Estrogen response elements alter coactivator recruitment through allosteric modulation of estrogen receptor beta conformation.

Estrogen receptor beta (ERbeta) activates transcription by binding to estrogen response elements (EREs) and coactivator proteins that act as bridging proteins between the receptor and the basal transcription machinery. Although the imperfect vitellogenin B1, pS2, and oxytocin (OT) EREs each differ from the consensus vitellogenin A2 ERE sequence by a single base pair, ERbeta activates transcription of reporter plasmids containing A2, pS2, B1, and OT EREs to different extents. To explain how these differences in transactivation might occur, we have examined the interaction of ERbeta with these EREs and monitored recruitment of the coactivators amplified in breast cancer (AIB1) and transcription intermediary factor 2 (TIF2). Protease sensitivity, antibody interaction, and DNA pull-down assays demonstrated that ERbeta undergoes ERE-dependent changes in conformation resulting in differential recruitment of AIB1 and TIF2 to the DNA-bound receptor. Overexpression of TIF2 or AIB1 in transient transfection assays differentially enhanced ERbeta-mediated transcription of reporter plasmids containing the A2, pS2, B1, and OT EREs. Our studies demonstrate that individual ERE sequences induce changes in conformation of the DNA-bound receptor and influence coactivator recruitment. DNA-induced modulation of receptor conformation may contribute to the ability of ERbeta to differentially activate transcription of genes containing divergent ERE sequences.

Interaction of estrogen receptors alpha and beta with estrogen response elements.

To understand how estrogen-responsive genes are regulated, we compared the abilities of estrogen receptors (ERs) alpha and beta to bind to and activate transcription through the consensus vitellogenin A2 ERE and the imperfect pS2, vitellogenin B1, and oxytocin (OT) EREs. Transient transfection experiments demonstrated that ERalpha and ERbeta induced the highest levels of transcription with the A2 ERE, intermediate levels of transcription with the OT ERE, and low levels of transcription with the pS2 and B1 EREs. ERalpha and ERbeta had higher affinities for the A2 ERE than for any of the three imperfect EREs but similar affinities for the pS2, B1, and OT EREs in gel mobility shift assays. ERalpha had a higher affinity and was a more potent activator of transcription than ERbeta. Interestingly, protease sensitivity assays demonstrated that A2, pS2, B1, and OT EREs induced distinct changes in ERalpha and ERbeta conformation thereby providing different functional surfaces for interaction with regulatory proteins involved in control of estrogen-responsive genes.

Allosteric modulation of estrogen receptor conformation by different estrogen response elements.

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.

Sp1 binding sites and an estrogen response element half-site are involved in regulation of the human progesterone receptor A promoter.

Progesterone receptor gene expression is induced by estrogen in MCF-7 human breast cancer cells. Although it is generally thought that estrogen responsiveness is mediated through estrogen response elements (EREs), the progesterone receptor gene lacks an identifiable ERE. The progesterone receptor A promoter does, however, contain a half-ERE/Sp1 binding site comprised of an ERE half-site upstream of two Sp1 binding sites. We have used in vivo deoxyribonuclease I (DNase I) footprinting to demonstrate that the half-ERE/Sp1 binding site is more protected when MCF-7 cells are treated with estrogen than when cells are not exposed to hormone, suggesting that this region is involved in estrogen-regulated gene expression. The ability of the half-ERE/Sp1 binding site to confer estrogen responsiveness to a simple heterologous promoter was confirmed in transient cotransfection assays. In vitro DNase I footprinting and gel mobility shift assays demonstrated that Sp1 present in MCF-7 nuclear extracts and purified Sp1 protein bound to the two Sp1 sites and that the estrogen receptor enhanced Sp1 binding. In addition to its effects on Sp1 binding, the estrogen receptor also bound directly to the ERE half-site. Taken together, these findings suggest that the estrogen receptor and Sp1 play a role in activation of the human progesterone receptor A promoter.

