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Comparison of transcriptional synergy of estrogen receptors α and β from multiple tandem estrogen response elements
Molecular and Cellular Endocrinology 165 (2000) 151 – 161 www.elsevier.com/locate/mce
Comparison of transcriptional synergy of estrogen receptors a and b from multiple tandem estrogen response elements Valentyn V. Tyulmenkov, Sarah C. Jernigan, Carolyn M. Klinge * Department of Biochemistry and Molecular Biology, Uni6ersity of Louis6ille School of Medicine, Louis6ille, KY 40292, USA Received 22 December 1999; accepted 4 April 2000
Abstract Estrogen receptors a and b (ERa and ERb) act as ligand-dependent transcriptional enhancers. We reported that ERa induces synergistic activation of luciferase reporter gene activity in response to E2 from three or four tandem copies of a consensus estrogen response element (ERE) in transiently transfected MCF-7 cells. Here we addressed three questions: (1) is the synergistic activation of reporter gene activity from multiple tandem EREs by ERa restricted to MCF-7 cells?; (2) does ERb induce synergistic activation of reporter activity from multiple tandem EREs?; and (3) does ERb bind cooperatively to multiple tandem EREs? To address the first two questions, ER-negative CHO-K1 cells were co-transfected with ERa or ERb and ERE-driven reporter plasmids. Both ERa and ERb activated ERE-driven luciferase gene activity in an estradiol-dependent manner. Induction by ERb was lower than ERa from each ERE. We demonstrate that both ERa and ERb induce transcriptional synergy with three or four, but not two, tandem copies of an ERE. Electrophoretic mobility shift assays (EMSA) indicated an increase in ER–ERE binding affinity associated with cooperative binding of ERa and ERb to multiple EREs that may be responsible for transcriptional synergy in transiently transfected cells. We also postulate that interaction of ERa and ERb with coactivators may also play a role in transcriptional synergy. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Estrogen receptor; Estrogen response element; Synergy; Cooperativity; Estradiol; 4-Hydroxytamoxifen
1. Introduction Estrogens exert a wide variety of effects on growth, development, and differentiation, including important regulatory functions within the reproductive systems of both females and males, in mammary gland development and differentiation, in central nervous system functions, in the hypothalamic-gonadal axis, and as anti-atherosclerotic agents. Estrogens mediate these activities through binding to a specific intranuclear receptor protein, the estrogen receptor (ER). ER is a member of the steroid/thyroid superfamily of proteins that act as hormone-inducible transcription factors (Beato and Sanchez-Pacheco, 1996). ER is encoded by two genes i.e. a and b (ERa and ERb) (Kuiper et al., 1996). The term ER will refer to both ERa and ERb, whereas ERa and ERb refer specifically to that iso* Corresponding author. Tel.: +1-502-8523668; fax: + 1-5028526222. E-mail address: [email protected] (C.M. Klinge).
form. While ERa and ERb form homodimers, they can also form ERa:ERb heterodimers in vitro and in vivo (Ogawa et al., 1998). ERa and ERb show different patterns of tissue expression and show different affinities for binding phytoestrogens, indicating that ERa and ERb may play distinct roles in vivo. Ligands, e.g. estradiol (E2), enter the cell nucleus and bind to the ligand binding domain (LBD) of ER inducing conformational changes in ER, including dimerization and phosphorylation (Weigel, 1996), leading to high affinity ER binding to specific DNA sequences: estrogen response elements (EREs). The current model to account for cell-specific regulation of estrogen target gene expression suggests that target cells express different levels of coactivators and corepressors which, along with the amounts of ERa, ERb, and ligand, allows fine-tuning of target gene transcription in response to estrogens. Recent Northern blot analysis confirmed the idea that different rat tissues (Misiti et al., 1998) and cell lines (Folkers et al., 1998) express different amounts of mRNA for the coactivators SRC-1, RIP-140, p300,
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and CBP, as well as the corepressors SMRT, and NCoR. Nuclear receptor coactivators and corepressors were recently reviewed (McKenna et al., 1999). In earlier work, we reported that estradiol (E2)-liganded ERa bound to three or four tandem copies of a consensus ERE (EREc38) in a cooperative manner (Klinge et al., 1992b). E2 – ER binding to one or two EREs was non-cooperative. When ER was liganded by the antiestrogen 4-hydroxy-tamoxifen (4-OHT), ER– ERE binding was not cooperative, regardless of the number of EREs (Klinge et al., 1992a, 1996a,b). More recently, we evaluated how binding to EREc38 affects ER conformation in the LBD as reflected in the dissociation kinetics of [3H] ligand from the ER (Klinge, 1999a). Binding of ERa to EREc38 slowed the rate of E2 dissociation, indicating that DNA allosterically modulates the LBD conformation creating a tighter fit between E2 and ERa (Klinge, 1999a). Synergistic E2-dependent activation of a reporter gene was detected from three and four, but not one or two, tandem copies of EREc38 in MCF-7 cells (Sathya et al., 1997; Klinge, 1999a). Since the number of tandem copies of EREc38 did not alter E2 dissociation kinetics, transcriptional (also called ‘functional’) synergy in MCF-7 cells must involve cellular factors in addition to the ER ligand. To separately evaluate the contributions of ERa and ERb to transcriptional synergy from multiple EREs, transient transfection assays were performed in ER negative CHO-K1 cells co-transfected with mammalian expression vectors for ERa or ERb. Here we report that both ERa and ERb act synergistically to activate reporter gene expression from three and four tandem EREs.
2. Materials and methods
2.1. Preparation of ERE containing plasmids The sequence of the synthetic consensus ERE oligonucleotide, called EREc38, used in these experiments is: 5%-CCAGGTCAGAGTGACCTGAGCTAAAATAACACATTCAG-3% (Peale et al., 1988). Single (EREc38) or multiple, head-to-tail, tandem copies of double stranded EREc38 oligomers (n (EREc38) where n=the number of tandem copies) were cloned into the plasmids pGEM-7Zf(+ ) or pGL3-Promoter (Promega, Madison, WI) as described (Klinge et al., 1992b, 1997).
2.2. Cell transfection CHO-K1 cells were purchased from ATCC (Manassas, VA). CHO cells (2.5× 105) were plated in each well of a 12-well Corning plate in IMDM (all cell culture reagents were from Life Technologies) medium without
phenol red, supplemented with 10% stripped fetal bovine serum (FBS) and 1% Pen-Strep. After 24 h, the cells were transfected using liposome-mediated transfection (Transfast, Promega). CHO-K1 cells were cotransfected with 10 ng of either pCMV-rh ERa or pCMV-rrERb (kindly provided by Dr Benita Katzenellenbogen (Reese and Katzenellenbogen, 1991) and Dr J.-A. Gustafsson (Kuiper et al., 1996), respectively), pGL3-pro-luciferase plasmid containing 1, 2, 3, or 4 tandem copies of EREc38 (0.6 mg), pCMVbgal (0.1 mg) (Clonetech), and 0.5 mg of carrier DNA using Transfast (Promega) essentially as described in (Klinge et al., 1997). The pCMV-rrERb has three different possible translation products of 485, 530, and 549 aa, respectively (Enmark, E. personal communication). Twentyfour hours after transfection, the cells were treated with 10 nM 17b-estradiol (Sigma), 100 nM 4-OHT (Research Biochemicals International, Natick, MA), or an equal volume of ethanol (as vehicle control). Each treatment was performed in triplicate. The cells were maintained in IMDM medium containing 1% stripped FBS. The cells were lysed 24 h after treatment in 150 ml of 1X reporter lysis buffer (Promega) and the cleared extract was assayed for luciferase and b-gal activities as described (Klinge et al., 1997). The fold induction of luciferase activity was normalized for b-gal and is expressed as the ratio of RLU between treatment groups and the vehicle control (which was set to 1) (Klinge et al., 1997).
