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Molecular Endocrinology 15 (7): 1114-1126
Copyright © 2001 by The Endocrine Society

Allosteric Modulation of Estrogen Receptor Conformation by Different Estrogen Response Elements

Jennifer R. Wood, Varsha S. Likhite, Margaret A. Loven and Ann M. Nardulli

Department of Molecular and Integrative Physiology (J.R.W., M.A.L., A.M.N.) Department of Biochemistry (V.S.L.) University of Illinois Urbana, Illinois 61801


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen-regulated gene expression is dependent on interaction of the estrogen receptor (ER) with the estrogen response element (ERE). We assessed the ability of the ER to activate transcription of reporter plasmids containing either the consensus vitellogenin A2 ERE or the imperfect pS2, vitellogenin B1, or oxytocin (OT) ERE. The A2 ERE was the most potent activator of transcription. The OT ERE was significantly more effective in activating transcription than either the pS2 or B1 ERE. In deoxyribonuclease I (DNase I) footprinting experiments, MCF-7 proteins protected A2 and OT EREs more effectively than the pS2 and B1 EREs. Limited protease digestion of the A2, pS2, B1, or OT ERE-bound receptor with V8 protease or proteinase K produced distinct cleavage products demonstrating that individual ERE sequences induce specific changes in ER conformation. Receptor interaction domains of glucocorticoid receptor interacting protein 1 and steroid receptor coactivator 1 bound effectively to the A2, pS2, B1, and OT ERE-bound receptor and significantly stabilized the receptor-DNA interaction. Similar levels of the full-length p160 protein amplified in breast cancer 1 were recruited from HeLa nuclear extracts by the A2, pS2, B1, and OT ERE-bound receptors. In contrast, significantly less transcriptional intermediary factor 2 was recruited by the B1 ERE-bound receptor than by the A2 ERE-bound receptor. These studies suggest that allosteric modulation of ER conformation by individual ERE sequences influences the recruitment of specific coactivator proteins and leads to differential expression of genes containing divergent ERE sequences.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The hormone estrogen is critical for normal development and maintenance of the female reproductive system. In addition, estrogen plays a role in prevention of cardiovascular disease and osteoporosis, proliferation of breast cancer cells, and concentration of sperm in the male reproductive tract (1, 2, 3, 4). Estrogen’s actions are mediated by the estrogen receptor (ER). The ER belongs to a large superfamily of nuclear receptors that function as ligand-inducible transcription factors (5). The ER activates transcription by binding to estrogen response elements (EREs), which are generally located in the 5'-flanking region of estrogen-responsive genes. The consensus ERE (GGTCAnnnTGACC), which is present in the Xenopus laevis vitellogenin A2 gene, is an inverted palindromic sequence separated by three intervening nucleotides (6). Numerous studies have examined the interaction of the ER with the A2 ERE in in vitro binding experiments, x-ray crystallographic studies, and transient transfection assays (6, 7, 8, 9, 10). If the ERE sequence deviates from the consensus sequence by even a single base pair, the receptor exhibits reduced affinity for the ERE and decreased ability to activate transcription (6, 7, 9, 11). However, relatively few studies have examined the ability of imperfect ERE sequences to mediate transcription activation. The fact that the majority of known estrogen-responsive genes contain imperfect EREs that differ from the consensus ERE sequence by one or more base pairs provides compelling impetus to better understand how ER interaction with these imperfect ERE sequences influences gene expression.

Multiple factors influence the ability of an ERE to activate transcription, including the recruitment of transcription factors to the DNA-bound receptor and to other cis elements present in target genes. Earlier studies suggested that ER interaction with the basal transcription factors TBP (TATA binding protein), TFIIB (transcription factor IIB), and TAFII30 may play a role in activating estrogen-responsive genes (12, 13, 14). However, since the interaction of these proteins with ER is not altered by ligand, they cannot confer estrogen responsiveness to target genes. Thus, direct interaction between the receptor and these general transcription factors may be necessary, but is not sufficient, for ligand-dependent regulation of target gene expression. Interaction of the receptor with coactivator proteins appears to be a crucial step in mediating estrogen-regulated gene expression. Estrogen-bound ER interacts directly with numerous coactivator proteins, including the p160 proteins SRC-1/NCoA-1, TIF2/GRIP1/NCoA-2, and pCIP/ACTR/AIB1/RAC3/TRAM-1 in in vitro transcription experiments and enhances expression of ERE-containing promoters (Refs. 15, 16 and references therein). Recruitment of the transcription adapter proteins CBP/p300 may also play a role in ligand-dependent activation by ER (17, 18).

Complementary techniques have demonstrated that ER conformation is different when the receptor is bound to estrogen or antiestrogen (19, 20, 21, 22). Our laboratory has also demonstrated that ER conformation is different when the receptor is bound to the A2 or pS2 ERE (23). Thus, ER conformation is dependent on both ligand- and DNA-induced conformational changes. It seems plausible that these ligand- and DNA-induced changes in ER conformation could lead to differential association of proteins with the receptor. DNA-induced conformational changes have also been observed with several trans acting factors including other nuclear receptors, NF{kappa}B (nuclear factor-{kappa}B), Pit-1, TATA-binding protein, and the yeast protein phermone/receptor transcription factor (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35).