Regulation of the estrogen-responsive pS2 gene in MCF-7 human breast cancer cells.

To understand how hormones and antihormones regulate transcription of estrogen-responsive genes, in vivo footprinting was used to examine the endogenous pS2 gene in MCF-7 cells. While the consensus pS2 estrogen response element (ERE) half site was protected in the absence of hormone, both the consensus and imperfect ERE half sites were protected in the presence of estrogen. 4-Hydroxytamoxifen and ICI 182,780 elicited distinct footprinting patterns, which differed from those observed with vehicle- or with estrogen-treated cells suggesting that the partial agonist/antagonist and antagonist properties of 4-hydroxytamoxifen or ICI 182,780, respectively, may be partially explained by modulation of protein-DNA interactions. Footprinting patterns in and around the TATA and CAAT sequences were identical in the presence and in the absence of estrogen suggesting that the basal promoter is accessible and poised for transcription even in the absence of hormone. In vitro DNase I footprinting experiments demonstrated that the estrogen receptor bound to the pS2 ERE and that adjacent nucleotides were protected by MCF-7 nuclear proteins. These findings indicate that transcription of the pS2 gene is modulated by alterations in protein binding to multiple sites upstream of the basal promoter, but not by changes in protein-DNA interactions in the basal promoter.

The high mobility group protein 1 enhances binding of the estrogen receptor DNA binding domain to the estrogen response element.

We have examined the ability of the high-mobility group protein 1 (HMG1) to alter binding of the estrogen receptor DNA-binding domain (DBD) to the estrogen response element (ERE). HMG1 dramatically enhanced binding of purified, bacterially expressed DBD to the consensus vitellogenin A2 ERE in a dose-dependent manner. The ability of HMG1 to stabilize the DBD-ERE complex resulted in part from a decrease in the dissociation rate of the DBD from the ERE. Antibody supershift experiments demonstrated that HMG1 was also capable of forming a ternary complex with the ERE-bound DBD in the presence of HMG1-specific antibody. HMG1 did not substantially affect DBD-ERE contacts as assessed by methylation interference assays, nor did it alter the ability of the DBD to induce distortion in ERE-containing DNA fragments. Because HMG1 dramatically enhanced estrogen receptor DBD binding to the ERE, and the DBD is the most highly conserved region among the nuclear receptor superfamily members, HMG1 may function to enhance binding of other nuclear receptors to their respective response elements and act in concert with coactivator proteins to regulate expression of hormone-responsive genes.

Estrogen response elements function as allosteric modulators of estrogen receptor conformation.

The estrogen receptor (ER) is a ligand-dependent transcription factor that regulates the expression of estrogen-responsive genes. ER-mediated transcriptional changes are brought about by interaction of the ER with the estrogen response element (ERE). In this study, we examined the interaction of the Xenopus laevis ER DNA binding domain (DBD) and the intact ER with the X. laevis vitellogenin A2 ERE and the human pS2 ERE. Using gel mobility shift, DNase I footprinting, and methylation interference assays, we demonstrated that the DBD bound only as a dimer to the A2 ERE. However, the DBD bound as a monomer to the consensus pS2 ERE half site at lower DBD concentrations and then as a homodimer to the consensus and imperfect pS2 ERE half site at higher DBD concentrations. Antibody supershift experiments carried out with partially purified, yeast-expressed full-length ER demonstrated that three ER-specific antibodies interacted differentially with A2 and pS2 ERE-bound ER, indicating that receptor epitopes were differentially exposed. Furthermore, partial digestion of the A2 and pS2 ERE-bound ER with chymotrypsin or trypsin produced distinct protease cleavage patterns. Taken together, these data provide evidence that differential interaction of the DBD with the A2 and pS2 EREs brings about global changes in ER conformation. The conformational changes in ER induced by individual ERE sequences could lead to association of the receptor with different transcription factors and assist in the differential modulation of estrogen-responsive genes in target cells.

Equilibrium binding of estrogen receptor with DNA using fluorescence anisotropy.