2.2.1. Western blotting To address the question of whether ERa and ERb are expressed at equal levels in the transfected CHO-K1 cells, whole cell extracts were prepared and the proteins slot-blotted onto a PVDF membrane (Klinge et al., 1997). The membranes were immunoblotted with ERaspecific Ab-10 (Neomarkers) or ERb-specific antiserum Y-19 (Santa Cruz). Western blotting was performed using NEN Renaissance chemiluminescence reagent as described (Klinge, 1999b). Densitometric analysis of the resulting film indicated that comparable protein expression levels of ERa and ERb were achieved in these cells (Western blot data not shown). 2.2.2. Electrophoretic mobility shift assay Nuclear extracts containing recombinant human (rh) ERa were prepared from baculovirus-infected IPLBSf21AE insect cells as described (Klinge et al., 1998). A similar procedure was used for the preparation of recombinant rat (rr) ERb from baculovirus-infected IPLB-Sf21AE insect cells (Klinge et al., 1999). EREcontaining oligomers were labeled with [32P]a-dTTP (800 Ci/mmol from NEN) (Klinge et al., 1997). The size of the ERE oligomers used was 77, 115, 153, and 191 bp for one, two, three, and four tandem copies of the consensus EREc38 sequence (hereafter referred to as
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1EREc38, 2(EREc38), 3(EREc38), and 4(EREc38), respectively). The DNA concentrations were measured using PicoGreen fluorescence assay (Molecular Probes, Eugene, OR). ER– ERE binding reactions included a series of increasing concentrations (40 – 2800 nM) of [32P] labeled oligomer, a constant amount (0.699 0.06 nM, as measured by ER-1(EREc38) binding capacity to obviate the difference between [3H]E2 binding and ERE binding capacities (Driscoll et al., 1998)) of liganded-ER, 1 mM E2, 0.75 mg/ml purified BSA (New England Biolabs), 20 mg/ml poly d(I-C) (Midland Certified Reagent Company, Midland, TX), and 15% (v/v) glycerol in a final volume of 25 ml. ERa-specific monoclonal antibody H222, generously provided by Abbott Laboratories (Abbott Park, IL). ERb-specific polyclonal antisera PA1-310 and Y-19 were purchased from Affinity Bioreagents (Golden, CO) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Reaction and electrophoresis conditions have been described (Klinge et al., 1997). All bands supershifted by an ER-specific antibody were classified as specifically bound DNA. The free DNA band migrated at the bottom of the gel and other bands were non-specifically bound DNA. Counts of ER-bound and free DNA were determined using a Packard Instruments InstantImager and associated software, Packard Imager for Windows v 2.04 (Packard Instrument Company, Meriden, CT). The DNA amount in each band was calculated by multiplying the amount of DNA in the reaction by the percentage of the total radioactivity/lane in that band. Each reaction was repeated in three to four independent assays. The higher ERE concentrations reached saturation binding, allowing the concentration of functional ER to be estimated in each assay from four to five gel lanes. The Hill equation is used to estimate the stoichiometric coefficient (n). However, Hill coefficients (nH) are not reliable for analysis of the stoichiometry of ER– ERE binding. Assume that each receptor molecule (R) binds n ligand molecules (L) with a constant affinity. The concentrations at equilibrium will fit to equation [R]× [L]n =KD [RLn ]
(1)
where [R] and [L] are the concentrations of free receptor and ligand, [RLn ] is that of bound receptor (or ligand), KD is the equilibrium dissociation constant. This equation may be rearranged [RLn ]/(Rtotal − [RLn ])=[L]n/KD so that logarithmic transformation will give the Hill equation log
B = n× log(F)− log(KD) Rtotal − B
(2)
where Rtotal is the total receptor concentration, B is the concentration of bound ligand, F is that of free ligand. In contrast, DNA with multiple EREs is a ligand that
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can bind more than one receptor molecule, and the equation at equilibrium is different from Eq. (1): [R]n × [L] =KD [RnL]
(3)
This equation may be rearranged: [L]/[RnL]= (Rtotal − [RnL])n/KD to be linearized by logarithmic transformation log
B = n× log(Rtotal − B)− log(KD) F
(4)
However, the assumption that KD is constant may be not met. Binding of ER to multiple EREs with varying KDs may be fit to Eq. (4), giving an average estimate for KD and an erroneous estimate for n, which deviates from the true value. This is illustrated by the comparison between the number of available O2 binding sites on the hemoglobin A molecule, i.e. 4, and the stoichiometric coefficient 3, as measured by the Hill analysis (Tyuma et al., 1973). According to this report, occupation of the four available binding sites by O2 is associated with a dramatic increase in affinity. This indicates that the assumption that KD is constant is not valid and results in a discrepancy between the estimated and expected stoichiometric coefficients (Tyuma et al., 1973). Thus, such a discrepancy may indicate interaction between adjacent binding sites or between molecules bound to these sites.
3. Results
3.1. E2 induces transcriptional synergy from multiple tandem copies EREs with ERa and ERb In order to determine the transcriptional responsiveness of each ER isoform independently, CHO-K1 (CHO) cells were co-transfected with the human ERa expression vector pCMV-ERa (Reese and Katzenellenbogen, 1991) or the rat ERb expression vector pCMVERb (Kuiper et al., 1996) for transient transfection experiments. First, the amount of ER expression vector required to activate luciferase activity from two tandem copies of EREc38 in CHO cells was examined (Fig. 1). The amount of luciferase activity increased with the amount of ER expression vector co-transfected, reaching a plateau at approximately 10 ng of plasmid and showing decreased responsiveness with more than 100 ng expression plasmid cotransfected. Based on these results, CHO cells were co-transfected with 10 ng of the expression vector for ERa or ERb. E2-induced luciferase activity from one or multiple tandem copies of EREc38 was assayed in ERa-expressing CHO cells. The relative induction of luciferase activity by E2 was dependent on the number of copies of EREc38 (Fig. 2A). As anticipated based on results
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from MCF-7 cells (Klinge, 1999a), synergistic activation was detected for 3(EREc38) and 4(EREc38). Cotreatment of the cells with E2 plus 4-OHT completely
Fig. 1. Optimization of amount of ER expression vector in transiently transfected CHO cells. CHO cells were co-transfected with the indicated amounts, in ng, of pCMV-ERa (A) or pCMV-ERb (B) plus two-tandem copies of EREc38 in the pGL3-Promoter vector (2(EREc38)) and pCMV-bgal as described in Section 2. Twenty-four hours after plating, the cells were treated with ethanol, 10 nM E2, or 10 nM E2 plus 1 mM 4-OHT. The cells were harvested 24 h after treatment and the cell extracts were assayed for luciferase and b-gal activities (Klinge et al., 1997). Data are displayed as fold induction of luciferase activity, which is the luciferase activity, divided by the b-gal activity in each well. These data are the mean 9 S.E.M. of three separate determinations.
Fig. 2. E2 induces transcriptional synergy from three or four tandem copies of EREc38. CHO cells were co-transfected with pCMV-ERa or pCMV-ERb plus the indicated pGL3-Promoter vector and pCMVbgal as described in Section 2. Twenty-four hours after plating, the cells were treated with ethanol, 10 nM E2, or 10 nM E2 plus 1 mM 4-OHT. The cells were harvested 24 h after treatment and the cell extracts were assayed for luciferase and b-gal activities (Klinge et al., 1997). (A) Data are displayed as fold induction of luciferase activity, which is the luciferase activity, divided by the b-gal activity in each well. The fill symbols are indicated in the inset. The fold induction of luciferase activity is expressed relative to the activity detected upon addition of ethanol (EtOH) which was set to 1; (B) the data presented in Fig. 1 are displayed as fold synergy that was determined by dividing the relative luciferase activity by the number of copies of EREc38 (Mattick et al., 1997). The data shown are presented as mean 9 S.E.M. for 12 independent experiments, in which each perturbation was performed in triplicate. Fill symbols are indicated in the inset and are identical to those in (A).
blocked E2-stimulated luciferase activity from all EREc38 multimers. Inhibition by 4-OHT indicates that the induction of luciferase in response to E2 was mediated by ER. The fold synergy was determined by dividing the relative luciferase activity by the number of copies of EREc38 (Mattick et al., 1997). Comparison of the fold synergy showed 1.4 fold synergy for 2(EREc38), indicating less than additive induction, versus 5.7 and 6.9 fold synergy for 3(EREc38) or 4(EREc38), respectively (Fig. 1B). Thus, cooperative binding of E2-ERa to three or four tandem copies of EREc38 (Klinge et al., 1992b) is reflected in synergistic activity of reporter gene expression in CHO cells transiently expressing ERa. ERb was reported to show lower activity compared to ERa in response to identical concentrations of E2 in transient transfection assays using two (Cowley and Parker, 1999) or three (McInerney et al., 1998) EREs linked to the pS2 gene promoter in CEF and CHO cells, respectively. Similarly, we found that ERb was less active than ERa with one, two, or three tandem copies of EREc38 in transiently transfected CHO cells (Fig. 1A). Interestingly, there was no significant difference in the fold induction of luciferase activity induced by ERa or ERb from 4(EREc38). Comparison of the fold synergy induced by ERb showed 1.4 fold synergy for 2(EREc38), indicating less than additive induction, versus 4.1 and 7.8 fold synergy for 3(EREc38) or 4(EREc38), respectively (Fig. 1B). Thus, despite lower induction of reporter gene activity, ERb synergistically activates reporter gene expression from three and four tandem copies of EREc38, a result similar to that for induction of reporter activity from the same EREs by ERa. One possible explanation for the similar induction of luciferase from three and four tandem copies of EREc38 with ERa is that all available transcription machinery, including coactivators, is saturated when cotransfecting the cells with 10 ng of ER expression vector. To examine this possibility, the amount of ERa transfected was reduced to 5 ng. As anticipated, less luciferase activity was induced from each ERE with 5 ng of ERa cotransfected. The relative induction of luciferase activity by E2 was dependent on the number of copies of EREc38 (Fig. 3). Interestingly, in contrast to the data shown in Fig. 2, E2 synergistically induced luciferase activity from 2(EREc38) as well from 3(EREc38) and 4(EREc38) when 5 ng was cotransfected. Significantly higher luciferase activity was detected from 4(EREc38) than 3(EREc38) (PB 0.05). A similar experiment was performed transfecting CHO cells with 5 ng pCMV-ERb (Fig. 3). In contrast to the synergistic induction of reporter activity from 2(EREc38) seen with 5 ng ERa, the activity induced by ERb was additive. However, in results similar to those shown in Fig. 2, ERb synergistically activated reporter
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Fig. 3. Subsaturating amounts of ERa and ERb reveal transcriptional synergy from multiple tandem copies of EREc38. CHO cells were co-transfected with 5 ng pCMV-ERa (open bars) or pCMV-ERb (shaded bars) plus the indicated pGL3-Promoter vector and pCMVbgal as described in Section 2. Cell treatments were identical to those described in Fig. 2. The fill symbols are identical to those described in Fig. 2. The fold induction of luciferase activity is expressed relative to the activity detected upon addition of EtOH, which was set to 1. The data shown are presented as mean 9 SEM from three separate experiments.
gene transcription from 3(EREc38) and 4(EREc38). Comparison of the data in Figs. 2 and 3 show no difference in the fold synergy from 3(EREc38) and 4(EREc38). However, the fold synergy was greater for 2(EREc38) when less ERa or ERb was transfected. These data indicate that the extent of transcriptional
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synergy is influenced by the intracellular levels of ERa or ERb. To address the question of whether ERa and ERb are expressed at equal levels in the transfected CHO-K1 cells, whole cell extracts were immunoblotted with ERa-specific Ab-10 or ERb-specific antiserum Y-19 (Western blot data not shown). Densitometric analysis of the resulting film indicated that comparable protein expression levels of ERa and ERb were achieved in these cells (data not shown).