Two receptors, ER{alpha} and ERß, have been described and may be involved in differential expression of target genes (36, 37). We have examined the interaction of one of these receptors, ER{alpha}, with the vitellogenin A2 ERE and three imperfect EREs. The Xenopus laevis B1 ERE2 [AGTCAnnnTGACC (38)] and the human oxytocin ERE [GGTGAnnnTGACC (39)] differ from the A2 ERE sequence in the 5'-half site while the human pS2 ERE [GGTCAnnnTGGCC (40)] differs from the A2 ERE sequence in the 3'-half site. We find that ER conformation is different when the receptor is bound to these four different ERE sequences and demonstrate that these alterations in receptor conformation influence the association of transcriptional intermediary factor 2 (TIF2) with the ERE-bound receptor.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ability of the A2, pS1, B1, and Oxytocin (OT) EREs to Activate Transcription
Transient cotransfection assays were carried out to assess the abilities of the A2, pS2, B1, and OT EREs to serve as enhancer elements for ER-mediated transcription activation. HeLa cells were cotransfected with a chloramphenicol acetyltransferase (CAT) reporter plasmid, a human ER{alpha} expression vector, and a ß-galactosidase reporter plasmid and exposed to 10 nM E2 or ethanol. The CAT reporter plasmids contained a single A2, pS2, B1, or OT ERE upstream of a TATA sequence. The most potent activator of transcription was the A2 ERE. CAT activity was 10.5-fold higher when cells transfected with the A2 ERE reporter plasmid were exposed to E2 than when cells were treated with ethanol (Fig. 1Go). When cells were transfected with a pS2, B1, or OT ERE-containing reporter plasmid, CAT activity was 2.7-, 1.6-, and 9.5-fold higher, respectively, when cells were exposed to E2 compared with ethanol controls. Although the A2 ERE consistently activated transcription to higher levels than the OT ERE, the increased basal CAT expression with the A2 ERE resulted in similar fold induction with these two EREs. CAT activity was unaffected by hormone treatment when the parent vector, which contained a TATA sequence but no ERE, was used (Fig. 1Go, -), demonstrating that the EREs were responsible for the increased CAT activity in the presence of E2. These data indicate that the three imperfect EREs, which have sequences that differ from the A2 sequence, but similar affinities for the receptor (11, 41), have very different abilities to activate transcription.



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  		Figure 1. E2-Dependent Activation of the A2, pS2, B1, and OT ERE

A, HeLa cells were cotransfected with a human ER expression vector, a ß-galactosidase expression plasmid, and a CAT reporter plasmid containing a TATA sequence alone (-) or in combination with an A2, pS2, B1, or OT ERE. Cells were treated with ethanol vehicle or 10 nM E2. Values are presented as the mean ± SEM. Student’s t tests demonstrated that all E2-treated samples were statistically different from the corresponding ethanol-treated samples, except for the parent plasmid. B, Sequences of the A2, pS2, B1, and OT ERE are shown. ERE half -sites are capitalized. Nucleotides that differ from the A2 ERE half-sites are underlined. The 3-bp spacer and nucleotides at the 5'- and 3'-ends of each ERE are from the endogenous gene sequences.

 
Protection of the A2, pS2, B1, and OT EREs by MCF-7 Nuclear Proteins
Although the promoters of the four reporter plasmids used in the transient transfection assays were identical in nucleotide sequence except for the ERE sequence, the reporter plasmids containing the A1, pS2, B1, and OT EREs were activated to very different extents. To understand how this might occur, we examined the interaction of the ERE-containing promoters with MCF-7 nuclear proteins in DNase I footprinting experiments. MCF-7 nuclear extracts contain ER as well as other nuclear proteins involved in estrogen-mediated transactivation. 32P- labeled DNA fragments containing a TATA sequence and either the A2, pS2, B1, or OT ERE were incubated with increasing amounts of MCF-7 nuclear extract and exposed to limited DNase I digestion. Since competition assays with purified ER have demonstrated that the affinity of the receptor is 2-fold higher for the A2 ERE than its affinity for any of the imperfect EREs (41), twice as much nuclear extract was included in the binding reactions with the imperfect EREs than with the A2 ERE shown in Fig. 2AGo. The ERE sequences were protected by proteins in the ER-containing nuclear extracts, but the extent of the protection was dependent on the sequence of the ERE (Fig. 2AGo). Phosphorimager analysis demonstrated that both A2 consensus ERE half-sites were effectively protected when increasing amounts of nuclear proteins were included in the binding reactions (Fig. 2BGo). Surprisingly, the OT consensus and imperfect ERE half-sites were also equally protected. In contrast, only the consensus ERE half-site was substantially protected in the B1 ERE, and the two pS2 ERE half-sites were minimally protected. The enhanced protection of the A2 and OT EREs compared with the pS2 and B1 EREs was most apparent when 10 µg of MCF-7 proteins were included in the binding reaction. In addition to the protection of the EREs, regions of DNase I hypersensitivity were observed flanking the EREs. This hypersensitivity was most evident with the A2 and OT EREs. Only minimal hypersensitivity was observed in the area flanking the pS2 ERE. Significant protection was also observed at the TATA sequence with each of the four DNA fragments. Similar protection and hypersensitivity patterns were observed with two different MCF-7 nuclear extracts in four independent footprinting experiments. In addition, similar footprinting patterns were obtained when the other DNA strand was exposed to DNase I digestion in the presence of MCF-7 nuclear extracts (data not shown).



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  		Figure 2. DNase I Footprinting with A2, pS2, B1, or OT ERE-Containing DNA Fragments and MCF-7 Nuclear Extracts

A, 32P-labeled DNA fragments containing a TATA sequence and an A2, pS2, B1, or OT ERE were combined with the indicated amounts of MCF-7 nuclear extracts (NE), exposed to limited DNase I digestion, and run on a sequencing gel. The positions of the TATA box, consensus ERE half-sites (open rectangles), and imperfect ERE half-sites (shaded rectangles) were determined by piperidine cleavage of DMS-treated, 32P-labeled DNA fragments (G). B, Phosphorimager analysis was used to quantitate the fraction of 5'- (open squares) and 3'- (closed squares) ERE half-sites protected by 0, 5, 10, or 50 µg MCF-7 nuclear extract. Data from four independent experiments were combined and each value is expressed as the mean ± SEM.

