Effects of Multiple Estrogen Responsive Elements, Their Spacing, and Location on Estrogen Response of Reporter Genes

G. Sathya, Wenzhuo Li, Carolyn M. Klinge, Jennifer H. Anolik, Russell Hilf and Robert A. Bambara

Department of Biochemistry and Biophysics and the Cancer Center (G.S., W.L., J.H.A., R.H., R.A.B.) University of Rochester School of Medicine and Dentistry Rochester, New York 14642
Department of Biochemistry (C.M.K.) University of Louisville School of Medicine Louisville, Kentucky 40292


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Most highly estrogen-responsive genes possess multiple estrogen-responsive elements (EREs) that act synergistically to activate expression. Synergism between EREs appears to depend on structural features of the EREs and the promoter. To examine the activation process, we cloned single or multiple tandem copies of the consensus ERE into reporter plasmids. These plasmids contained either a chloramphenicol acetyl transferase reporter gene driven by a minimal promoter or a luciferase reporter gene driven by the Simian virus 40 (SV40) promoter. Using MCF-7 human breast cancer cells, we demonstrate that synergism among EREs depends on the number of EREs, their spacing, and the distance of the EREs from the promoter. The induction capacity of EREs falls off slowly with distance from the promoter. Remarkably, multiple EREs can induce effectively and synergize even when they are located more than 2000 nucleotides from the promoter. For EREs located immediately upstream of the promoter, both the distance separating the EREs and the distance to the promoter have to be optimal for synergy. Altering either distance changes the response from synergistic to additive. For distant EREs, presumed to interact by a looping mechanism at the promoter, the length of DNA between the EREs and the promoter is not critical. Synergy among closely spaced EREs that are far from the promoter only requires an optimal distance separating the ERE centers of symmetry. Interestingly, very widely separated EREs can also synergize, presumably also because of their ability to interact by looping.

The estrogen response from single or multiple tandem copies of ERE half-palindromes near the SV40 promoter was also tested. The negligible induction capacity of a single half-site was not significantly increased in multiple sites. The biological role of half-EREs is not apparent in the system employed here.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogens belong to a family of classic steroid hormones that include progesterone, androgens, glucocorticoids, and mineralocorticoids. The major site of synthesis and secretion of estrogens is the ovaries. Estrogens control a variety of physiological processes by regulating the expression of specific genes at their target sites. The hormone acts via the estrogen receptor protein, ER, located predominantly in the nucleus. The hormone-activated receptor binds to estrogen responsive elements (EREs) present in the regulatory region of the target gene. By interacting with the basal transcription machinery, the activated receptor modulates gene expression (1, 2, 3). The minimal ERE that confers estrogen inducibilty in transfection assays has been reported to be the 13-bp 5'-GGTCAnnnTGACC-3' (4, 5, 6). We derived a consensus ERE from three highly estrogen-responsive genes. It is a 38-bp sequence, comprising a 17-bp inverted repeat 5'-CAGGTCA nnn TGACCTG-3' followed by an AT-rich sequence (7).

Most of the highly estrogen-responsive genes, including the vitellogenins and the progesterone receptor, have multiple, variably spaced EREs within their regulatory regions (8, 9). The presence of multiple EREs results in synergistic activation of these genes. Synergism in the vitellogenin B1 gene was reported to result from the cooperative binding of the receptor to the two ERE sites (10). However, Ponglikitmongkol et al. (11) reported that synergism may not be primarily due to cooperative binding. The stereoalignment of ER- bound EREs and their spacing influence synergistic response to estradiol (11). Interestingly, another highly estrogen-responsive gene, ovalbumin, is synergistically activated by a series of four half-palindromic sites, all of which appear to be necessary for induction (12, 13). The ovalbumin EREs appear to be cell type-specific, indicating the requirement of certain additional factors.

In this report, we investigated how multiple EREs mediate reporter gene expression in response to estradiol (E2), and whether placing them in tandem or separating them from one another affects the level of induction. We examined whether estrogen response from multiple EREs was affected by the strength of the promoter and the relative distance of the EREs from the promoter. We compared the levels of induction by E2 in reporter genes having single or multiple copies of EREs located upstream or downstream of the transcribed region and positioned in tandem or widely separated.