Interaction of estrogen receptor (ER) with DNA sequences known as estrogen response elements (ERE) is required for estrogen regulation of the expression of target genes. To characterize the affinity and specificity of ER interaction with ERE sequences in vitro under equilibrium conditions, fluorescence anisotropy assays were performed using recombinant, purified ER and a fluorescein-labeled 35-base pair oligonucleotide bearing an idealized palindromic ERE. In buffer containing 100 mM KCl, the baculovirus-expressed, purified human ER bound with similar affinity to the consensus ERE and a mutant ERE with a single base pair change per half-site. Above 225 mM KCl, ER exhibited discrimination between the consensus and mutated ERE targets. Between 225 and 275 mM KCl, binding to the consensus ERE was independent of salt concentration and occurred with an equilibrium dissociation constant (Kd) of 1.8 +/- 0.6 nM, whereas binding to the mutant ERE was not detected at ER concentrations below 100 nM under the same conditions. At 300 mM KCl, the Kd for the consensus ERE increased approximately 25-fold, suggesting complex salt concentration dependence. Both estrogen-occupied and unoccupied ER bound to the consensus ERE sequence with similar affinity, indicating that estrogen affects ER activity at a step other than DNA binding. Unlike the full-length ER, the recombinant DNA binding domain of ER did not discriminate between the consensus and mutated ERE sequences even at buffer salt concentrations greater than 200 mM NaCl, suggesting that ER sequences outside the DNA binding domain may be important in promoting specific binding.

Mechanistic aspects of estrogen receptor activation probed with constitutively active estrogen receptors: correlations with DNA and coregulator interactions and receptor conformational changes.

The estrogen receptor (ER) belongs to a large family of nuclear receptors, many of whose members function as ligand-dependent transcriptional activators. The mechanism by which the receptor is converted from an inactive into an activated state is not yet completely understood. To investigate the kind of changes in receptor conformation and interactions that are involved in this activation, we have used the wild type ER and a set of constitutively active ER point mutants that show from 20% to nearly 100% activity in the absence of estrogen. These mutants are of particular interest as they could mimic, in the absence of ligand, the activated state of the wild type receptor. We have analyzed several transcriptional steps that could be involved in the activation: the ability of these receptors 1) to interact with several coactivators (steroid receptor coactivator-1, SRC-1; transcription intermediary factor-1, TIF-1; and estrogen receptor-associated protein 140, ERAP 140) and with members of the preinitiation complex [TATA box-binding protein (TBP), transcription factor IIB (TFIIB)]; 2) to exhibit conformational changes revealed by proteolytic digest patterns similar to those observed for the wild type hormone-occupied ER; and 3) to bend estrogen response element-containing DNA, which is thought to be one of the important phenomena triggering transcriptional activation. Our results demonstrate that the interaction of these mutant receptors with coactivators is likely to be one of the features of the activated step, as the mutant receptors interacted with some coactivators in a ligand-independent manner in proportion to their extent of constitutive activity. However, the different degrees of ligand-independent interaction of the mutant ERs with the three coactivators suggest that SRC-1, TIF-1, and ERAP 140 may play different roles in receptor activity. Limited proteolytic digest experiments reveal that the activated state of the receptor corresponds to a particular conformation of the receptor, which is fully observed with the mutant ER showing the highest activity in the absence of estrogen. Finally, it appears that in inactive or active states, the receptor exhibits distinctly different DNA-bending abilities. Addition of estradiol is able to modify the bending ability of only the wild type receptor, whereas estradiol has no influence on the constitutive receptors, which exhibited the same bending ability as that observed for the ligand-occupied wild type receptor. These data document that the ER undergoes major changes in its conformation and also in its functional properties when it is turned from an inactive into an active state and that mutational changes in the ER protein that result in constitutive, hormone-independent activation mimic many of the changes in ER properties that are normally under hormone regulation.

Prebending the estrogen response element destabilizes binding of the estrogen receptor DNA binding domain.