 
Interaction of the ER with the A2, pS2, B1, and OT EREs
To further define how the A2, pS2, B1, and OT EREs differentially activate transcription, we initiated a series of studies to examine the ER-ERE interaction in detail using highly purified receptor. Baculovirus-expressed ER, which was fused at the amino terminus to a flag epitope, was purified by immunoadsorption. The ability of the purified ER to bind to each of the four EREs was assessed in gel mobility shift assays. Inclusion of increasing concentrations of ER in the binding reaction resulted in a dose-dependent increase in the formation of the receptor-DNA complex (Fig. 3Go). Thus, the baculovirus-expressed, purified receptor bound effectively to each of the EREs tested.



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  		Figure 3. Binding of ER to the A2, pS2, B1, and OT EREs

Increasing amounts of purified ER (0, 15, or 30 fmol) were combined with 32P-labeled DNA fragments containing the A2, pS2, B1, or OT ERE and fractionated on a nondenaturing acrylamide gel.

 
Protease Sensitivity of ERE-Bound Receptor
We previously demonstrated that the conformation of partially purified, yeast-expressed human ER was different when the receptor was bound to the A2 and pS2 EREs (23) and hypothesized that this change in ER conformation might influence the ability of the receptor to activate transcription. To determine whether a DNA-induced change in ER conformation was a common characteristic of the ER-DNA interaction and whether changes in receptor conformation might influence transcription activation, protease sensitivity assays were carried out to examine the conformation of the receptor when it was bound to the A2, pS2, B1, or OT ERE. Protease sensitivity assays rely on the ability of a protease to cleave a protein at accessible amino acids. Thus, if the DNA-bound receptor is subjected to limited proteolysis and the resulting receptor-DNA complexes migrate with different mobilities, one can infer that different amino acids are exposed and that ER conformation varies when the receptor is bound to different ERE sequences.

Purified ER was combined with 32P-labeled DNA fragments containing the A2, pS2, B1, or OT ERE and digested with increasing concentrations of Staphylococcus aureus V8 protease, which cleaves at aspartic and glutamic acids. Digestion of the A2 ERE-bound receptor with V8 protease produced three major (V2, V3, and V4) and two minor (V5 and V6) receptor-DNA complexes (Fig. 4Go). This digestion pattern was distinctly different from the digestion pattern formed with the OT ERE-bound receptor (V1, V2, V5, and V6). While digestion of the pS2 ERE-bound receptor with V8 protease produced four equally represented complexes (V2, V3, V5, and V6), digestion of the B1 ERE-bound receptor produced three major (V2, V3, and V5) and two minor (V4 and V6) complexes.



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  		Figure 4. V8 Protease Digestion Patterns of the ERE-Bound Receptor

32P-labeled DNA fragments containing the A2, pS2, B1, or OT ERE were combined with 100 fmol purified ER, digested with 0, 0.25, 0.5, 1.0, 2.5, 3.75, or 5.0 µg V8 protease, and fractionated on a nondenaturing acrylamide gel. The V8 protease-digested receptor-DNA complexes are indicated (V1–V6).

 
To determine whether a protease with different specificity could produce different digestion patterns when the receptor was bound to each of the four EREs, protease sensitivity assays were carried out with proteinase K, which preferentially cleaves at aliphatic and aromatic amino acids. The digestion patterns of the A2 and OT ERE-bound ER differed significantly from each other and from the digestion patterns of the pS2 and B1 ERE-bound receptor (Fig. 5Go). Digestion of the A2 ERE-bound receptor with proteinase K produced one major (P5) and two minor (P4 and P7) receptor-DNA complexes. In contrast, when the receptor was bound to the OT ERE, six receptor-DNA complexes were observed (P1, P2, P3, P4, P6, and P7). Digestion of the pS2 and B1 ERE-bound receptor resulted in formation of protein-DNA complexes with similar mobilities (P3, P4, P5, P6, and P7). However, protein-DNA complexes P4 and P5 were more prominent with the B1 ERE than with the pS2 ERE. Since our protease sensitivity assays used highly purified ER and DNA fragments that were identical in size and nucleotide composition, except for the ERE sequence, these findings demonstrate that the differences observed in receptor-DNA complex formation were due to differences in conformation of the receptor when bound to the A2, pS2, B1, and OT EREs. In spite of the fact that five different proteases were used to examine conformation of unoccupied, E2-occupied receptor and antiestrogen-occupied receptor bound to the A2 ERE, no differences in digestion patterns were observed (data not shown).



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  		Figure 5. Proteinase K Digestion Patterns of the ERE-Bound Receptor

32P-labeled DNA fragments containing the A2, pS2, B1, or OT ERE were combined with 100 fmol purified ER, digested with 0, 1.0, 2.5, 5.0, 10, 25, or 50 ng proteinase K, and fractionated on a nondenaturing acrylamide gel. The proteinase K-digested receptor-DNA complexes are indicated (P1–P7).

 
Association of ER with Receptor Interaction Domains (RIDs)
It has become increasingly clear that the ER does not function in isolation but requires the participation of other proteins to effectively regulate transcription of target genes. Since we had demonstrated that the ERE sequence could alter the accessibility of amino acids in the DNA-bound receptor, we next wanted to determine whether DNA-induced changes in receptor conformation could alter the ability of the receptor to associate with two coactivators that are known to interact with ER, glucocorticoid receptor interacting protein 1 (GRIP1, Ref. 42) and steroid receptor coactivator 1 (SRC-1, Ref. 43). Both of these coactivators have three central RIDs (Fig. 6AGo, {blacksquare}), which contain LXXLL motifs and are essential for interaction with nuclear receptors. SRC-1, unlike GRIP1, contains an additional RID in the carboxy terminus (31, 44).