Additionally, we examined whether ERE half-site sequences could have a biological role when present as single or multiple copies, by measuring the levels of induction by E2 in reporters having single or multiple copies of half-ERE sequences. Based on our observations, we propose generalizations that describe the behavior of multiple EREs in regulating gene expression.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Trimeric EREs Synergize Upstream of a Minimal Promoter
To understand how single or multiple EREs affected reporter gene activation in response to E2, we used chloramphenicol acetyltransferase (CAT) reporter constructs that contained a single copy, or multiple tandem copies, of EREc38 upstream of a minimal promoter containing the Xenopus vitellogenin B1 TATA box. The EREs were located 52 nucleotides upstream of the TATA box. The center-to-center distance between the EREs was 38 nucleotides. As shown in Fig. 1Go, the ratio of normalized CAT activity with/without 1 nM of estradiol for one and two copies of EREc38 was 1.2 and 1.88, respectively. For three copies of the ERE we observed a very large increase in the ratio representing a 16-fold induction. Addition of a fourth ERE more than doubled the ratio again. The specificity of the response to E2 was demonstrated by the lack of reporter activation in response to 4-hydroxytamoxifen (4-OHT) even at concentrations up to 10-6 M (data not shown). The E2-induced response was reduced to basal levels with the addition of 100-fold excess of 4-OHT (Fig. 2Go). 4-OHT is a metabolite of tamoxifen, a type I antiestrogen used to treat patients with breast cancer (14, 15). Our results demonstrate that 4-OHT inhibits E2-induced reporter gene activation by single and multiple EREs in MCF-7 cells.



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Figure 1. Trimeric EREc38 Synergize Upstream of a Minimal Promoter

pATC- 1, 2, 3, 4EREc38 constructs contain one, two, three, or four tandem copies of the consensus EREc38 upstream of the vitellogenin B1 TATA box in the CAT reporter vector. 1.5 µg of each vector was cotransfected with 0.3 µg of pCMVß-galactosidase internal control plasmid into MCF-7 cells. Three hours after transfection, 1 nM E2 or an equal volume of ethanol (as control) was added in duplicate. CAT and ß-galactosidase assays on the cell lysates were performed after 24 h. Ratios of the mean normalized CAT activities, with and without estradiol, from six individual experiments are plotted; error bars are SEM.

 


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Figure 2. 4-OHT Does Not Induce Expression from the CAT Reporters

CAT reporter constructs containing one, two, three, or four tandem copies of EREc38 upstream of the minimal promoter were cotransfected with pCMVß-gal internal control plasmid into MCF-7 cells. Three hours after transfection, 1 nM E2 (gray bars), 1 nM E2 plus 100 nM 4-OHT (white bars), or an equal volume of ethanol as control (black bars) were added in duplicate. CAT and ß-galactosidase assays were performed after 24 h. The normalized CAT activity (mean with range) for each construct obtained from a single experiment is illustrated.

 
Multiple EREs Upstream of a Promoter Can Respond Linearly to Estrogen
Single or multiple tandem copies of EREc38 were cloned 83 nucleotides upstream of the SV40 promoter (described in Materials and Methods and Fig. 3AGo). The center-to-center spacing between the EREs in these constructs was 38 nucleotides. The ratio of normalized luciferase activity with and without estrogen was 2.0 for 1EREc38, 3.7 for 2EREc38, 7.6 for 3EREc38, and 5.1 for 4EREc38, as shown in Fig. 3BGo. The numbers indicate a slightly synergistic response for each increase in the number of EREs in the construct up to three. That response was slightly reduced with addition of a fourth ERE, showing that four leads to diminishing returns. The overall appearance of the increase is approximately linear with the number of EREs. This observation clearly differs from that obtained with the CAT reporters, where we found a distinct synergistic increase for three, and even four, copies of EREs. The altered pattern of induction in the two reporters could result from differences in either the promoter structures, or the distance of the EREs from the promoter. The SV40 promoter contains several other cis acting elements including six GC boxes, which bind Sp1 upstream of the TATA box, causing higher basal activity than with the minimal promoter. However, as considered below, we attribute differences in synergy to the distance relationships among EREs and the TATA box.



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Figure 3. Multiple EREs Upstream of a Complex Promoter Respond Additively to Estrogen

A, pGL3–1EREc38, 2EREc38, 3EREc38, and 4EREc38 contain one, two, three, or four copies, respectively, of EREc38 at 83 nucleotides upstream of the SV40 promoter. The center-to-center distance between the EREs is 38 nucleotides. K, KpnI; S, SacI; black box, SV40 promoter; gray box, luciferase-coding region; EREc38 is represented by a white rectangle, and the bar in the center of the rectangle denotes the center nucleotide. B, Luciferase reporter constructs containing one, two, three, or four tandem copies of EREc38, 83 nucleotides upstream of the SV40 promoter, were cotransfected with pCMVß-gal internal control plasmid into MCF-7 cells. Three hours after transfection, 1 nM E2, or an equal volume of ethanol as control, was added in duplicate. Luciferase and ß-galactosidase assays were performed after 24 h. The ratio of normalized luciferase activity, with and without estradiol, from three separate experiments is shown with SEM.