Binding of many eukaryotic transcription regulatory proteins to their DNA recognition sequences results in conformational changes in DNA. To test the effect of altering DNA topology by prebending a transcription factor binding site, we examined the interaction of the estrogen receptor (ER) DNA binding domain (DBD) with prebent estrogen response elements (EREs). When the ERE in minicircle DNA was prebent toward the major groove, which is in the same direction as the ER-induced DNA bend, there was no significant effect on ER DBD binding relative to the linear counterparts. However, when the ERE was bent toward the minor groove, in a direction that opposes the ER-induced DNA bend, there was a four- to eightfold reduction in ER DBD binding. Since reduced binding was also observed with the ERE in nicked circles, the reduction in binding was not due to torsional force induced by binding of ER DBD to the prebent ERE in covalently closed minicircles. To determine the mechanism responsible for reduced binding to the prebent ERE, we examined the effect of prebending the ERE on the association and dissociation of the ER DBD. Binding of the ER DBD to ERE-containing minicircles was rapid when the EREs were prebent toward either the major or minor groove of the DNA (k(on) of 9.9 x 10(6) to 1.7 x 10(7) M(-1) s(-1)). Prebending the ERE toward the minor groove resulted in an increase in k(off) of four- to fivefold. Increased dissociation of the ER DBD from the ERE is, therefore, the major factor responsible for reduced binding of the ER DBD to an ERE prebent toward the minor groove. These data provide the first direct demonstration that the interaction of a eukaryotic transcription factor with its recognition sequence can be strongly influenced by altering DNA topology through prebending the DNA.

DNA bending is induced by binding of the glucocorticoid receptor DNA binding domain and progesterone receptors to their response element.

Circular permutation analysis was used to determine the degree of DNA bending induced by binding of the glucocorticoid receptor (GR) DNA binding domain (DBD), the human progesterone receptor (PR) DBD, PR-A:A and PR-B:B homodimers, and PR-A:B heterodimers to the glucocorticoid response element/progesterone response element (GRE/PRE). The bending angles induced by the GR DBD and the PR DBD were approximately 28 degrees and 25 degrees, respectively. The PR-B:B and PR-A:A homodimers and the PR-A:B heterodimers all induced similar DNA bending angles of 72-77 degrees. The substantially greater DNA bend induced by full-length PR compared to the PR DBD indicates that sequences outside the classic zinc finger DNA binding domain may play an important role in the interaction of PR with the GRE/PRE. Because PR-A:A and PR-B:B homodimers and the PR-A:B heterodimers induce similar DNA bends, the different abilities of the PR-A and PR-B isoforms to activate transcription are not due to differences in their abilities to distort DNA structure.

Estrogen receptor accessory proteins augment receptor-DNA interaction and DNA bending.

Increasing evidence suggests that accessory proteins play an important role in the ability of the estrogen receptor (ER) and other nuclear hormone receptors to modulate transcription when bound to cis-acting hormone response elements in target genes. We have previously shown that four proteins, hsp70, protein disulfide isomerase (PDI) and two unknown proteins (p48 and p45), copurify with ER that has been isolated by site-specific DNA chromatography (BERE) and influence the interaction of ER with DNA in vitro. To better define the nature of these effects, we used filter binding and electrophoretic mobility shift assays to study the ability of these proteins to alter the kinetics of ER-DNA interaction and to influence the ability of ER to bend DNA when bound to an estrogen response element (ERE). The results of both assays indicate that ERE-purified ER, with its four associated proteins (hsp70, PDI, p48, p45), has a greater ability to bind to the vitellogenin A2 ERE than ER purified by estradiol-Sepharose chromatography in the absence (ESeph) or presence (EATP) of ATP, in which p48, p45 (ESeph) and hsp70 (EATP) are removed. Surprisingly, the rates of association and dissociation of ER and ERE were essentially the same for all three mixtures, suggesting that one or more ER-associated proteins, especially p45 and p48, may be required for ER to attain maximum DNA binding activity. In addition, circular permutation and phasing analyses demonstrated that the same ER-associated proteins produced higher order ER-DNA complexes that significantly increased the magnitude of DNA distortion, but did not alter the direction of the ER-induced bend of ERE-containing DNA fragments, which was toward the major groove of the DNA helix. These results suggest that p45 and/or p48 and possibly hsp70, play an important role both in the specific DNA binding and bending activities of ER and thus contribute to the overall stimulation of transcription in target genes that contain cis-acting EREs.