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  		Figure 6. Interaction of GRIP1 and SRC-1 GST Fusion Proteins with A2, pS2, B1, or OT ERE-Bound ER

A, Schematic representation of GRIP1, SRC-1, and GST fusion proteins. Corresponding amino acid numbers and RIDs ({blacksquare}) are indicated. B, Purified ER (57 fmol) was incubated with 32P-labeled DNA fragments containing an A2, pS2, B1, or OT ERE in the presence of 100 nM E2. GST or GRIP1 (GRIP15-766, GRIP1563-1121, and GRIP1730-1121) and SRC-1 (SRC1595-780 and GST-SRC-11237-1440) GST fusion proteins were included in the binding reactions as indicated, and samples were run on nondenaturing acrylamide gels.

 
GRIP1 and SRC-1 glutathione-S-transferase (GST) fusion proteins were tested for their abilities to interact with the DNA-bound ER in gel mobility shift assays. Three GRIP1 GST fusion proteins, which contained either three or one of the central RIDs and varying amounts of amino- or carboxy-terminal flanking sequence, and two SRC-1 GST fusion proteins, which contained either the three central RIDs or the carboxy-terminal RID, were used. When the SRC-1 or GRIP1 GST fusion proteins were combined with 32P-labeled ERE-containing DNA fragments and purified ER, each of the RID-containing GST fusion proteins interacted with the DNA-bound ER and supershifted the receptor-DNA complexes (Fig. 6BGo). The receptor-DNA complex was unaffected by inclusion of GST alone in the binding reaction. Interestingly, the amount of coactivator-receptor-DNA complex formed was significantly greater than the amount of receptor-DNA complex, suggesting that interaction of the RID-containing peptides with the receptor stabilized the ER-ERE interaction.

Association of Coactivators with the ERE-Bound Receptor
Although the GRIP1 and SRC-1 RIDs failed to distinguish between the A2, pS2, B1, and OT ERE-bound receptor, it seemed possible that coactivator regions outside the RIDs might be required to detect subtle changes in ER conformation. Therefore, we examined the abilities of the ERE-bound receptors to recruit full-length p160 proteins from HeLa nuclear extracts. As seen in Fig. 7AGo, although AIB1 (amplified in breast cancer 1) and TIF2, the human homolog of GRIP1, were readily detected in HeLa nuclear extracts, SRC-1 was barely detectable. As expected, no ER was present in the HeLa nuclear extracts. The association of these p160 proteins with the ERE-bound receptor was assessed in DNA pull-down assays. Biotinylated oligos containing either the A2, pS2, B1, or OT ERE or a nonspecific nucleotide sequence were adsorbed to streptavidin magnetic beads and incubated with the baculovirus-expressed, purified ER and HeLa nuclear extracts. After washing away nonspecifically bound proteins, the DNA-bound ER and associated proteins were eluted, run on an SDS acrylamide gel, transferred to nitrocellulose, and probed with antibodies. As seen in Fig. 7BGo, ER bound to each of the four ERE-containing oligos, but very little ER was bound to the oligo containing a nonspecific sequence (NS). When the same blot was probed with an AIB1-specific monoclonal antibody, significant levels of AIB1 were detected when the oligos contained an ERE, but not when the oligo lacked an ERE sequence. When a TIF2-specific monoclonal antibody was used, the levels of TIF2 recruited to the A2, pS2, B1, and OT ERE-bound ER were again significantly more than observed in the absence of the ERE. However, the levels of TIF2 associated with the four different EREs appeared to vary. To determine whether different levels of AIB1 or TIF2 were recruited to the four different EREs, data from five independent pull-down experiments were combined and quantitated. To account for any differences in the affinity of the receptor for consensus and imperfect EREs (41) and to ensure that the level of coactivator protein did not simply reflect the level of ERE-bound receptor, all data were expressed as the relative ratio of coactivator to ER. Although all four of the ERE-bound receptors recruited similar amounts of AIB1, the B1 ERE-bound receptor recruited statistically lower levels of TIF2 than the A2 ERE-bound receptor (Fig. 7CGo). ER binding to the nonspecific DNA was extremely low and resulted in nearly undetectable levels of AIB1 and TIF2. Thus, recruitment of AIB1 and TIF2 required an ERE and the ER. No significant binding of SRC-1, CBP, p300, TFIIB, TBP, or nuclear receptor corepressor (NCoR) was detected with any of the four EREs (data not shown).



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  		Figure 7. Interaction of the ERE-Bound Receptor with HeLa Nuclear Proteins

A, HeLa nuclear extracts were run on an SDS acrylamide gel, transferred to nitrocellulose, probed with a monoclonal antibody to AIB1, TIF2, SRC-1, or ER, and visualized with a chemiluminescent detection system. The position of mol wt standards run on the same gel are indicated. B, Immobilized oligos containing a nonspecific (NS) sequence or the A2, pS2, B1, or OT ERE were incubated with baculovirus-expressed, purified ER and HeLa nuclear extracts. ER and associated proteins were eluted and subjected to Western blot analysis using antibodies to ER, AIB1, or TIF2. C, Results from five independent pull-down experiments were combined, and the ability of the ERE-bound receptors to recruit AIB1 and TIF2 were assessed. The coactivator/ER ratios are presented as the mean ± SEM. Different letters indicate significant differences in the ability of the ERE-bound receptor to recruit coactivators as determined by ANOVA (P < 0.05) followed by Bonferroni correction.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
To determine whether ERE-induced changes in receptor conformation could play a role in differential expression of estrogen-responsive genes, we assessed the ability of the ER to activate transcription of reporter plasmids containing four different EREs, examined the interaction of the receptor with these consensus and imperfect EREs, and tested the ability of the ERE-bound receptors to recruit coactivator proteins.