 
ERE Positioning and Spacing Influence Induction Levels
In the luciferase reporter constructs, we made two alterations in an attempt to determine whether the additive estradiol response of upstream dimeric EREs could be enhanced to a synergistic response. One copy or two tandem copies of EREc38 were cloned into the NheI site, 28 nucleotides upstream of the promoter, creating the constructs pUP1 and pUP2, respectively (Fig. 4AGo). The ERE centers for pUP1 and each of pUP2 were 37, 60, and 83 nucleotides from the promoter, respectively. The two EREs in pUP2 had a center-to-center spacing of 23 nucleotides rather than 38 nucleotides as in pGL3–2EREc38. The ratios of luciferase activity with and without estradiol were 1.7 for pUP1 and 4.5 for pUP2, with 1.0 being the basal uninduced level (Fig. 4BGo). The ratio of the induction level of pUP2 over pUP1 was considerably greater than the ratio of the induction level for pGL3–2EREc38 over pGL3–1EREc38 (Fig. 3BGo). These observations led us to conclude that moving two EREs with respect to each other and the promoter can alter synergy. We also point out that the EREs in pGL3–2EREc38, separated by 38 nucleotides, are on opposite faces of the DNA helix, whereas in pUP2, the two EREs are on the same face of the helix. The latter orientation might have promoted synergistic interaction between the receptor molecules bound at these sites.



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Figure 4. Appropriately Spaced Dimeric EREc38 Elements Synergize Upstream of the SV40 Promoter

A, pUP1 contains a single copy of EREc38 at the NheI site, 28 nucleotides upstream of the SV40 promoter. pUP2 contains two tandem copies of EREc38 at the NheI site. The center-to-center distance between the EREs is 23 nucleotides. N, NheI; black box, SV40 promoter; gray box, luciferase coding region; EREs are shown by white rectangles with the bar inside representing the center nucleotide. B, pUP1 and pUP2 constructs were cotransfected with pCMVß-gal internal control plasmid into MCF-7 cells. Three hours after transfection, 1 nM E2, or an equal volume of ethanol as control, was added in duplicate to the cells. Luciferase and ß-galactosidase assays were performed after 24 h. The ratio (±SEM) of normalized luciferase activity with/without estradiol from three separate experiments is shown.

 
EREs Are Functional when Present Downstream of the Target Gene
We tested the ability of single or multiple EREs to activate an estrogen-inducible gene by cloning EREs at the SalI site downstream of the luciferase gene. At this position, the EREs are more than 2000 nucleotides away from the promoter (Fig. 5AGo). Figure 5BGo shows the ratio of normalized luciferase activity with and without 1 nM estradiol for pLL1 and pLL2, which contain one and two copies of EREs, respectively, downstream of the luciferase gene. The ratios for pLL1 and pLL2 are 1.3 and 3.0, respectively, where 1 represents the basal level in the absence of estrogen. Therefore, the fold induction by estrogen is 0.3 and 2.0, respectively. This indicates a synergistic response for two copies of EREs. The response from pLL2 is lower than the response from two EREs located upstream of the transcribed region. Evidently, EREs may function well at a downstream site and may synergize when the distance separating them is optimal, but proximity to the promoter improves their induction capacity.



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Figure 5. EREs Placed Downstream of the Gene Are Functional

A, One copy or two tandem copies of EREc38 were cloned into the pGL3-Promoter vector at the downstream SalI site to generate pLL1 and pLL2 constructs. The center-to-center distance between the two EREs of pLL2 was 23 nucleotides. S, SalI; black box, SV40 promoter; gray box, luciferase-coding region; EREs are shown by white rectangles with the bar inside representing the center nucleotide. B, pLL1 and pLL2 constructs were cotransfected with pCMVß-gal internal control plasmid into MCF-7 cells. Three hours after transfection, 1 nM E2, or an equal volume of ethanol as control, was added in duplicate to the cells. Luciferase and ß-galactosidase assays were performed after 24 h. The ratio (±SEM) of normalized luciferase activity with and without estradiol from four to five separate experiments is plotted in the graph.