3.2. Cooperati6e binding of ERa and ERb to multiple tandem copies of EREc38 Since we reported that calf uterine E2-liganded ERa binds cooperatively to three or four tandem copies of EREc38, but not to 1(EREc38) or two tandem copies of EREc38 (Klinge et al., 1992b), it was of interest to determine if ERb binds cooperatively to multiple tandem EREs. The kinetics of baculovirus-expressed rh ERa and rr ERb binding to one, two or three tandem copies of EREc38 were examined using EMSA. Although baculovirus-expressed ERa shows a single band on a Western blot (data not shown) two bands were detected for ERa binding to 1(EREc38) (Fig. 4).
Fig. 4. ERa binds cooperatively to multiple tandem copies of EREc38. EMSA was used to quantitate the kinetics of ERa binding to 1(EREc38) (panel A); 2(EREc38) (panel B); 3(EREc38) (panel C); or 4(EREc38) (panel D). In each gel, a constant amount (0.7 nM) ERa was incubated with 1 mM E2 for 1-h on ice. Then, the respective [32P]-labeled EREc38 (0.04, 0.08, 0.15, 0.26, 0.42, 0.66, 1.04, 1.71, and 2.52 nM in lanes 1–9, respectively) was added to the reaction, and samples were incubated for 20 min at room temperature prior to fractionation on 4% polyacrylamide gels electrophoresed at 4°C. Details of EMSA are described in Section 2. The specificity of ERa– ERE binding was confirmed by supershift with antibody H222 (lane 10 in each panel which included 0.42 nM DNA). These gels are representative of the three to four gels run for each EREc38 oligomer.
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Table 1 Comparison of ERa and ERb binding parametersa 1(EREc38) Receptor ERa ERb
KD (pM) 88.5 98.5 145.7 913.7e
2(EREc38) n 0.9 90.1 1.09 0.1
KD (pM) 37.99 8.6b 19.19 3.2f
3(EREc38) n 3.0 90.2c 2.3 90.2
KD (pM) 2.4 90.1b 0.7 90.4f
4(EREc38) n 3.1 90.5 6.1 90.6g
KD (pM) 17.6 91.6b 13.8 95.3f
n 1.2 90.1d 4.0 90.4
a ERa and ERb binding parameters, KD and the stoichiometric coefficient n, were calculated using values obtained from EMSA experiments as described in Section 2. Each value is the mean 9S.E.M. of three separate experiments with each data point performed in duplicate within each experiment. b Significantly different (PB0.0001) from the KD for interaction of ERa with 1(EREc38). c Significantly different (PB0.001) from the anticipated nH value of 2, i.e. two ER dimers bound to two EREs. d Significantly different (PB0.001) from the anticipated nH value of 4. e Significantly different (PB0.001) from the KD for interaction of ERa with 1(EREc38). f Significantly different (PB0.001) from the KD for interaction of ERb with 1(EREc38) g Significantly different (PB0.001) from the anticipated nH value of 3.
These bands are specific for ERa since both are fully supershifted by ERa-specific antibody H222. Protein– protein interaction between ERa and other proteins present in the partially purified NE from SF21 cells along with reversible conformational changes in ERa during non-denaturing gel electrophoresis may account for the two bands. To evaluate ERa – ERE binding stoichiometry, EMSA data were quantitated and then analyzed by a modification of Hill analysis described in Section 2. The apparent KD values and apparent stoichiometric coefficients (n) for ERa and ERb binding to one, two, three, or four tandem copies of EREc38 are given in Table 1. The apparent affinity of ERa binding to two, three, and four copies of EREc38 significantly exceeded that of binding to one EREc38 (Table 1). The estimated stoichiometric coefficients (n) for ERa binding to 1(EREc38) and 3(EREc38) did not deviate significantly from the expected values of one and three, i.e. the number of ERa dimers anticipated to bind to one or three EREs, respectively (Table 1). The agreement of the estimated and expected stoichiometry of ERa binding to one and three tandem copies of EREc38 indicates the validity of Eq. (1). Therefore, the affinity of ERa binding to any ERE within 3(EREc38) appears to be independent of ERa binding to the adjacent ERE site (Section 2 for a kinetic explanation as to how differences in binding affinity may cause a discrepancy between the estimated and expected n values). The apparent stoichiometry of ERa binding to 2(EREc38), i.e. 3, was higher than the expected value of 2 (Table 1). This suggests either that ERa binding to one of two adjacent EREs changes the affinity of ERa interaction with the unoccupied ERE, or three ERa dimers bind 2(EREc38). The band count of five ERa – 2(EREc38) complexes is compatible with both of these suggestions (Fig. 4). In contrast, the estimated stoichiometry of ERa binding to 4(EREc38), i.e. 1.2, was significantly lower than the expected value of 4 (Table 1). This suggests
either that ERa binding to one of the 4 EREs changes the affinity of ERa interaction with the unoccupied EREs, or that only one ERa dimer binds to 4(EREc38). Since five bands are visible for ERa – 4(EREc38) (Fig. 4), the data eliminate the possibility that only one ERa dimer is bound. Instead, the discrepancy between the measured and expected stoichiometry may reflect a change in affinity of unoccupied EREs caused by ERa binding to the adjacent ERE sites. Fig. 5 presents quantitative data obtained with ERa binding to each EREc38 multimer and plotted according to Eq. (4). As shown, these data fit the expected linear model with correlation coefficients exceeding 80%. In contrast to the two bands seen for ERa – 1(EREc38) binding (Fig. 4A), a single ERb –1(EREc38) band was detected (Fig. 6A). ERb bound to a single copy of EREc38 with lower affinity compared with ERa (Table 1). ERb bound to multiple copies of EREc38 sites with a significantly higher affinity than to a single EREc38. The number of bands of ERb – EREc38 complex formed corresponded to the number of tandem copies of the ERE for 1–3(EREc38), but only three bands were detected for ERb –4(EREc38). The data obtained for ERb binding to multiple tandem copies of EREc38 also fit the expected linear model based on Eq. (4) and gave regression coefficients exceeding 80% (Fig. 7). The calculated stoichiometric coefficient (n) for ERb binding to one, two, and four (EREc38) was not significantly different from the expected values of one, two, and four. The agreement between the estimated and expected stoichiometry of ERb binding to two and four tandem copies of EREc38 again indicates the validity of Eq. (1). Therefore, these n values indicate that the affinity of ERb binding to remaining unoccupied ERE(s) is independent of ERb binding to the adjacent EREs. In contrast, the stoichiometric coefficient of 6.1 for ERb binding to 3(EREc38) was significantly higher
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than the expected value of 3 (Table 1). Since ERb binding to 3(EREc38) formed three major and one minor bands recognized by an ERb-specific antibody (Fig. 6C), the stoichiometry is not expected to be greater than four. The discrepancy between the measured n value, on one hand, and both the expected stoichiometry and the band count, on the other hand, appears not to be due to six ER dimers bound, but may instead reflect a change in the affinity of unoccupied EREs caused by ERb binding to the adjacent ERE sites.
4. Discussion Differences in ligand binding, tissue distribution, and transcriptional activation of reporter constructs indicate that ERa and ERb play overlapping, but different roles in vivo (Kuiper et al., 1997). Here we examined whether ERb displayed transcriptional synergism at multiple tandem copies of a consensus ERE sequence. Earlier we reported that ERa synergistically activated reporter gene activity from multiple tandem copies of an ERE consensus sequence in MCF-7 cells (Klinge, 1999a; Sathya et al., 1997). Although E2 treatment of CHO
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cells resulted in a significantly lower induction of luciferase activity by ERb than by ERa, there was no difference in the fold-synergy induced by ERa or ERb. These data may indicate that transcriptional synergy is mediated by a common mechanism for both ERa and ERb. The observation that ERb shows lower induction of ERE-driven reporter gene activity than ERa in transiently transfected CHO cells is similar to that reported by other investigators using a single or multiple tandem copies of consensus ERE driving reporter gene expression in COS-1, HeLa, and HepG2 cells (Cowley and Parker, 1999; Tremblay et al., 1999). With one ERE-tk-Luc in HeLa cells, ERb activity was 52% of ERa; in COS-1 cells, ERb activity was 19% of ERa; and in HepG2 cells, ERb was 4% of ERa (Cowley and Parker, 1999). We observed that ERb activity was 53% of ERa from 1(EREc38) in CHO cells. This result is similar to that detected in COS-1 cells. Although there was a species difference in the source of recombinant ERa and ERb used in our experiments, i.e. rhERa and rrERb, we note that the differences in transcriptional activation seen between rhERa and rrERb are similar to those reported recently for rhERa and rhERb (Hall and McDonnell, 1999). The latter report showed that rhERb was about one third as
Fig. 5. ERa binds cooperatively to multiple tandem copies of EREc38. ERa– ERE complexes were quantitated as described in Section 2 and expressed as the concentrations of ERa-bound and free ERE. The near-saturation measurements at the higher ERE concentrations were used to estimate the functional ERa concentration (Rtotal in Eq. (4)) for each gel. After transformation of the data according to Eq. (4), data from three to four gels were pooled. Linear regression analysis was applied to the data obtained with 1(EREc38) (panel A); 2(EREc38) (panel B); 3(EREc38) (panel C); or 4(EREc38) (panel D). The regression coefficients and their standard errors expressed according to Eq. (4) are shown in Table 1.