Allosteric Modulation of ER Conformation
Using highly purified ER{alpha}, protease sensitivity assays were carried out to demonstrate that the conformation of the receptor was different when it was bound to four different ERE sequences. These findings suggest that individual EREs function as allosteric modulators of receptor conformation. Previous x-ray crystallographic studies have documented that a localized change in the ER DNA binding domain must occur when it binds to the A2 and B1 EREs (10, 45). Our protease sensitivity assays suggest that changes must also occur in the DNA binding domain of the receptor when it is bound to the pS2 and OT EREs and that the changes in the DNA binding domain must be translated into more global changes in the full-length receptor. The differential association of antibodies with the DNA binding domain and the amino and carboxy termini of the A2 and pS2 ERE-bound receptor lend further support to this idea (23). Our studies provide strong evidence that the ER displays substantial structural flexibility and that a change in one region of the receptor may alter conformation in other regions of the receptor. These findings emphasize the importance of examining the full-length, DNA-bound receptor when defining mechanisms by which the ER modulates transcription activation.

A number of studies have examined ligand-induced conformational changes in the ER ligand binding domain and in the full-length receptor (19, 20, 46). Interestingly, we were unable to detect ligand-induced differences in conformation of the A2 ERE-bound receptor in our protease sensitivity assays using five different proteases (data not shown). This could mean that the DNA-induced change in receptor conformation obscures the ligand-induced change in receptor conformation, that DNA occludes amino acids that are exposed in the absence of DNA, or that the assay used does not have the sensitivity required to detect ligand-induced changes in receptor conformation.

Receptor-DNA interactions
Distinct differences in the sensitivity of the A2, pS2, B1, and OT EREs to DNase I digestion were detected in our in vitro footprinting experiments. These differences most likely result from changes in amino acid-nucleotide interactions that occur when the nucleotide sequence deviates from the A2 ERE. The presence of an adenine in the vitellogenin B1 ERE 5' half-site (AGTCAnnnTGACC), rather than the guanine found in the A2 ERE 5'-half-site, requires the rearrangement of a local hydrogen bond network (10, 45). Similar amino acid rearrangement may be required when the adenine in the A2 ERE 3'-half-site (GGTCAnnnTGACC), which forms a hydrogen bond with an glutamic acid in the ER DNA binding domain, is replaced by a guanine in the pS2 ERE 3'-half-site (GGTCAnnnTGGCC). Although the cytosine present in the A2 ERE 5'-half-site does not form a hydrogen bond with the ER DNA binding domain in x-ray crystallographic studies, the complementary guanine nucleotide does form a hydrogen bond with an arginine. This 1-bp change in the OT ERE 5'-half-site (GGTGAnnnTGACC) apparently represents a less detrimental change in nucleotide sequence than those found in the imperfect pS2 and B1 ERE half-sites and does not substantially decrease the protection of the OT ERE compared with the A2 ERE. The effective protection of both A2 and OT ERE half -sites may partially account for the enhanced ability of these two sequences to function as more potent transcriptional enhancers.

DNase I hypersensitivity was observed flanking each of the ERE sequences. We have previously shown that ER binding induces conformational changes in DNA fragments containing A2 and imperfect EREs (11, 47). These ER-induced changes in DNA structure could increase the accessibility of sequences flanking the ERE to DNase I cleavage. Taken together, our combined results indicate that the ER-ERE interaction is a dynamic process involving changes in receptor conformation and in DNA structure.

Interaction of ER with Coactivators
A number of coactivator proteins have been identified that interact with nuclear hormone receptors in a hormone and AF-2-dependent manner including the highly related p160 family members SRC-1/NCoA-1, TIF2/GRIP1/NCoA-2, and pCIP/ACTR/AIB1/RAC3/TRAM-1 (15, 48). We have demonstrated that SRC-1 and GRIP1 GST fusion proteins interacted with purified ER when it was bound to A2, pS2, B1, and OT EREs. Thus, DNA-induced changes in receptor conformation did not interfere with the ability of the receptor to interact with GRIP1 and SRC-1 RIDs. In fact, the SRC-1 and GRIP1 fusion proteins substantially increased the amount of receptor-DNA complex formed. The stabilization of the ER-ERE interaction may, in part, help explain the abilities of these coactivators to enhance estrogen-mediated transcription activation. While we have not addressed the abilities of individual SRC-1 and GRIP1 RIDs to stabilize the ER-DNA interaction, others have demonstrated that the second of the three central SRC-1 and GRIP1 RIDs have a higher affinity for ER and an increased capacity to activate transcription than the other two central RIDs (44, 49). We found the fusion proteins containing the three central GRIP1 RIDs or the third of the central GRIP1 RIDs (GRIP1730-1121) were both quite effective in stabilizing the ER-DNA interaction.