 
Multiple EREs Separated by Large Distances from Each Other Can Contribute to Induction of a Single Promoter
The results presented above show that tandem copies of EREs with a center-to-center distance of 23 nucleotides were synergistic when placed downstream of the luciferase gene. EREs located upstream and closer to the promoter were even more effective, but not by a large amount. Since EREs can act at a large distance from the promoter, we wanted to measure the effect of separating EREs from each other by more than 2000 nucleotides. To test this, we cloned in EREs at distant sites upstream and downstream of the luciferase gene as shown in Fig. 6AGo. We compared the estrogen induction in pUP1 (one upstream), pUP1-down1 (one upstream and one downstream), pUP1-down2 (one up and two down), and pLL2 (two down). The ratios of the normalized luciferase activity with and without estradiol for these constructs were 1.7, 2.8, 5.8, and 3.0, respectively (Fig. 6BGo). The induction from two EREs was almost the same in the two constructs that contain the EREs in tandem (pLL2) and nontandem (pUP1-down1) locations. This shows that EREs separated by a large distance can both contribute to induction of the same promoter. The pUP1-down2 construct has three EREs, one located upstream and two tandem EREs located downstream of the gene. The response from this construct (4.8-fold) was more than additive of the induction produced by the single upstream ERE (0.7-fold) plus the dual downstream EREs (2.0-fold). This shows that EREs are capable of synergizing even when separated by very large distances.



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Figure 6. EREs Synergize at Very Large Distances of Separation

A, pUP1-down1 contains one ERE at the NheI site and one ERE at the SalI site downstream of the luciferase gene. pUP1-down2 contains a single ERE upstream and two tandem EREs downstream. The center-to-center distance of the downstream EREs is 23 nucleotides. N, NheI; S, SalI; black box, SV40 promoter; gray box, luciferase-coding region; all EREs are shown as white rectangles. The bar inside the rectangle represents the center nucleotide. B, The pUP1 luciferase construct contained a single copy of the consensus EREc38 at 28 nucleotides upstream of the SV40 promoter in the PGL3-Promoter. A single or two copies of EREc38 were inserted into the downstream SalI site of pUP1 to generate pUP1-down1 and pUP1-down2 constructs. pLL2 had two tandem copies of EREc38 at the SalI site but no EREs upstream. These constructs were cotransfected with pCMVß-gal internal control plasmid into MCF-7 cells. Three hours after transfection, 1 nM E2, or an equal volume of ethanol as control, was added in duplicate to the cells. Luciferase and ß-galactosidase assays were performed after 24 h. The ratio (±SEM) of normalized luciferase activity with/without estradiol from three to five separate experiments is illustrated.

 
Multiple Copies of Half-EREs Produce a Negligible Estrogen Response
Genes regulated by estrogens often contain multiple EREs, having sequences that vary from the core consensus palindrome by one or two nucleotide changes (16, 17, 18). These nonconsensus EREs were reported to have lower affinity for liganded ER in vitro (19). Half-palindromic sequences are also frequently found near full-length EREs in the regulatory regions of responsive genes. Measurements made in vitro have also shown that individual half-sites have very low affinity for the receptor; however, certain genes are regulated by half-sites in vivo (20, 21, 22, 23). We next tested whether half-EREs are functional in our transfection assay and whether multiple tandem copies of half-EREs have any significant effect on estrogen-induced gene expression. Single or multiple tandem copies of half-ERE sequences were cloned upstream of the SV40 promoter in the luciferase reporter plasmid. The distance between the half-sites was 38 nucleotides. Figure 7Go shows the response from single or multiple tandem copies of half-ERE sequences upstream of the SV40 promoter of the luciferase gene. The ratios of normalized luciferase activities with and without estrogen for one, two, and three tandem copies of half-EREc38 are 1.4, 1.42, and 1.6, respectively. These results show that as the number of half- EREs is increased from one to three, there is little increase in the response. Also, the levels of induced expression by half-sites are significantly lower than expression levels from multiple consensus EREs.