Effects of wild type and mutant estrogen receptors on DNA flexibility, DNA bending, and transcription activation.

We examined the ability of wild type (WT) estrogen receptor (ER) and mutant ERs to induce distortion and directed bends in DNA fragments containing estrogen response elements and then monitored the ability of these receptors to activate transcription. The ER mutants had either 108 (109-595 ER) or 175 (delta AB ER) amino acids deleted from the amino terminus; 42 (delta F ER) or 65 (1-530 ER) amino acids deleted from the carboxy terminus; or a single point mutation at amino acid 380 (glu-->gln) in the ER hormone binding domain (E380Q ER). Circular permutation analysis was used to determine the degree of distortion induced in estrogen response element-containing DNA fragments (65 degrees for WT ER and E380Q ER, 56 degrees for 109-595 ER, 54 degrees for delta AB ER, 63 degrees for delta F ER, and 60 degrees for 1-530 ER). Phasing analysis delineated the magnitude of directed DNA bends (8.3 degrees for WT ER, 6.9 degrees for 109-595 ER, 6.5 degrees for delta AB ER, 10.6 degrees for delta F ER, 12.4 degrees for 1-530 ER, and 10.2 degrees for E380Q ER) and demonstrated that the direction of the bend was always toward the major groove of the DNA helix. The ability of each receptor to induce transcription of an estrogen-responsive reporter plasmid (E380Q ER > WT ER = delta F ER > 109-595 ER > delta AB ER > 1-530 ER) was related to the ability of the receptor to induce appropriate distortion (63 degrees-65 degrees) and directed DNA bending (8 degrees-10 degrees) angles and the presence of transcription activation functions AF-1 and AF-2. These studies suggest that ER-induced DNA bending is one part of a multistep process involved in regulating estrogen-responsive genes.

Estrogen receptor affinity and location of consensus and imperfect estrogen response elements influence transcription activation of simplified promoters.

We have examined the ability of estrogen receptor (ER) to bind and bend DNA fragments containing the Xenopus laevis vitellogenin A2 estrogen response element (ERE), which contains a palindromic, consensus ERE sequence, the X. laevis vitellogenin B1 ERE2, which contains a 1-bp mismatch in the 5'-end of the half-palindrome, and the human pS2 ERE, which contains a 1-bp mismatch in the 3'-end of the half-palindrome. ER binding induced a 65 degrees bend in DNA fragments containing the consensus ERE, the vitellogenin B1 ERE2, or the pS2 ERE. However, ER affinity for the consensus ERE was 2-fold greater than for either the vitellogenin B1 ERE2 or the pS2 ERE. When Chinese hamster ovary (CHO) cells were transfected with reporter plasmids containing either the consensus ERE, the vitellogenin B1 ERE2, or the pS2 ERE separated from the TATA sequence by 26 helical turns, exposure to 10 nm 17 beta-estradiol increased transcription 12.7-, 2.4-, and 3.8-fold, respectively. Increasing the spacing between the ERE and TATA sequence to three helical turns decreased the ability of the consensus ERE to activate transcription by 55% and increased the ability of the pS2 ERE to activate transcription by 35% but had no significant effect on vitellogenin B1 ERE2 activity. Further increasing the distance between the ERE and TATA sequence to 3.6 helical turns restored the activity of promoters containing the consensus ERE and pS2 ERE but decreased the activity of the promoter containing the relatively weak vitellogenin B1 ERE2. These data support the idea that 1) the affinity of ER for the ERE, 2) the location of an ERE within the promoter, and 3) the magnitude and orientation of DNA bends induced by binding of ER or other proteins are important in transcription activation of estrogen-responsive genes.