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Fig. 6. ERb binds cooperatively to multiple tandem copies of EREc38. EMSA was used to quantitate the kinetics of ERb binding to 1(EREc38) (panel A); 2(EREc38) (panel B); 3(EREc38) (panel C); or 4(EREc38) (panel D). In each gel, a constant amount (0.7 nM) of ERb was incubated with 1 mM E2 for 1-h on ice. Then the respective [32P]-labeled EREc38 (0.06, 0.11, 0.20, 0.35, 0.55, 0.83, 1.24, 1.93, and 2.76 nM in lanes 1–9, respectively) was added to the reaction, and samples were incubated as described in Fig. 4. Details of EMSA are described in Section 2. The specificity of ERb– ERE binding was confirmed by supershift with antibody Y-19 (lane 10 in each panel which also included 0.55 nM DNA). These gels are representative of four gels run for each EREc38 oligomer.
active as rhERa at inducting luciferase reporter gene activity from three tandem copies of the vitellogenin ERE in transiently transfected HepG2 cells treated with 10 nM E2 in cells expressing rhERb was approximately 33% of that for rhERa (Hall and McDonnell, 1999). These results are in good agreement with the data shown in Fig. 2 and indicate that the small percentage in aa change between the rat and human ERb sequences (Kuiper et al., 1996; Enmark et al., 1997; Moore et al., 1998; Ogawa et al., 1998) do not appear to result in large differences in transcriptional activity in transiently transfected cells. No one has examined whether ERb synergistically activated transcription from multiple EREs. Transcriptional synergism from multiple hormone response elements has been reported for glucocorticoid, mineralocorticoid, and androgen receptors (IngiguezLluhi et al., 1999), and for ERa (Klinge, 1999a; Mattick et al., 1997; Sathya et al., 1997). Although the exact mechanism for ERa transcriptional synergism is unknown, transcriptional synergism by GR requires an intact DNA binding domain (DBD), LBD, and the DBD dimer interface (Ingiguez-Lluhi et al., 1999). The data presented here and in our previous work (Klinge, 1999a; Klinge et al., 1996a; Sathya et al., 1997) indicate that synergy depends on the ligand bound to ERa,
implicating the LBD, and on the ERE sequence, thus also implicating the DBD in transcriptional synergy. Indeed our observation that ERb synergistically transactivates gene expression from multiple tandem EREs despite the fact that the N-terminal AF-1 domain of ERb is non-functional (McInerney et al., 1998), indicates that the ER A/B domain region is not involved in functional synergy. Most of the genes that are highly induced by estrogens, including the vitellogenins, PR, and ovalbumin contain multiple, variably spaced EREs (Martinez et al., 1987; Kraus et al., 1994; Kato et al., 1995) which result in synergistic activation of these genes in estrogen-treated target cells. Transcriptional synergy may result from several possible mechanisms. These include cooperative recruitment of a coactivator, action at distinct rate-limiting steps in transcription initiation, cooperative DNA binding (Herschlag and Johnson, 1993), and/or direct protein–protein interactions between ERa dimers. Among the possible mechanisms for the transcriptional synergism observed here, ER might cause changes in DNA topology that are transmitted to another ER bound nearby. ERa has been shown to bend DNA (Nardulli et al., 1993 Nardulli et al., 1995). Thus, one may speculate that the distinct local topologies
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induced by binding of one ERa dimer have differential allosteric effects on ERa conformation and activity at adjacent sites. There are no reports as to whether ERb bends DNA. We and others have demonstrated that the stereoalignment of EREs and their spacing influence synergistic responses to estradiol (Ponglikitmongkol et al., 1990; Klinge et al., 1992a,b, 1996a; Anolik et al., 1995, 1996 Sathya et al., 1997; Nordeen et al., 1998). We reported that calf uterine, E2-liganded ERa binds cooperatively to three or four tandem copies of EREc38, but not to 1(EREc38) or 2(EREc38) (Klinge et al., 1992b). These results fit the pattern of transcriptional synergism seen here and in our previous studies with MCF-7 cells (Sathya et al., 1997; Klinge, 1999a). Here we compared the binding of recombinant human ERa and rat ERb to these EREs in vitro. Oligomers containing single or multiple copies of EREc38 show an identical pattern of decreasing binding affinity for both ERa and ERb: 3(EREc38) \ 4(EREc38)\ 2(EREc38)\1(EREc38). We suggest that the increased binding affinity may contribute to the synergistic activation of reporter gene expression from 3(EREc38) and 4(EREc38) (Fig. 2). The changes in binding affinity of ER in response to the increase in number of tandem EREs demonstrate
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both similarities and differences between ERa and ERb. To understand the functional role of each ER isoform, it is necessary to examine data on their differences. For ERa, the requirements of the model described by Eq. (1) were met only for ERa binding to three tandem copies of EREc38 but not to two and four tandem copies. This indicates a change in affinity of the unoccupied ERE sites associated with ERa binding to the adjacent ERE(s). We showed previously that the helix structure of DNA makes the adjacent ERE sites stereochemically different in a way that may contribute to variations in the affinity of ERa binding to different numbers of tandem ERE sites (Klinge et al., 1992b, 1996a). Here we observed an increase in binding affinity for both ERa and ERb to two or more tandem copies of EREc38. However, in contrast to ERa, ERb appears to bind with comparable affinity to two and four tandem copies of EREc38 and with higher affinity to 3(EREc38). Differences in the conformation of ERa and ERb bound to these EREs may account for the observed differences in KD values. The differences in binding affinity in response to occupation of adjacent ERE sites with ERa and ERb indicates that these two ER species, when bound to ERE, may have different conformations, altering their
Fig. 7. ERb binds cooperatively to multiple tandem copies of EREc38. Data on ERb– ERE complexes were quantitated and processed as described in Fig. 5. Linear regression analysis was applied to the pooled data obtained with 1(EREc38) (panel A); 2(EREc38) (panel B); 3(EREc38) (panel C); or 4(EREc38) (panel D). The regression coefficients and their standard errors expressed according to Eq. (4) are shown in Table 1.
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interaction with other ERE-bound ER dimers, or recruit different cofactors, or both. We speculate that ERb –ERE binding is stabilized by the binding of other ERb dimers at adjacent sites. Since transcriptional synergy by ERa depends on the cell type used in transient transfection (Mattick et al., 1997), it seems likely that cell-specific cofactors influence transcriptional synergy by ERb as well. Further studies are necessary to examine these possibilities.
Acknowledgements We thank Rosemary L. Sims and Sheetal J. Mehta for their assistance in preparing plasmids used transient transfection assays. We thank Dr J-A. Gustafsson for supplying the pCMV-ERb vector and Abbott Labs for the gift of H222. We thank Dr Peter C. Kulakosky for preparing ERa and ERb and for his thoughtful suggestions on this manuscript.This study was also supported by NIH R01 DK 53220 and a University of Louisville Research on women Grant to C.M.K.
References Anolik, J.H., Klinge, C.M., Hilf, R., Bambara, R.A., 1995. Cooperative binding of estrogen receptor to DNA depends on spacing of binding sites, flanking sequence, and ligand. Biochemistry 34, 2511 – 2520. Anolik, J.H., Klinge, C.M., Brolly, C.L., Bambara, R.A., Hilf, R., 1996. Stability of the ligand of estrogen response element-bound estrogen receptor depends on flanking sequences and cellular factors. J. Steroid Biochem. Mol. Biol. 59, 413–429. Beato, M., Sanchez-Pacheco, A., 1996. Interaction of steroid hormone receptors with the transcription initiation complex. Endocr. Rev. 17, 587 – 609. Cowley, S.M., Parker, M.G., 1999. A comparison of transcriptional activation by ERa and ERb. J. Steroid Biochem. Mol. Biol. 69, 165 – 175. Driscoll, M.D., Sathya, G., Muyan, M., Klinge, C.M., Hilf, R., Bambara, R.A., 1998. Sequence requirements for estrogen receptor binding to estrogen response elements. J. Biol. Chem. 273, 29321 – 29330. Enmark, E., Pelto-Huikko, M., Grandien, K., Lagercrantz, S., Lagercrantz, J., Fried, G., Nordenskjold, M., Gustafsson, J.A., 1997. Human estrogen receptor b-gene structure, chromosomal localization, and expression pattern. J. Clin. Endocrinol. Metab. 82, 4258 – 4265. Folkers, G.E., van der Burg, B., van der Saag, P.T., 1998. Promoter architecture, cofactors, and orphan receptors contribute to cellspecific activation of the retinoic acid receptor b2 promoter. J. Biol. Chem. 273, 32200–32212. Hall, J.M., McDonnell, D.P., 1999. The estrogen receptor b-isoform (ERb) of the human estrogen receptor modulates ERa transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology 140, 5566–5578. Herschlag, D., Johnson, F.B., 1993. Synergism in transcriptional activation: a kinetic view. Genes Dev. 7, 173–179. Ingiguez-Lluhi, J.A., Yamamoto, K.R., Pearce, D., 1999. A short N-terminal motif defines a novel transcriptional synergy regulatory surface in steroid receptors. Abstract OR47-4, Program
Abstracts of the 81st Annual Meeting of the Endocrine Society, San Diego, CA., June 12 – 15, 1999. Kato, S., Sasaki, J.H., Suzawa, M., Masushige, S., Tora, L., Chambon, P., Gronemeyer, H., 1995. Widely spaced, directly repeated PuGGTCA elements act as promiscuous enhancers for different classes of nuclear receptors. Mol. Cell. Biol. 15, 5858 – 5867. Klinge, C.M., 1999a. Estrogen receptor binding to estrogen response elements slows ligand dissociation and synergistically activates reporter gene expression. Mol. Cell. Endocrinol. 150, 99–111. Klinge, C.M., 1999b. Role of estrogen receptor ligand and estrogen response element sequence on interaction with chicken ovalbumin upstream promoter transcription factor (COUP-TF). J. Steroid. Biochem. Mol. Biol. 71, 1 – 19. Klinge, C.M., Bambara, R.A., Hilf, R., 1992a. Antiestrogen-liganded estrogen receptor interaction with estrogen responsive element DNA in vitro. J. Steroid Biochem. Mol. Biol. 43, 249 –262. Klinge, C.M., Peale, F.V. Jr., Hilf, R., Bambara, R.A., Zain, S., 1992b. Cooperative estrogen receptor interaction with consensus or variant estrogen responsive elements in vitro. Cancer Res. 52, 1073 – 1081. Klinge, C.M., Traish, A.M., Bambara, R.A., Hilf, R., 1996a. Dissociation of 4-hydroxytamoxifen, but not estradiol or tamoxifen aziridine, from the estrogen receptor when the receptor binds estrogen response element DNA. J. Steroid Biochem. Mol. Biol. 57, 51 – 66. Klinge, C.M., Traish, A.M., Driscoll, M.D., Hilf, R., Bambara, R.A., 1996b. Site-directed estrogen receptor antibodies stabilize 4-hydroxytamoxifen ligand, but not estradiol, and indicate ligand-specific differences in the recognition of estrogen response element DNA in vitro. Steroids 61, 278 – 289. Klinge, C.M., Silver, B.F., Driscoll, M.D., Sathya, G., Bambara, R.A., Hilf, R., 1997. COUP-TF interacts with estrogen receptor, binds to estrogen response elements and half-sites, and modulates estrogen-induced gene expression. J. Biol. Chem. 272, 31465– 31474. Klinge, C.M., Studinski-Jones, A.L., Kulakosky, P.C., Bambara, R.A., Hilf, R., 1998. Comparison of tamoxifen ligands on estrogen receptor interaction with estrogen response elements. Mol. Cell. Endocrinol. 143, 79 – 90. Klinge, C.M., Bowers, J.L., Kulakosky, P.C., Kamboj, K.K., Swanson, H.I., 1999. The aryl hydrocarbon receptor (AHR)/AHR nuclear translocator (ARNT) heterodimer interacts with naturally occurring estrogen response elements. Mol. Cell. Endocrinol. 157, 105 – 119. Kraus, W.L., Montano, M.M., Katzenellenbogen, B.S., 1994. Identification of multiple, widely spaced estrogen-responsive regions in the rat progesterone receptor gene. Mol. Endocrinol. 8, 952–969. Kuiper, G.G., Enmark, E., Pelto-Huikko, M., Nilsson, S., Gustafsson, J.-A., 1996. Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc. Natl. Acad. Sci. USA. 93, 5925– 5930. Kuiper, G.G., Carlsson, B., Grandien, J., Enmark, E., Haggblad, J., Nilsson, S., Gustafsson, J.-A., 1997. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors a and b. Endocrinology 138, 863 – 870. Martinez, E., Givel, F., Wahli, W., 1987. The estrogen-responsive element as an inducible enhancer: DNA sequence requirements and conversion to a glucocorticoid responsive element. EMBO J. 6, 3719 – 3727. Mattick, S., Glenn, K., deHaan, G., Shapiro, D.J., 1997. Analysis of ligand dependence and hormone response element synergy in transcription by estrogen receptor. J. Steroid Biochem. Mol. Biol. 60, 285 – 294. McInerney, E.M., Weis, K.E., Sun, J., Mosselman, S., Katzenellenbogen, B.S., 1998. Transcription activation by the human estrogen receptor subtype b (ERb) studied with ERb and ERa receptor chimeras. Endocrinology 139, 4513 – 4522.