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Figure 7. Half-EREs Minimally Affect Estrogen Response

One, two, or three tandem copies of 1/2 EREc38 were cloned 83 nucleotides upstream of the SV40 promoter in the pGL3-Promoter vector. There is a 38-nucleotide separation between the half-sites. Cotransfections with pCMVß-gal internal control plasmid into MCF-7 cells were performed. Three hours after transfection, 1 nM E2, or an equal volume of ethanol as control, was added in duplicate to the cells. Luciferase and ß-galactosidase assays were performed after 24 h. The ratio (±SEM) of normalized luciferase activity with and without estradiol from three separate experiments is presented.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The main objective of our study was to analyze the influence of selected factors on the induction of gene expression by multiple EREs. The parameters considered were: the number of EREs, the distance separating them, their stereoalignment; the distance between the EREs and the promoter; the presence of additional cis-acting elements; and the effect of adjacent ERE half-sites. We used two reporter systems containing different promoters to ascertain and compare the effects of single or multiple tandem copies of consensus EREs on estrogen-induced gene expression. The data show that the percent induction varies based on the strength of the promoter and the presence of additional cis-acting elements. The E2-induced reporter gene activity was lower in the presence of the SV40 promoter because of its higher basal activity.

When two EREs were placed at 83 nucleotides from the SV40 promoter and their center-to-center distance was 38 nucleotides, they responded approximately additively to the hormone. When they were moved closer, to a distance of 28 nucleotides from the promoter and their spacing reduced to 23 nucleotides, they activated expression synergistically. When the EREs and promoter are close, linear separation on the DNA has a major influence on the process of synergy. In this situation, it appears that physical structure and bending constraints of the protein-DNA complexes control the ability of components to interact appropriately for synergy.

We then tested whether EREs were functional when located downstream of the gene, at a distance of about 2400 nucleotides from the promoter. As expected, the response levels from the downstream EREs were not as high as seen with the upstream EREs. However, the response from downstream tandem copies of EREs separated by 23 nucleotides remained synergistic. This suggests that when EREs are located close to each other, and spaced at an optimal distance, they can synergize at a promoter located far away.

In the situation in which multiple EREs were located upstream and downstream of the reporter gene, there was remarkably effective interaction among them for induction of the promoter. There was moderate synergy in the construct having one ERE upstream and one downstream. The fold of induction in this construct (1.8) was more than the sum of induction from a single upstream ERE in pUP1 (0.7-fold) and a single downstream ERE in pLL1 (0.3). The presence of one additional ERE downstream in pUP1-down2 construct demonstrated a strong synergy with the upstream ERE.

Based on these observations, can we define rules that describe the efficiency of induction of gene expression by multiple EREs? Certainly, two or more EREs can synergize for induction. Synergistic interactions among EREs can occur at distances close to the promoter or at more than 2000 nucleotides from the promoter. EREs located close to the promoter are constrained by two parameters: the distance between the ERE centers and the distance between the EREs and the TATA box. Both of these parameters must be optimal for synergy. EREs located far away from the promoter presumably interact with the promoter by a looping mechanism (24). In this way, the ER-ERE complexes could physically interact with the promoter, irrespective of their distances from the promoter along the DNA. Therefore, for EREs located far away from the promoter, the distance separating the EREs is the only requirement for synergy. Presumably, two EREs located far from each other, could also interact for synergy by looping.

When EREs are located both close to and far away from the promoter of a single gene, the situation is more complex but still completely consistent with the rules described above. The EREs can interact with each other for synergy, because looping can bring them together. The ERE that is far away interacts with the promoter by looping. However, the ERE that is close to the promoter is constrained by its position on the DNA. The capacity of this ERE for synergy with the downstream promoter depends on whether or not it is at the optimal distance from the promoter. This reasoning would explain why we observed strong synergy in the pUP1-down2 construct, which had a single ERE at 28 nucleotides upstream of the promoter and two tandem EREs separated by a center-to-center distance of 23 nucleotides located downstream of the luciferase gene. The downstream EREs were at an optimal distance to interact with each other and could loop to the promoter. The pUP1 ERE was at an optimal distance from the promoter to enable it to interact with both it and the looped EREs.

Our model explains synergy in other constructs as well. The EREs in the CAT reporter were located at about five helical turns (52 nucleotides) from the TATA box, which was apparently an optimal distance. In the trimer construct, the first ERE and the third ERE were separated by 76 nucleotides, with the observation of synergy defining this as an optimal distance. The first ERE of the dimer construct was located at the same distance (52 nucleotides) from the promoter. However, the distance between the two EREs of this construct was a nonoptimal 38 nucleotides. The result was a linear, nonsynergistic response. The luciferase reporter had single or multiple copies of EREc38 at 83 nucleotides from the start of the SV40 promoter region. The SV40 TATA box was located at 191 nucleotides from the EREs. Although the distance separating the EREs in the luciferase reporter was identical to that in the CAT reporter, the distance between EREs and the TATA box was suboptimal. This resulted in a response that was not highly synergistic from three EREs upstream of the luciferase gene. The response from four copies of EREs also appeared to be linear instead of synergistic. Although the complex structure of the SV40 promoter increased the basal levels of expression in the constructs, the requirement for synergism between multiple EREs was consistent for the two kinds of promoters.