Many groups have reported that the interaction of ER with p160 proteins is ligand dependent and plays an important role in transcription activation (Refs. 15, 48 and references therein). However, the effect of DNA binding on the receptor-coactivator interaction has generally not been addressed. Although AIB1 was recruited equally to all four of the ERE-bound receptors, significantly less TIF2 was recruited to the B1 ERE-bound receptor compared with the A2 ERE-bound receptor. Increased expression and recruitment of TIF2 has been correlated with enhanced activation of estrogen-responsive reporter plasmids (50). Thus, it is quite intriguing that the B1 ERE was the least potent transcriptional enhancer in HeLa cells in vivo and that the B1ERE- bound receptor was the least efficient in recruiting TIF2 from HeLa nuclear extracts in vitro. These findings suggest that ER conformation and the association of the receptor with coactivator proteins are influenced by DNA binding. Although the differential recruitment of TIF2 to the ERE-bound receptor may help to enhance gene expression, it seems likely that the exposure of distinct receptor epitopes would also lead to the recruitment of other ERE-specific coregulatory proteins and thereby assist in modulating transactivation. This is, to our knowledge, the first demonstration that differences in ERE sequence alter the association of ER with a coregulatory protein. However, DNA-induced effects on recruitment of coregulatory proteins are not restricted to the ER. Takeshita et al. (31) have suggested that DNA binding influences the association of the thyroid hormone receptor with SRC1.

Regulation of Estrogen-Responsive Genes
Our transfection studies used simple promoters containing a single ERE and a TATA sequence. Certainly naturally occurring estrogen-responsive promoters contain numerous cis elements and require the participation of multiple trans acting factors to effectively regulate transcription. However, our transfection experiments have clearly demonstrated that the abilities of A2 and imperfect EREs to regulate transcription varied substantially. The affinity of the ER is 2-fold higher for the A2 ERE than for the pS2, B1, or OT EREs (11, 41). Thus, the decreased affinity of the receptor for the imperfect EREs may partially account for the decreased ability of the imperfect EREs to activate transcription. However, since the affinity of the receptor for the imperfect EREs is similar (11, 41), differences in affinities of the receptor for the imperfect EREs could not explain the differences in the abilities of the three imperfect EREs to activate transcription. We propose that ERE-induced changes in receptor conformation and the differential recruitment of coregulatory proteins by the ERE-bound receptor may lead to differential expression of genes possessing distinct ERE sequences. Additional regulatory versatility could be provided by the ability of the receptor to detect subtle differences in ERE sequence and bind preferentially to specific ERE half-sites. These studies have identified mechanisms that could mediate differential expression of estrogen-responsive genes in a single cell and provided new insight to define how imperfect EREs regulate transcription activation.

DNA-induced changes in receptor conformation have now been documented with a number of nuclear receptor family members. Estrogen, glucocorticoid, vitamin D, progesterone, retinoic acid, retinoid X, and thyroid hormone (23, 27, 28, 29, 30, 31, 32, 33, 34) receptors undergo conformational changes on binding to their cognate recognition sequences. Given the high degree of structural and functional homology of nuclear receptor superfamily members, it seems plausible that DNA-induced changes in receptor conformation and sequence-specific recruitment of nuclear proteins could play a role in regulating transcription of other hormone-responsive genes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture
HeLa cells were maintained in DMEM/F12 containing 10% FCS and transferred to media containing 10% charcoal dextran (CD)-treated (51) FCS 2 days before harvest. MCF-7 cells were maintained in phenol red free Eagle’s MEM containing 5% CD-treated (51) calf serum and transferred to serum-free media (52) 3 days before harvest.

Plasmids
OT ERE oligos 5'-CTAGATTACCGGTGACCTTGACCCTACTCA-3' and 5'-GATCTGAGTAGGGTCAAGGTCACCGGTAAT-3' were annealed and inserted into pCY7 (53) as previously described for the B3ERE circular permutation vectors (11) to produce B3OT ERE. B3consERE, B3pS2ERE, B3ERE2 (11), and B3OT ERE contained identical nucleotide sequence, except for the ERE sequence. Annealed OT ERE oligos were also inserted into TATA-CAT (54) as described previously (11) to construct the CAT reporter plasmid OT ERE TATA-CAT. OT ERE+10 TATA-CAT was prepared as described for the ERE+10 TATA-CAT vectors (11). The CAT reporter plasmids contained promoters that were identical in nucleotide sequence except for the ERE sequence. Thus, the sequence flanking the EREs and position of the promoters were identical and would not influence transactivation. All plasmids were sequenced and purified on two cesium chloride gradients.

SRC-1 (pGEX NBD1and pGEX NBD2) and GRIP1 (pGEX-T1GRIP1 5-766, pGEX GRIP1563-1121, pGEX GRIP1730-1121) GST expression vectors were generously provided by Akira Takashita (Toranomon Hospital, Tokyo, Japan) and Michael Stallcup (University of Southern California, Los Angeles, CA), respectively.

Preparation of Expressed Proteins and Nuclear Extracts
Viral stock for production of ER{alpha} in Sf9 cells was generously provided by James Kadonaga (University of California, San Diego, CA) and Lee Kraus (Cornell University, Ithaca, NY). Cells were infected with the ER{alpha}-containing recombinant baculovirus for 72 h, exposed to 10 nM E2 17ß-estradiol (E2) 20 min before harvest and immunopurified essentially as described (55). Flag-tagged ER was eluted from the M2 antibody resin with elution buffer [20 mM Tris, pH 7.5, 100 mM NaCl, 0.2 mM EDTA, 10% glycerol, 0.1% NP-40, and 2 mM dithiothreitol (DTT)] containing 0.5 mg/ml ovalbumin and 0.2 mg/ml flag peptide (University of Illinois Biotechnology Center, Urbana, IL). To determine ER concentration, purified ER was combined with 30 nM [6,7-3H] estradiol (52 Ci/mmol, Amersham Pharmacia Biotech, Piscataway, NJ) with or without a 150-fold excess of unlabeled E2 in PTGG buffer (4 mM Na2HPO4, pH 7.4, 0.08% mono-thioglycerol, 10% glycerol) with protease inhibitors (50 µg/ml leupeptin, 5 µg/ml PMSF, 1 µg/ml pepstatin, and 5 µg/ml aprotinin) and incubated at room temperature for 30 min. Hydroxylapatite resin (100 µl) was added and incubated for 25 min at 4 C. The resin was washed with PTGG buffer four times and resuspended in 1 ml ethanol. The ethanol-solubilized 3H E2 was quantitated and the level of bound ER was determined by subtracting nonspecific counts per min from total counts per min.