V.V. Tyulmenko6 et al. / Molecular and Cellular Endocrinology 165 (2000) 151–161 McKenna, N.J., Lanz, R.B., O’Malley, B.W., 1999. Nuclear receptor coregulators: cellular and molecular biology. Endocr. Rev. 20, 321 – 344. Misiti, S., Schomburg, L., Yen, P.M., Chin, W.W., 1998. Expression and hormonal regulation of coactivator and corepressor genes. Endocrinology 139, 2493–2500. Moore, J.T., McKee, D.D., Slentz-Kesler, K., Moore, L.B., Jones, S.A., Horne, E.L., Su, J.L., Kliewer, S.A., Lehmann, J.M., Willson, T.M., 1998. Cloning and characterization of human estrogen receptor b isoforms. Biochem. Biophys. Res. Commun. 247, 75 – 78. Nardulli, A.M., Greene, G.L., Shapiro, D.J., 1993. Human estrogen receptor bound to an estrogen response element bends DNA. Mol. Endocrinol. 7, 331–340. Nardulli, A.M., Grobner, C., Cotter, D., 1995. Estrogen receptor-induced DNA bending: Orientation of the bend and replacement of an estrogen response element with an intrinsic DNA bending sequence. Mol. Endocrinol. 9, 1064–1076. Nordeen, S.K., Ogden, C.A., Taraseviciene, L., Lieberman, B.A., 1998. Extreme position dependence of a canonical hormone response element. Mol. Endocrinol. 12, 891–898. Ogawa, S., Inoue, S., Watanabe, T., Hiroi, H., Orimao, A., 1998. The complete primary structure of human estrogen receptorb (hER b) and its heterodimerization with ERa in vivo and in vitro. Biochem. Biophys. Res. Commun. 243, 122–126.
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Comparison of transcriptional synergy of estrogen receptors a and b from multiple tandem estrogen response elements Valentyn V. Tyulmenkov, Sarah C. Jernigan, Carolyn M. Klinge * Department of Biochemistry and Molecular Biology, Uni6ersity of Louis6ille School of Medicine, Louis6ille, KY 40292, USA Received 22 December 1999; accepted 4 April 2000
Abstract Estrogen receptors a and b (ERa and ERb) act as ligand-dependent transcriptional enhancers. We reported that ERa induces synergistic activation of luciferase reporter gene activity in response to E2 from three or four tandem copies of a consensus estrogen response element (ERE) in transiently transfected MCF-7 cells. Here we addressed three questions: (1) is the synergistic activation of reporter gene activity from multiple tandem EREs by ERa restricted to MCF-7 cells?; (2) does ERb induce synergistic activation of reporter activity from multiple tandem EREs?; and (3) does ERb bind cooperatively to multiple tandem EREs? To address the first two questions, ER-negative CHO-K1 cells were co-transfected with ERa or ERb and ERE-driven reporter plasmids. Both ERa and ERb activated ERE-driven luciferase gene activity in an estradiol-dependent manner. Induction by ERb was lower than ERa from each ERE. We demonstrate that both ERa and ERb induce transcriptional synergy with three or four, but not two, tandem copies of an ERE. Electrophoretic mobility shift assays (EMSA) indicated an increase in ER–ERE binding affinity associated with cooperative binding of ERa and ERb to multiple EREs that may be responsible for transcriptional synergy in transiently transfected cells. We also postulate that interaction of ERa and ERb with coactivators may also play a role in transcriptional synergy. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Estrogen receptor; Estrogen response element; Synergy; Cooperativity; Estradiol; 4-Hydroxytamoxifen
1. Introduction Estrogens exert a wide variety of effects on growth, development, and differentiation, including important regulatory functions within the reproductive systems of both females and males, in mammary gland development and differentiation, in central nervous system functions, in the hypothalamic-gonadal axis, and as anti-atherosclerotic agents. Estrogens mediate these activities through binding to a specific intranuclear receptor protein, the estrogen receptor (ER). ER is a member of the steroid/thyroid superfamily of proteins that act as hormone-inducible transcription factors (Beato and Sanchez-Pacheco, 1996). ER is encoded by two genes i.e. a and b (ERa and ERb) (Kuiper et al., 1996). The term ER will refer to both ERa and ERb, whereas ERa and ERb refer specifically to that iso* Corresponding author. Tel.: +1-502-8523668; fax: + 1-5028526222. E-mail address: [email protected] (C.M. Klinge).
form. While ERa and ERb form homodimers, they can also form ERa:ERb heterodimers in vitro and in vivo (Ogawa et al., 1998). ERa and ERb show different patterns of tissue expression and show different affinities for binding phytoestrogens, indicating that ERa and ERb may play distinct roles in vivo. Ligands, e.g. estradiol (E2), enter the cell nucleus and bind to the ligand binding domain (LBD) of ER inducing conformational changes in ER, including dimerization and phosphorylation (Weigel, 1996), leading to high affinity ER binding to specific DNA sequences: estrogen response elements (EREs). The current model to account for cell-specific regulation of estrogen target gene expression suggests that target cells express different levels of coactivators and corepressors which, along with the amounts of ERa, ERb, and ligand, allows fine-tuning of target gene transcription in response to estrogens. Recent Northern blot analysis confirmed the idea that different rat tissues (Misiti et al., 1998) and cell lines (Folkers et al., 1998) express different amounts of mRNA for the coactivators SRC-1, RIP-140, p300,
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and CBP, as well as the corepressors SMRT, and NCoR. Nuclear receptor coactivators and corepressors were recently reviewed (McKenna et al., 1999). In earlier work, we reported that estradiol (E2)-liganded ERa bound to three or four tandem copies of a consensus ERE (EREc38) in a cooperative manner (Klinge et al., 1992b). E2 – ER binding to one or two EREs was non-cooperative. When ER was liganded by the antiestrogen 4-hydroxy-tamoxifen (4-OHT), ER– ERE binding was not cooperative, regardless of the number of EREs (Klinge et al., 1992a, 1996a,b). More recently, we evaluated how binding to EREc38 affects ER conformation in the LBD as reflected in the dissociation kinetics of [3H] ligand from the ER (Klinge, 1999a). Binding of ERa to EREc38 slowed the rate of E2 dissociation, indicating that DNA allosterically modulates the LBD conformation creating a tighter fit between E2 and ERa (Klinge, 1999a). Synergistic E2-dependent activation of a reporter gene was detected from three and four, but not one or two, tandem copies of EREc38 in MCF-7 cells (Sathya et al., 1997; Klinge, 1999a). Since the number of tandem copies of EREc38 did not alter E2 dissociation kinetics, transcriptional (also called ‘functional’) synergy in MCF-7 cells must involve cellular factors in addition to the ER ligand. To separately evaluate the contributions of ERa and ERb to transcriptional synergy from multiple EREs, transient transfection assays were performed in ER negative CHO-K1 cells co-transfected with mammalian expression vectors for ERa or ERb. Here we report that both ERa and ERb act synergistically to activate reporter gene expression from three and four tandem EREs.
2. Materials and methods
2.1. Preparation of ERE containing plasmids The sequence of the synthetic consensus ERE oligonucleotide, called EREc38, used in these experiments is: 5%-CCAGGTCAGAGTGACCTGAGCTAAAATAACACATTCAG-3% (Peale et al., 1988). Single (EREc38) or multiple, head-to-tail, tandem copies of double stranded EREc38 oligomers (n (EREc38) where n=the number of tandem copies) were cloned into the plasmids pGEM-7Zf(+ ) or pGL3-Promoter (Promega, Madison, WI) as described (Klinge et al., 1992b, 1997).