Estrogen receptor-induced DNA bending: orientation of the bend and replacement of an estrogen response element with an intrinsic DNA bending sequence.

The estrogen receptor (ER) binds to DNA fragments containing estrogen response elements (EREs) and causes them to bend. To characterize this ER-induced DNA bend and determine if it is involved in transcription activation, three different lines of investigation were used. Using MCF-7 human breast cancer cell extracts and circular permutation analysis, it was determined that molybdate-stabilized, unoccupied cytosolic ER was unable to bind to ERE-containing DNA fragments when maintained at 4 C, but that thermal activation enabled the cytosolic receptor to bind and bend ERE-containing DNA fragments to the same extent as ER present in whole cell extracts. DNA phasing analysis was utilized to determine that ER binding induced DNA fragments containing EREs to bend toward the major groove of the DNA helix. The orientation of this bend was the same with thermally activated, unoccupied cytosolic ER and with unoccupied ER, 17 beta-estradiol-occupied ER, and 4-hydroxytamoxifen-occupied ER present in whole cell extracts. Using transient cotransfection assays, the ability of an intrinsically bent DNA sequence to replace an ERE was tested. When a single consensus ERE, which is induced to bend 56 degrees on ER binding, was replaced with a 54 degrees intrinsic DNA bending sequence, transcription was effectively activated. Similar levels of transcription were also observed when promoters contained either a 108 degrees intrinsic DNA bending sequence or two consensus EREs. However, the 54 degrees DNA bending sequence and a single ERE were unable to cooperatively activate transcription. Because the magnitude and orientation of ER-induced DNA bends are the same with the unoccupied and occupied receptor, DNA bending alone probably does not function as a transcriptional switch to turn on gene transcription. However, DNA bending may be required to provide the architecture needed for modulation of target genes.

Human estrogen receptor bound to an estrogen response element bends DNA.

We have used gel mobility shift assays to examine changes in DNA bending induced by binding of human estrogen receptor (hER) to a series of estrogen response element (ERE) containing DNA fragments. Competition experiments with ERE-containing DNA fragments and antibody supershift experiments demonstrated that ER in crude extracts from MCF-7 human breast cancer cells exhibited specific interaction with the ERE. Using DNA bending standards, we found that binding of ER to a single ERE induced a reproducible DNA bend of 56 degrees. This was 1.65-fold greater than the 34 degrees bending angle we recently reported for binding of bacterially expressed ER DNA binding domain. The DNA bending angle induced was the same whether the salt-extracted receptor was unoccupied, occupied by 17 beta-estradiol, or occupied by trans-hydroxytamoxifen. To determine if proteins associated with ER in MCF-7 cells affect the degree of bending, we examined the ability of partially purified hER expressed in yeast to bend DNA. The degree of bending induced by the partially purified yeast ER was the same as the bending induced by crude MCF-7 cell ER. More highly purified ER from yeast extracts did not bind to an ERE-containing DNA fragment, suggesting that additional proteins may play an important role in the interaction of the ER with the ERE. When two EREs were present in the DNA fragment, a small but reproducible increase in bending was observed. Our demonstration that binding of hER to the ERE induces DNA bending suggests a possible role for DNA bending in ER-induced transcription activation.

DNA bending by nuclear receptors.