SRC-1 and GRIP1 GST fusion proteins were expressed in the BL21DE3 pLys S strain of Escherichia coli. Cells were induced with 1 mM isopropylthio-ß-D-galactoside for 3 h at 37 C, pelleted, frozen, and lysed in 3 volumes of TEGND (50 mM Tris, pH 7.9, 1 mM EDTA, 10% glycerol, 0.5 M NaCl, 5 mM DTT) with protease inhibitors. Sodium deoxycholic acid was added to 0.05% and rotated at 4 C for 15 min. The cell lysate was clarified by centrifugation at 180,000 x g for 30 min. The supernatant was incubated with glutathione sepharose beads (Amersham Pharmacia Biotech) for 30 min at 4 C. After washing beads with PBS containing 0.1% Triton X-100, 5 mM DTT, and protease inhibitors, the GST-fusion proteins were eluted with 10 mM reduced glutathione in 50 mM Tris, pH 8, and 0.2% Triton X-100.

To prepare MCF-7 nuclear extracts, cells were harvested, exposed to10 nM E2 for 20 min at 37 C, and homogenized in TEG buffer (50 mM Tris, pH 7.9, 7.5 mM EDTA, and 10% glycerol) containing protease inhibitors. Nuclei were pelleted and resuspended in TEG buffer containing 0.5 M KCl with protease inhibitors and incubated for 20 min at 4 C with vortexing at 10-min intervals. Nuclear lysates were spun 180,000 x g for 30 min at 4 C. The supernatant containing ER and other nuclear proteins was aliquoted and stored at -80 C. Protein concentrations of the MCF-7 nuclear extracts were determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Richmond, CA) with BSA as a standard. HeLa nuclear extracts were prepared in similar fashion except that cells were not exposed to E2.

HeLa Cell Transfections
HeLa cells were seeded in six-well plates at a density of 850,000 cells per well. After 16 h, the cells were changed to serum free media and transfected by combining 5 µg ERE+10 CAT reporter plasmid (11), 250 ng cytomegalovirus (CMV)ß-gal (CLONTECH Laboratories, Inc., Palo Alto CA), and 5 ng of the human ER expression vector pCMV5 hER (56) with 10 µg lipofectin (Life Technologies, Inc., Gaithersburg, MD) and 32 µg transferrin (Sigma, St. Louis MO) in HBSS. The DNA/lipofectin/transferrin mixture was incubated for 10 min at ambient temperature and added to the cells. After 6–7 h at 37 C, the DNA/lipofectin/transferrin mixture was removed and the cells were maintained on DMEM/F12 with 10% CDFCS ± 10 nM E2 for 24 h. Cells were harvested in TNE (40 mM Tris, pH 7.5, 140 mM NaCl, and 1.5 mM EDTA), pelleted, and resuspended in 250 mM Tris-HCl. The cells were lysed during three freeze/thaw cycles and the supernatant was cleared by centrifugation.

ß-Galactosidase activity was determined (57) and used to normalize each sample for transfection efficiency. To determine CAT activity, 130 µl cell extract were combined with 41.4 µg acetyl CoA and 0.1 µCi [14C] chloramphenicol, adjusted to a final volume of 150 µl with 20 mM Tris, pH 7.5, and incubated 1.5 h at 37 C. Acetylated chloramphenicol was separated from nonacetylated chloramphenicol on Sil G TLC plates (Alltech, Deerfield, IL) using 5.3% methanol in chloroform. Levels of nonacetylated and acetylated chloramphenicol were quantitated on a Molecular Dynamics Phosphorimager with ImageQuant software (Molecular Dynamics, Inc., Sunnyvale CA). Student’s t tests were used to determine whether statistical differences between ethanol and E2-treated groups existed.

DNase I Footprinting
The CAT reporter vectors consERE-CAT, pS2ERE-CAT, ERE2-CAT (11), and OT ERE-CAT, which contained the A2, pS2, B1, and OT EREs, respectively, were digested with BamHI. The 1.8-kb ERE-containing DNA fragments were gel purified, labeled as described (23), and digested with NcoI. The 128-bp 32P-labeled ERE-containing DNA fragments were gel purified and combined with the indicated amounts of MCF-7 nuclear extract in binding reaction buffer (15 mM Tris, pH 7.9, 0.2 mM EDTA, 10% glycerol, and 4 mM DTT) containing 45 mM KCl, 1 µg poly(dI-dC), 1.25 mM MgCl2, and 0.5 mM CaCl2 in a final reaction volume of 50 µl. Ovalbumin was included as needed to maintain constant protein concentrations. The binding reactions were incubated for 15 min at room temperature and then digested with RQ1 ribonuclease RNase-free DNase (Promega Corp., Madison WI) for 0.5–9 min. The resulting DNA fragments were separated on a sequencing gel and visualized by autoradiography. The protection of 5'- and 3'-ERE half-sites was quantitated using the method of Brenowitz et al. (58). The 5'- and 3'-ERE half-site boundaries were determined using the dimethylsulfate (DMS)-treated, piperidine-cleaved 32P-labeled DNA fragments, and the levels of radioactivity in each 5'- and 3'-ERE half -site were quantitated from four independent experiments using a phosphorimager and ImageQuant software (Molecular Dynamics, Inc.). To account for differences in sample loading, the amounts of radioactivity present in each 5'- and 3'-half site were normalized to a region of the gel that was unaffected by the addition of nuclear proteins. The same region of each lane was used for normalization purposes. The level of protection, fraction protected, was calculated by comparing the radioactivity remaining in each ERE half-site after addition of MCF-7 nuclear proteins with the level of radioactivity present in each ERE half -site in the absence of nuclear proteins.