2.2. Cell transfection CHO-K1 cells were purchased from ATCC (Manassas, VA). CHO cells (2.5× 105) were plated in each well of a 12-well Corning plate in IMDM (all cell culture reagents were from Life Technologies) medium without
phenol red, supplemented with 10% stripped fetal bovine serum (FBS) and 1% Pen-Strep. After 24 h, the cells were transfected using liposome-mediated transfection (Transfast, Promega). CHO-K1 cells were cotransfected with 10 ng of either pCMV-rh ERa or pCMV-rrERb (kindly provided by Dr Benita Katzenellenbogen (Reese and Katzenellenbogen, 1991) and Dr J.-A. Gustafsson (Kuiper et al., 1996), respectively), pGL3-pro-luciferase plasmid containing 1, 2, 3, or 4 tandem copies of EREc38 (0.6 mg), pCMVbgal (0.1 mg) (Clonetech), and 0.5 mg of carrier DNA using Transfast (Promega) essentially as described in (Klinge et al., 1997). The pCMV-rrERb has three different possible translation products of 485, 530, and 549 aa, respectively (Enmark, E. personal communication). Twentyfour hours after transfection, the cells were treated with 10 nM 17b-estradiol (Sigma), 100 nM 4-OHT (Research Biochemicals International, Natick, MA), or an equal volume of ethanol (as vehicle control). Each treatment was performed in triplicate. The cells were maintained in IMDM medium containing 1% stripped FBS. The cells were lysed 24 h after treatment in 150 ml of 1X reporter lysis buffer (Promega) and the cleared extract was assayed for luciferase and b-gal activities as described (Klinge et al., 1997). The fold induction of luciferase activity was normalized for b-gal and is expressed as the ratio of RLU between treatment groups and the vehicle control (which was set to 1) (Klinge et al., 1997).
2.2.1. Western blotting To address the question of whether ERa and ERb are expressed at equal levels in the transfected CHO-K1 cells, whole cell extracts were prepared and the proteins slot-blotted onto a PVDF membrane (Klinge et al., 1997). The membranes were immunoblotted with ERaspecific Ab-10 (Neomarkers) or ERb-specific antiserum Y-19 (Santa Cruz). Western blotting was performed using NEN Renaissance chemiluminescence reagent as described (Klinge, 1999b). Densitometric analysis of the resulting film indicated that comparable protein expression levels of ERa and ERb were achieved in these cells (Western blot data not shown). 2.2.2. Electrophoretic mobility shift assay Nuclear extracts containing recombinant human (rh) ERa were prepared from baculovirus-infected IPLBSf21AE insect cells as described (Klinge et al., 1998). A similar procedure was used for the preparation of recombinant rat (rr) ERb from baculovirus-infected IPLB-Sf21AE insect cells (Klinge et al., 1999). EREcontaining oligomers were labeled with [32P]a-dTTP (800 Ci/mmol from NEN) (Klinge et al., 1997). The size of the ERE oligomers used was 77, 115, 153, and 191 bp for one, two, three, and four tandem copies of the consensus EREc38 sequence (hereafter referred to as
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1EREc38, 2(EREc38), 3(EREc38), and 4(EREc38), respectively). The DNA concentrations were measured using PicoGreen fluorescence assay (Molecular Probes, Eugene, OR). ER– ERE binding reactions included a series of increasing concentrations (40 – 2800 nM) of [32P] labeled oligomer, a constant amount (0.699 0.06 nM, as measured by ER-1(EREc38) binding capacity to obviate the difference between [3H]E2 binding and ERE binding capacities (Driscoll et al., 1998)) of liganded-ER, 1 mM E2, 0.75 mg/ml purified BSA (New England Biolabs), 20 mg/ml poly d(I-C) (Midland Certified Reagent Company, Midland, TX), and 15% (v/v) glycerol in a final volume of 25 ml. ERa-specific monoclonal antibody H222, generously provided by Abbott Laboratories (Abbott Park, IL). ERb-specific polyclonal antisera PA1-310 and Y-19 were purchased from Affinity Bioreagents (Golden, CO) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Reaction and electrophoresis conditions have been described (Klinge et al., 1997). All bands supershifted by an ER-specific antibody were classified as specifically bound DNA. The free DNA band migrated at the bottom of the gel and other bands were non-specifically bound DNA. Counts of ER-bound and free DNA were determined using a Packard Instruments InstantImager and associated software, Packard Imager for Windows v 2.04 (Packard Instrument Company, Meriden, CT). The DNA amount in each band was calculated by multiplying the amount of DNA in the reaction by the percentage of the total radioactivity/lane in that band. Each reaction was repeated in three to four independent assays. The higher ERE concentrations reached saturation binding, allowing the concentration of functional ER to be estimated in each assay from four to five gel lanes. The Hill equation is used to estimate the stoichiometric coefficient (n). However, Hill coefficients (nH) are not reliable for analysis of the stoichiometry of ER– ERE binding. Assume that each receptor molecule (R) binds n ligand molecules (L) with a constant affinity. The concentrations at equilibrium will fit to equation [R]× [L]n =KD [RLn ]
(1)
where [R] and [L] are the concentrations of free receptor and ligand, [RLn ] is that of bound receptor (or ligand), KD is the equilibrium dissociation constant. This equation may be rearranged [RLn ]/(Rtotal − [RLn ])=[L]n/KD so that logarithmic transformation will give the Hill equation log
B = n× log(F)− log(KD) Rtotal − B
(2)
where Rtotal is the total receptor concentration, B is the concentration of bound ligand, F is that of free ligand. In contrast, DNA with multiple EREs is a ligand that
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can bind more than one receptor molecule, and the equation at equilibrium is different from Eq. (1): [R]n × [L] =KD [RnL]
(3)
This equation may be rearranged: [L]/[RnL]= (Rtotal − [RnL])n/KD to be linearized by logarithmic transformation log
B = n× log(Rtotal − B)− log(KD) F
(4)
However, the assumption that KD is constant may be not met. Binding of ER to multiple EREs with varying KDs may be fit to Eq. (4), giving an average estimate for KD and an erroneous estimate for n, which deviates from the true value. This is illustrated by the comparison between the number of available O2 binding sites on the hemoglobin A molecule, i.e. 4, and the stoichiometric coefficient 3, as measured by the Hill analysis (Tyuma et al., 1973). According to this report, occupation of the four available binding sites by O2 is associated with a dramatic increase in affinity. This indicates that the assumption that KD is constant is not valid and results in a discrepancy between the estimated and expected stoichiometric coefficients (Tyuma et al., 1973). Thus, such a discrepancy may indicate interaction between adjacent binding sites or between molecules bound to these sites.
3. Results
3.1. E2 induces transcriptional synergy from multiple tandem copies EREs with ERa and ERb In order to determine the transcriptional responsiveness of each ER isoform independently, CHO-K1 (CHO) cells were co-transfected with the human ERa expression vector pCMV-ERa (Reese and Katzenellenbogen, 1991) or the rat ERb expression vector pCMVERb (Kuiper et al., 1996) for transient transfection experiments. First, the amount of ER expression vector required to activate luciferase activity from two tandem copies of EREc38 in CHO cells was examined (Fig. 1). The amount of luciferase activity increased with the amount of ER expression vector co-transfected, reaching a plateau at approximately 10 ng of plasmid and showing decreased responsiveness with more than 100 ng expression plasmid cotransfected. Based on these results, CHO cells were co-transfected with 10 ng of the expression vector for ERa or ERb. E2-induced luciferase activity from one or multiple tandem copies of EREc38 was assayed in ERa-expressing CHO cells. The relative induction of luciferase activity by E2 was dependent on the number of copies of EREc38 (Fig. 2A). As anticipated based on results
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from MCF-7 cells (Klinge, 1999a), synergistic activation was detected for 3(EREc38) and 4(EREc38). Cotreatment of the cells with E2 plus 4-OHT completely
Fig. 1. Optimization of amount of ER expression vector in transiently transfected CHO cells. CHO cells were co-transfected with the indicated amounts, in ng, of pCMV-ERa (A) or pCMV-ERb (B) plus two-tandem copies of EREc38 in the pGL3-Promoter vector (2(EREc38)) and pCMV-bgal as described in Section 2. Twenty-four hours after plating, the cells were treated with ethanol, 10 nM E2, or 10 nM E2 plus 1 mM 4-OHT. The cells were harvested 24 h after treatment and the cell extracts were assayed for luciferase and b-gal activities (Klinge et al., 1997). Data are displayed as fold induction of luciferase activity, which is the luciferase activity, divided by the b-gal activity in each well. These data are the mean 9 S.E.M. of three separate determinations.
