 0, 1, 2.5, 5, 10, and 25 µg/reaction trypsin or chymotrypsin in a total of 10 µl reaction mixture for 10 min at room temperature. Reactions were terminated by the addition of 2x sample buffer containing 5% ß-mercaptoethanol, boiled for 5 min, and subjected to 8–16% gradient SDS-PAGE.

To probe the conformational changes of ER ligand-ER complexes upon binding EREs, 15 nM ERs were incubated without or with 10-6 M E2, 4-OHT, or ICI for 15 min at room temperature. End-labeled ERE (0.125 nM) was added into the reaction and incubated further for 15 min at room temperature. Reaction mixtures were then subjected to 0, 1, 2.5, 5, 10, and 25 µg/reaction trypsin or chymotrypsin, or 0, 2.5, 5, 10, 25, and 50 µg/reaction Endoproteinase Glu-C (Calbiochem, La Jolla, CA) for 10 min at room temperature. Samples, in a total volume of 10 µl, were immediately subjected to 8% nondenaturing PAGE.

Cell Culture and Transfection
Maintenance and transfection of COS-1, CHO, HeLa, and HepG2 cells were described previously (84). Because the transcriptional response from single ERE sequence is very low (data not shown), we used reporter plasmids that contain two copies of EREs in tandem, with 38 bp center-to-center distance, located upstream of a promoter. The promoter we used was a simple TATA box or TK that drives the firefly luciferase cDNA as the reporter enzyme. After transfection, the cells were incubated in fresh medium with or without E2, 4-OHT, or ICI for 24 h. An expression vector carrying the ß-galactosidase cDNA driven by the cytomegalovirus promoter was also cotransfected as the control for transfection efficiency in the amount of 200 ng. Processing of cell lysates for the reporter enzymes was described previously (84). Results are the mean ± SEM of three independent experiments in duplicate. In all transfections, normalized luciferase values are presented as the fold-change over the enzyme activity induced by the parent expression vector bearing no cDNA in the absence of E2 (data not shown).


    ACKNOWLEDGMENTS
 
We thank Drs. Simak Ali, Ronald M. Evans, Hinrich Gronemeyer, Paul Meltzer, and Bert W. O’Malley and acknowledge the late Angelo Notides for providing plasmids for ER and cofactor cDNAs. We also thank Drs. Jeffrey J. Hayes and Eric M. Phizicky 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.) and an American Cancer Society Institutional Research Grant 98-276-02 (to M.M.). P.Y. is a recipient of Predoctoral Fellowship DAMD 179717227 from the Department of Defense Breast Cancer Research Program.

1 Present address: Molecular Staging, Inc., 300 George Street, Suite 701, New Haven, Connecticut 06511. Back

Abbreviations: AF-1, AF-2, Activation functions 1 and 2; AIB-1, amplified in breast cancer-1; CHO, Chinese hamster ovary; DBD, DNA-binding domain; ERE, estrogen-responsive element; GRIP-1, GR-interacting protein; GST, glutathione-S-transferase; HRA, hydroxyl radical assay; ICI, ICI 182,780; LBD, ligand-binding domain; 4-OHT, trans-4-hydroxytamoxifen; pBS-KS, pBluescript II KS (+); SRC-1, steroid receptor coactivator-1; TIF-1 and TIF-2, transcriptional intermediary factor-1 and -2; TK, thymidine kinase.

Received for publication August 6, 2001. Accepted for publication December 17, 2001.


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