Gel Mobility Shift and Protease Sensitivity Assays
For characterization of the purified ER, the circular permutation vectors B3consERE, B3pS2ERE, B3ERE2 (11), and B3OTERE containing the A2, pS2, B1, and OT EREs, respectively, were digested with EcoRI and BamHI. DNA fragments were 32P labeled as described previously (23). The 55-bp ERE-containing DNA fragments were isolated and combined with purified, E2-occupied ER in binding reaction buffer, 20 mM KCl, and 50 ng of poly(dI-dC) in a final volume of 20 µl and incubated 15 min at room temperature. For the coactivator studies, 55 bp 32P-labeled ERE-containing DNA fragments were incubated with 57 fmol of purified, E2-occupied ER for 10 min. Ovalbumin (20 µg) was added to all binding reactions and GRIP1 and SRC-1 GST fusion proteins or GST were added as indicated. Partial proteolysis of DNA-bound ER was carried out with 55 bp ERE-containing DNA fragments and 100 fmol of purified, E2-occupied ER{alpha} as described (23). After a 10-min incubation, the indicated amounts of S. aureus V8 protease (Worthington Biochemical Corp., Freehold, NJ) or proteinase K (Promega Corp.) were added to the binding reactions. Free and complexed DNAs were separated on low ionic strength nondenaturing acrylamide gels (59).

DNA Pull-Down Assays
The 34-bp oligos used in pull-down assays were prepared by annealing a 5'-biotinylated forward strand to the reverse strand. Assays were carried out essentially as described (60). Four picomoles of annealed oligos containing either the A2, pS2, B1, or OT ERE, or a nonspecific sequence were immobilized on 100 µg of streptavidin paramagnetic beads (Dynal, Lake Success, NY) in 10 µl of buffer T (10 mM Tris, pH 7.5, 1 mM EDTA, 1 M NaCl, 0.003% NP40) for 1 h at room temperature with constant agitation. After one wash with buffer T at 1 mg beads/ml buffer and one wash with transcription buffer (10 mM HEPES, pH 7.6, 100 mM potassium glutamate, 2.5 mM DTT, 10 mM magnesium acetate, 5 mM EGTA, 3.5% glycerol) with 0.003% NP40, the immobilized DNA was incubated with transcription buffer containing 2.5 mg/ml BSA, 5 mg/ml polyvinylpyrrolidone, and 2.5 mM DTT for 30 min at room temperature. The immobilized DNA was washed twice with transcription buffer containing 0.5 mg/ml BSA and 0.05% NP40 and incubated with 750 fmol of purified ER in 50 µl of transcription buffer containing 0.001% NP40, 5 µg of BSA, and 10-6 M E2. 50 µl transcription buffer containing 100 µg of HeLa nuclear extract, 250 ng poly(dIdC), 1 mM ATP, 0.001% NP40, 80 mM KCl, and 10-6 M E2 was incubated for 10 min at 4 C and spun in a microfuge for 2 min at 4 C. The supernatant was added to the ER-DNA mixture. After rotation for 4 h at 4 C, the nonadsorbed proteins were removed and the DNA was washed three times with 300 µl of transcription buffer containing 0.5 mg/ml BSA, 0.05% NP40, and 10-7 E2. The ER and its associated proteins were eluted in 10 µl of SDS sample buffer, separated on 10% SDS gel, and electroblotted onto a nitrocellulose membrane. Western analysis was carried out with monoclonal antibodies directed against TIF2 (BD Transduction Laboratories, Inc. Lexington, KY), AIB1 (BD Transduction Laboratories, Inc.), SRC1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or the FLAG epitope (Sigma, St. Louis, MO). A chemiluminescent detection system (Pierce Chemical Co., Rockford, IL) was used to detect the proteins. Autoradiograms were scanned and quantitated using ImageQuant 5.0. Coactivator/ER ratios from five independent experiments were combined. To minimize inter experimental variation, each coactivator/ER ratio was divided by the mean coactivator/ER ratio for that experiment and multiplied by mean coactivator/ER ratio for all experiments. ANOVA was carried out using InStat 1.0 software (Louisiana State University, Baton Rouge, LA).


   ACKNOWLEDGMENTS
 
We thank James Kadonaga, Lee Kraus, Michael Stallcup, and Akira Takeshita for generously providing reagents used in these studies. We thank Penelope Pitch and Yvonne Ziegler for technical assistance, Kurt Kwast for assistance with statistical analysis, and Larry Petz for helpful comments during the preparation of this manuscript.


   FOOTNOTES
 
Address requests for reprints to: Ann M. Nardulli, Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, Illinois 61801. E-mail: anardull{at}life.uiuc.edu

This work was supported by NIH Grant DK-53884 (to A.M.N.) and an American Heart Association Predoctoral Fellowship (to J.R.W.).

Received for publication January 12, 2001. Revision received March 16, 2001. Accepted for publication April 2, 2001.


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Nucleic Acids Res., August 1, 2007; 35(15): 5028 - 5038.
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J. R. Schultz-Norton, K. A. Walt, Y. S. Ziegler, I. X. McLeod, J. R. Yates, L. T. Raetzman, and A. M. Nardulli
The Deoxyribonucleic Acid Repair Protein Flap Endonuclease-1 Modulates Estrogen-Responsive Gene Expression
Mol. Endocrinol., July 1, 2007; 21(7): 1569 - 1580.