Fig. 2. E2 induces transcriptional synergy from three or four tandem copies of EREc38. CHO cells were co-transfected with pCMV-ERa or pCMV-ERb plus the indicated pGL3-Promoter vector and pCMVbgal as described in Section 2. Twenty-four hours after plating, the cells were treated with ethanol, 10 nM E2, or 10 nM E2 plus 1 mM 4-OHT. The cells were harvested 24 h after treatment and the cell extracts were assayed for luciferase and b-gal activities (Klinge et al., 1997). (A) Data are displayed as fold induction of luciferase activity, which is the luciferase activity, divided by the b-gal activity in each well. The fill symbols are indicated in the inset. The fold induction of luciferase activity is expressed relative to the activity detected upon addition of ethanol (EtOH) which was set to 1; (B) the data presented in Fig. 1 are displayed as fold synergy that was determined by dividing the relative luciferase activity by the number of copies of EREc38 (Mattick et al., 1997). The data shown are presented as mean 9 S.E.M. for 12 independent experiments, in which each perturbation was performed in triplicate. Fill symbols are indicated in the inset and are identical to those in (A).
blocked E2-stimulated luciferase activity from all EREc38 multimers. Inhibition by 4-OHT indicates that the induction of luciferase in response to E2 was mediated by ER. The fold synergy was determined by dividing the relative luciferase activity by the number of copies of EREc38 (Mattick et al., 1997). Comparison of the fold synergy showed 1.4 fold synergy for 2(EREc38), indicating less than additive induction, versus 5.7 and 6.9 fold synergy for 3(EREc38) or 4(EREc38), respectively (Fig. 1B). Thus, cooperative binding of E2-ERa to three or four tandem copies of EREc38 (Klinge et al., 1992b) is reflected in synergistic activity of reporter gene expression in CHO cells transiently expressing ERa. ERb was reported to show lower activity compared to ERa in response to identical concentrations of E2 in transient transfection assays using two (Cowley and Parker, 1999) or three (McInerney et al., 1998) EREs linked to the pS2 gene promoter in CEF and CHO cells, respectively. Similarly, we found that ERb was less active than ERa with one, two, or three tandem copies of EREc38 in transiently transfected CHO cells (Fig. 1A). Interestingly, there was no significant difference in the fold induction of luciferase activity induced by ERa or ERb from 4(EREc38). Comparison of the fold synergy induced by ERb showed 1.4 fold synergy for 2(EREc38), indicating less than additive induction, versus 4.1 and 7.8 fold synergy for 3(EREc38) or 4(EREc38), respectively (Fig. 1B). Thus, despite lower induction of reporter gene activity, ERb synergistically activates reporter gene expression from three and four tandem copies of EREc38, a result similar to that for induction of reporter activity from the same EREs by ERa. One possible explanation for the similar induction of luciferase from three and four tandem copies of EREc38 with ERa is that all available transcription machinery, including coactivators, is saturated when cotransfecting the cells with 10 ng of ER expression vector. To examine this possibility, the amount of ERa transfected was reduced to 5 ng. As anticipated, less luciferase activity was induced from each ERE with 5 ng of ERa cotransfected. The relative induction of luciferase activity by E2 was dependent on the number of copies of EREc38 (Fig. 3). Interestingly, in contrast to the data shown in Fig. 2, E2 synergistically induced luciferase activity from 2(EREc38) as well from 3(EREc38) and 4(EREc38) when 5 ng was cotransfected. Significantly higher luciferase activity was detected from 4(EREc38) than 3(EREc38) (PB 0.05). A similar experiment was performed transfecting CHO cells with 5 ng pCMV-ERb (Fig. 3). In contrast to the synergistic induction of reporter activity from 2(EREc38) seen with 5 ng ERa, the activity induced by ERb was additive. However, in results similar to those shown in Fig. 2, ERb synergistically activated reporter
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Fig. 3. Subsaturating amounts of ERa and ERb reveal transcriptional synergy from multiple tandem copies of EREc38. CHO cells were co-transfected with 5 ng pCMV-ERa (open bars) or pCMV-ERb (shaded bars) plus the indicated pGL3-Promoter vector and pCMVbgal as described in Section 2. Cell treatments were identical to those described in Fig. 2. The fill symbols are identical to those described in Fig. 2. The fold induction of luciferase activity is expressed relative to the activity detected upon addition of EtOH, which was set to 1. The data shown are presented as mean 9 SEM from three separate experiments.
gene transcription from 3(EREc38) and 4(EREc38). Comparison of the data in Figs. 2 and 3 show no difference in the fold synergy from 3(EREc38) and 4(EREc38). However, the fold synergy was greater for 2(EREc38) when less ERa or ERb was transfected. These data indicate that the extent of transcriptional
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synergy is influenced by the intracellular levels of ERa or ERb. To address the question of whether ERa and ERb are expressed at equal levels in the transfected CHO-K1 cells, whole cell extracts were immunoblotted with ERa-specific Ab-10 or ERb-specific antiserum Y-19 (Western blot data not shown). Densitometric analysis of the resulting film indicated that comparable protein expression levels of ERa and ERb were achieved in these cells (data not shown).
3.2. Cooperati6e binding of ERa and ERb to multiple tandem copies of EREc38 Since we reported that calf uterine E2-liganded ERa binds cooperatively to three or four tandem copies of EREc38, but not to 1(EREc38) or two tandem copies of EREc38 (Klinge et al., 1992b), it was of interest to determine if ERb binds cooperatively to multiple tandem EREs. The kinetics of baculovirus-expressed rh ERa and rr ERb binding to one, two or three tandem copies of EREc38 were examined using EMSA. Although baculovirus-expressed ERa shows a single band on a Western blot (data not shown) two bands were detected for ERa binding to 1(EREc38) (Fig. 4).
Fig. 4. ERa binds cooperatively to multiple tandem copies of EREc38. EMSA was used to quantitate the kinetics of ERa binding to 1(EREc38) (panel A); 2(EREc38) (panel B); 3(EREc38) (panel C); or 4(EREc38) (panel D). In each gel, a constant amount (0.7 nM) ERa was incubated with 1 mM E2 for 1-h on ice. Then, the respective [32P]-labeled EREc38 (0.04, 0.08, 0.15, 0.26, 0.42, 0.66, 1.04, 1.71, and 2.52 nM in lanes 1–9, respectively) was added to the reaction, and samples were incubated for 20 min at room temperature prior to fractionation on 4% polyacrylamide gels electrophoresed at 4°C. Details of EMSA are described in Section 2. The specificity of ERa– ERE binding was confirmed by supershift with antibody H222 (lane 10 in each panel which included 0.42 nM DNA). These gels are representative of the three to four gels run for each EREc38 oligomer.
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Table 1 Comparison of ERa and ERb binding parametersa 1(EREc38) Receptor ERa ERb
KD (pM) 88.5 98.5 145.7 913.7e
2(EREc38) n 0.9 90.1 1.09 0.1
KD (pM) 37.99 8.6b 19.19 3.2f
3(EREc38) n 3.0 90.2c 2.3 90.2
KD (pM) 2.4 90.1b 0.7 90.4f
4(EREc38) n 3.1 90.5 6.1 90.6g
KD (pM) 17.6 91.6b 13.8 95.3f
n 1.2 90.1d 4.0 90.4
a ERa and ERb binding parameters, KD and the stoichiometric coefficient n, were calculated using values obtained from EMSA experiments as described in Section 2. Each value is the mean 9S.E.M. of three separate experiments with each data point performed in duplicate within each experiment. b Significantly different (PB0.0001) from the KD for interaction of ERa with 1(EREc38). c Significantly different (PB0.001) from the anticipated nH value of 2, i.e. two ER dimers bound to two EREs. d Significantly different (PB0.001) from the anticipated nH value of 4. e Significantly different (PB0.001) from the KD for interaction of ERa with 1(EREc38). f Significantly different (PB0.001) from the KD for interaction of ERb with 1(EREc38) g Significantly different (PB0.001) from the anticipated nH value of 3.
These bands are specific for ERa since both are fully supershifted by ERa-specific antibody H222. Protein– protein interaction between ERa and other proteins present in the partially purified NE from SF21 cells along with reversible conformational changes in ERa during non-denaturing gel electrophoresis may account for the two bands. To evaluate ERa – ERE binding stoichiometry, EMSA data were quantitated and then analyzed by a modification of Hill analysis described in Section 2. The apparent KD values and apparent stoichiometric coefficients (n) for ERa and ERb binding to one, two, three, or four tandem copies of EREc38 are given in Table 1. The apparent affinity of ERa binding to two, three, and four copies of EREc38 significantly exceeded that of binding to one EREc38 (Table 1). The estimated stoichiometric coefficients (n) for ERa binding to 1(EREc38) and 3(EREc38) did not deviate significantly from the expected values of one and three, i.e. the number of ERa dimers anticipated to bind to one or three EREs, respectively (Table 1). The agreement of the estimated and expected stoichiometry of ERa binding to one and three tandem copies of EREc38 indicates the validity of Eq. (1). Therefore, the affinity of ERa binding to any ERE within 3(EREc38) appears to be independent of ERa binding to the adjacent ERE site (Section 2 for a kinetic explanation as to how differences in binding affinity may cause a discrepancy between the estimated and expected n values). The apparent stoichiometry of ERa binding to 2(EREc38), i.e. 3, was higher than the expected value of 2 (Table 1). This suggests either that ERa binding to one of two adjacent EREs changes the affinity of ERa interaction with the unoccupied ERE, or three ERa dimers bind 2(EREc38). The band count of five ERa – 2(EREc38) complexes is compatible with both of these suggestions (Fig. 4). In contrast, the estimated stoichiometry of ERa binding to 4(EREc38), i.e. 1.2, was significantly lower than the expected value of 4 (Table 1). This suggests
either that ERa binding to one of the 4 EREs changes the affinity of ERa interaction with the unoccupied EREs, or that only one ERa dimer binds to 4(EREc38). Since five bands are visible for ERa – 4(EREc38) (Fig. 4), the data eliminate the possibility that only one ERa dimer is bound. Instead, the discrepancy between the measured and expected stoichiometry may reflect a change in affinity of unoccupied EREs caused by ERa binding to the adjacent ERE sites. Fig. 5 presents quantitative data obtained with ERa binding to each EREc38 multimer and plotted according to Eq. (4). As shown, these data fit the expected linear model with correlation coefficients exceeding 80%. In contrast to the two bands seen for ERa – 1(EREc38) binding (Fig. 4A), a single ERb –1(EREc38) band was detected (Fig. 6A). ERb bound to a single copy of EREc38 with lower affinity compared with ERa (Table 1). ERb bound to multiple copies of EREc38 sites with a significantly higher affinity than to a single EREc38. The number of bands of ERb – EREc38 complex formed corresponded to the number of tandem copies of the ERE for 1–3(EREc38), but only three bands were detected for ERb –4(EREc38). The data obtained for ERb binding to multiple tandem copies of EREc38 also fit the expected linear model based on Eq. (4) and gave regression coefficients exceeding 80% (Fig. 7). The calculated stoichiometric coefficient (n) for ERb binding to one, two, and four (EREc38) was not significantly different from the expected values of one, two, and four. The agreement between the estimated and expected stoichiometry of ERb binding to two and four tandem copies of EREc38 again indicates the validity of Eq. (1). Therefore, these n values indicate that the affinity of ERb binding to remaining unoccupied ERE(s) is independent of ERb binding to the adjacent EREs. In contrast, the stoichiometric coefficient of 6.1 for ERb binding to 3(EREc38) was significantly higher
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than the expected value of 3 (Table 1). Since ERb binding to 3(EREc38) formed three major and one minor bands recognized by an ERb-specific antibody (Fig. 6C), the stoichiometry is not expected to be greater than four. The discrepancy between the measured n value, on one hand, and both the expected stoichiometry and the band count, on the other hand, appears not to be due to six ER dimers bound, but may instead reflect a change in the affinity of unoccupied EREs caused by ERb binding to the adjacent ERE sites.