Although steroid hormone receptors constitute an intensively studied family of ligand-regulated transcription factors, the mechanism by which these receptors activate transcription has not been defined. Evidence has accumulated from prokaryotic and eukaryotic systems that many transcription factors are capable of binding to their cognate recognition sequences and causing DNA to bend. Therefore, it has been hypothesized that DNA bending and transcription activation may be functionally coupled. We have utilized circular permutation analysis to examine the ability of the estrogen receptor DNA binding domain and the intact estrogen receptor to bend DNA fragments containing estrogen response elements (EREs). The DNA binding domain, which is a less potent activator of transcription, bent ERE containing DNA fragments less (34 degrees) than the intact estrogen receptor (56 degrees), which is a more potent activator of transcription. In addition, when two EREs were present in a DNA fragment, the degree of DNA bending observed was greater than when one ERE was present. These data suggest that DNA bending may play a role in transcription activation of estrogen responsive genes.

Binding of the estrogen receptor DNA-binding domain to the estrogen response element induces DNA bending.

We have used circular permutation analysis to determine whether binding of purified Xenopus laevis estrogen receptor DNA-binding domain (DBD) to a DNA fragment containing an estrogen response element (ERE) causes the DNA to bend. Gel mobility shift assays showed that DBD-DNA complexes formed with fragments containing more centrally located EREs migrated more slowly than complexes formed with fragments containing EREs near the ends of the DNA. DNA bending standards were used to determine that the degree of bending induced by binding of the DBD to an ERE was approximately 34 degrees. A 1.55-fold increase in the degree of bending was observed when two EREs were present in the DNA fragment. These in vitro studies suggest that interaction of nuclear receptors with their hormone response elements in vivo may result in an altered DNA conformation.

The role of estrogen response elements in expression of the Xenopus laevis vitellogenin B1 gene.

We have used site-directed mutagenesis and a homologous transient transfection system to investigate the role of the two imperfect estrogen response elements (EREs) located at -302/-334 in the 5'-flanking region of the estrogen-regulated Xenopus laevis vitellogenin B1 gene. Deletion of either ERE effectively abolishes estrogen-dependent transcription of the vitellogenin promoter. Neither replacement of the two imperfect EREs with a single consensus ERE at -334, nor insertion of one or two consensus EREs at -359, restores full estrogen responsiveness to the mutant promoter. In competition gel mobility shift assays using the DNA binding domain of the Xenopus estrogen receptor, the consensus ERE was a severalfold more effective competitor than the two imperfect B1 EREs. These data suggest that flanking DNA sequences may exert a significant effect on the activity of EREs as hormone-dependent transcription activators. When the imperfect EREs at -302/-334 were present, an additional consensus ERE at -359 exhibited synergistic activation of transcription. However, two consensus EREs located close to the TATA box showed additive, not synergistic, activation of transcription. In contrast, synergistic activation of transcription was observed in synthetic promoters containing two EREs and either the vitellogenin activator element or the NF1 or AP1 upstream activator elements.

Purified estrogen receptor DNA binding domain expressed in Escherichia coli activates transcription of an estrogen-responsive promoter in cultured cells.

The region of the Xenopus laevis estrogen receptor responsible for interaction with DNA, the DNA binding domain (DBD), has been cloned and overexpressed in Escherichia coli using a T7 RNA polymerase expression system. Extracts from cells transformed with the DBD expression vector contain a single protein which reacts with polyclonal antibodies to estrogen receptor and exhibits sequence-specific binding to a DNA fragment containing a consensus estrogen response element. The DBD protein has been purified to near homogeneity. Determination of the rotational relaxation time of the dansylated DBD by fluorescence polarization and size fractionation by Superdex column chromatography indicate that the DBD is a monomer in solution. The DBD forms a single protein-estrogen response element complex in gel mobility shift assays at DBD concentrations of 18-3,600 nM, suggesting that the DBD is bound to both halves of the palindromic estrogen response element. To investigate the ability of the DBD expressed in bacteria to activate gene expression, we have developed a simple liposome-based system for delivery of protein into cultured cells. Transfected DBD protein elicited large, concentration-dependent increases in transcription of an estrogen receptor regulated reporter gene. These data demonstrate that the bacterially expressed DNA binding domain, which represents a small portion of the Xenopus laevis estrogen receptor, retains significant ability to activate transcription of an estrogen-responsive promoter in vertebrate cells.