4. Discussion Differences in ligand binding, tissue distribution, and transcriptional activation of reporter constructs indicate that ERa and ERb play overlapping, but different roles in vivo (Kuiper et al., 1997). Here we examined whether ERb displayed transcriptional synergism at multiple tandem copies of a consensus ERE sequence. Earlier we reported that ERa synergistically activated reporter gene activity from multiple tandem copies of an ERE consensus sequence in MCF-7 cells (Klinge, 1999a; Sathya et al., 1997). Although E2 treatment of CHO
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cells resulted in a significantly lower induction of luciferase activity by ERb than by ERa, there was no difference in the fold-synergy induced by ERa or ERb. These data may indicate that transcriptional synergy is mediated by a common mechanism for both ERa and ERb. The observation that ERb shows lower induction of ERE-driven reporter gene activity than ERa in transiently transfected CHO cells is similar to that reported by other investigators using a single or multiple tandem copies of consensus ERE driving reporter gene expression in COS-1, HeLa, and HepG2 cells (Cowley and Parker, 1999; Tremblay et al., 1999). With one ERE-tk-Luc in HeLa cells, ERb activity was 52% of ERa; in COS-1 cells, ERb activity was 19% of ERa; and in HepG2 cells, ERb was 4% of ERa (Cowley and Parker, 1999). We observed that ERb activity was 53% of ERa from 1(EREc38) in CHO cells. This result is similar to that detected in COS-1 cells. Although there was a species difference in the source of recombinant ERa and ERb used in our experiments, i.e. rhERa and rrERb, we note that the differences in transcriptional activation seen between rhERa and rrERb are similar to those reported recently for rhERa and rhERb (Hall and McDonnell, 1999). The latter report showed that rhERb was about one third as
Fig. 5. ERa binds cooperatively to multiple tandem copies of EREc38. ERa– ERE complexes were quantitated as described in Section 2 and expressed as the concentrations of ERa-bound and free ERE. The near-saturation measurements at the higher ERE concentrations were used to estimate the functional ERa concentration (Rtotal in Eq. (4)) for each gel. After transformation of the data according to Eq. (4), data from three to four gels were pooled. Linear regression analysis was applied to the data obtained with 1(EREc38) (panel A); 2(EREc38) (panel B); 3(EREc38) (panel C); or 4(EREc38) (panel D). The regression coefficients and their standard errors expressed according to Eq. (4) are shown in Table 1.
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Fig. 6. ERb binds cooperatively to multiple tandem copies of EREc38. EMSA was used to quantitate the kinetics of ERb binding to 1(EREc38) (panel A); 2(EREc38) (panel B); 3(EREc38) (panel C); or 4(EREc38) (panel D). In each gel, a constant amount (0.7 nM) of ERb was incubated with 1 mM E2 for 1-h on ice. Then the respective [32P]-labeled EREc38 (0.06, 0.11, 0.20, 0.35, 0.55, 0.83, 1.24, 1.93, and 2.76 nM in lanes 1–9, respectively) was added to the reaction, and samples were incubated as described in Fig. 4. Details of EMSA are described in Section 2. The specificity of ERb– ERE binding was confirmed by supershift with antibody Y-19 (lane 10 in each panel which also included 0.55 nM DNA). These gels are representative of four gels run for each EREc38 oligomer.
active as rhERa at inducting luciferase reporter gene activity from three tandem copies of the vitellogenin ERE in transiently transfected HepG2 cells treated with 10 nM E2 in cells expressing rhERb was approximately 33% of that for rhERa (Hall and McDonnell, 1999). These results are in good agreement with the data shown in Fig. 2 and indicate that the small percentage in aa change between the rat and human ERb sequences (Kuiper et al., 1996; Enmark et al., 1997; Moore et al., 1998; Ogawa et al., 1998) do not appear to result in large differences in transcriptional activity in transiently transfected cells. No one has examined whether ERb synergistically activated transcription from multiple EREs. Transcriptional synergism from multiple hormone response elements has been reported for glucocorticoid, mineralocorticoid, and androgen receptors (IngiguezLluhi et al., 1999), and for ERa (Klinge, 1999a; Mattick et al., 1997; Sathya et al., 1997). Although the exact mechanism for ERa transcriptional synergism is unknown, transcriptional synergism by GR requires an intact DNA binding domain (DBD), LBD, and the DBD dimer interface (Ingiguez-Lluhi et al., 1999). The data presented here and in our previous work (Klinge, 1999a; Klinge et al., 1996a; Sathya et al., 1997) indicate that synergy depends on the ligand bound to ERa,
implicating the LBD, and on the ERE sequence, thus also implicating the DBD in transcriptional synergy. Indeed our observation that ERb synergistically transactivates gene expression from multiple tandem EREs despite the fact that the N-terminal AF-1 domain of ERb is non-functional (McInerney et al., 1998), indicates that the ER A/B domain region is not involved in functional synergy. Most of the genes that are highly induced by estrogens, including the vitellogenins, PR, and ovalbumin contain multiple, variably spaced EREs (Martinez et al., 1987; Kraus et al., 1994; Kato et al., 1995) which result in synergistic activation of these genes in estrogen-treated target cells. Transcriptional synergy may result from several possible mechanisms. These include cooperative recruitment of a coactivator, action at distinct rate-limiting steps in transcription initiation, cooperative DNA binding (Herschlag and Johnson, 1993), and/or direct protein–protein interactions between ERa dimers. Among the possible mechanisms for the transcriptional synergism observed here, ER might cause changes in DNA topology that are transmitted to another ER bound nearby. ERa has been shown to bend DNA (Nardulli et al., 1993 Nardulli et al., 1995). Thus, one may speculate that the distinct local topologies
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induced by binding of one ERa dimer have differential allosteric effects on ERa conformation and activity at adjacent sites. There are no reports as to whether ERb bends DNA. We and others have demonstrated that the stereoalignment of EREs and their spacing influence synergistic responses to estradiol (Ponglikitmongkol et al., 1990; Klinge et al., 1992a,b, 1996a; Anolik et al., 1995, 1996 Sathya et al., 1997; Nordeen et al., 1998). We reported that calf uterine, E2-liganded ERa binds cooperatively to three or four tandem copies of EREc38, but not to 1(EREc38) or 2(EREc38) (Klinge et al., 1992b). These results fit the pattern of transcriptional synergism seen here and in our previous studies with MCF-7 cells (Sathya et al., 1997; Klinge, 1999a). Here we compared the binding of recombinant human ERa and rat ERb to these EREs in vitro. Oligomers containing single or multiple copies of EREc38 show an identical pattern of decreasing binding affinity for both ERa and ERb: 3(EREc38) \ 4(EREc38)\ 2(EREc38)\1(EREc38). We suggest that the increased binding affinity may contribute to the synergistic activation of reporter gene expression from 3(EREc38) and 4(EREc38) (Fig. 2). The changes in binding affinity of ER in response to the increase in number of tandem EREs demonstrate
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both similarities and differences between ERa and ERb. To understand the functional role of each ER isoform, it is necessary to examine data on their differences. For ERa, the requirements of the model described by Eq. (1) were met only for ERa binding to three tandem copies of EREc38 but not to two and four tandem copies. This indicates a change in affinity of the unoccupied ERE sites associated with ERa binding to the adjacent ERE(s). We showed previously that the helix structure of DNA makes the adjacent ERE sites stereochemically different in a way that may contribute to variations in the affinity of ERa binding to different numbers of tandem ERE sites (Klinge et al., 1992b, 1996a). Here we observed an increase in binding affinity for both ERa and ERb to two or more tandem copies of EREc38. However, in contrast to ERa, ERb appears to bind with comparable affinity to two and four tandem copies of EREc38 and with higher affinity to 3(EREc38). Differences in the conformation of ERa and ERb bound to these EREs may account for the observed differences in KD values. The differences in binding affinity in response to occupation of adjacent ERE sites with ERa and ERb indicates that these two ER species, when bound to ERE, may have different conformations, altering their
Fig. 7. ERb binds cooperatively to multiple tandem copies of EREc38. Data on ERb– ERE complexes were quantitated and processed as described in Fig. 5. Linear regression analysis was applied to the pooled data obtained with 1(EREc38) (panel A); 2(EREc38) (panel B); 3(EREc38) (panel C); or 4(EREc38) (panel D). The regression coefficients and their standard errors expressed according to Eq. (4) are shown in Table 1.
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interaction with other ERE-bound ER dimers, or recruit different cofactors, or both. We speculate that ERb –ERE binding is stabilized by the binding of other ERb dimers at adjacent sites. Since transcriptional synergy by ERa depends on the cell type used in transient transfection (Mattick et al., 1997), it seems likely that cell-specific cofactors influence transcriptional synergy by ERb as well. Further studies are necessary to examine these possibilities.
Acknowledgements We thank Rosemary L. Sims and Sheetal J. Mehta for their assistance in preparing plasmids used transient transfection assays. We thank Dr J-A. Gustafsson for supplying the pCMV-ERb vector and Abbott Labs for the gift of H222. We thank Dr Peter C. Kulakosky for preparing ERa and ERb and for his thoughtful suggestions on this manuscript.This study was also supported by NIH R01 DK 53220 and a University of Louisville Research on women Grant to C.M.K.
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