The synergistic response from dimeric EREs separated by 23 nucleotides placed 28 nucleotides upstream of the SV40 promoter was also consistent with our proposed rules for multiple EREs, as the structure of these EREs satisfied the two distance requirements for synergy. The requirements were also met by the dimeric EREs with optimal center-to-center distance located downstream of the luciferase gene.

The induction of gene expression by estradiol in each of our plasmids is normalized to the basal expression in the absence of added hormone, a value that we have set to 1.0. In fact, the basal level of expression from a promoter in this system is influenced by the presence and number of EREs in each plasmid. Generally, we find that the introduction of increasing numbers of EREs into a plasmid progressively increases the amount of reporter gene expression, even in the absence of added hormone. Furthermore, the increase in basal expression appears to show some synergy with the increase in the numbers of EREs. This phenomenon might result from a low level of receptor-mediated induction, occurring in the absence of added estradiol. The observed variations in basal expression do not affect the conclusions concerning synergism of estradiol-mediated induction. In fact, the estradiol-dependent synergy we measure would be enhanced in many cases if we had measured all induction against a single basal level of expression, e.g. that of a plasmid with no EREs.

Our observations, leading to the above generalizations, are consistent with the DNA-bending phenomenon proposed for ER-ERE interactions. Nardulli and co-workers (25, 26, 27, 28, 29) have shown that when ER binds to an ERE, it induces a conformational change in the DNA, resulting in a bend of about 65°. The helical separation between EREs and the TATA box may influence the bending induced by both ER binding to the ERE and the interaction of transcription factor IID with the TATA box (30, 31). In a situation of close proximity, the helix orientation of the binding sites for each protein and the degree of DNA bending are expected to exert a strong influence on the induction process. Incorrect positioning of proteins on the DNA helix may prevent interactions necessary for synergy. These structural considerations are not critical for EREs located far from the promoter region, since looping allows interactions to occur with much less constraint. Our results are also consistent with the observations of others that EREs can act at very large distances from the promoter (32, 33).

Many estrogen-regulated genes possess both full palindromic EREs and half-site EREs (12, 34, 35, 36, 37). We tested the effect of single and tandem copies of half-ERE sequences located upstream of the luciferase gene on enhancement of estrogen-induced gene expression. Half-sites were found to have negligible induction capacity. Comparison of multiple tandem copies of half-EREs did not show a progressively increasing effect on estrogen response. Also, when we inserted the half-site adjacent to dimeric consensus EREs downstream of the luciferase gene, we did not detect a significant increase in estrogen response from the half-ERE-containing construct (data not shown). It is unclear whether the orientation and spacing between half-sites and full consensus EREs can affect their induction capacity. If so, there may be induction from a configuration that we have not explored. Although there have been several reports on genes regulated by half-EREs, in this reporter system half-EREs are unable to significantly enhance estrogen response from full EREs.

One parameter not addressed in experiments presented here is the role of cooperative binding of the receptor to multiple nearby EREs. It is not known whether cooperative binding is the basis for the synergism of vitellogenin EREs (10, 11). We previously reported that trimeric and tetrameric head-to-tail tandem constructs of EREc38 appeared to bind estradiol-liganded ER (E2-ER) cooperatively (19). The measured cooperativity depended on stereoalignment of EREs, the presence of a natural AT-rich flanking sequence, and the nature of the ligand (38, 39, 40, 41, 42, 43). In transfection studies with CAT reporters, we detected synergistic response from a trimeric ERE containing plasmid, but not from a CAT reporter containing dimeric EREs that displayed cooperativity of binding (data not shown). From these observations, we cannot conclude whether cooperative binding is essential for synergistic gene expression from EREs.

The physiological sequelae in response to estrogens is modulated by the intracellular concentrations of ligand, ER, and ligand-specific coactivator proteins. Our data show that the degree of synergy between EREs is modulated by the physical distance between EREs and by the separation between EREs and the TATA box. It is also clear that multiple EREs in estrogen-responsive genes can collaborate to control the level of expression. They can carry out this collaboration at great linear distances from the promoter and each other. Hence, the cell can control the degree to which these elements synergize for induction of expression by altering their relative positions.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
MCF-7 cells were maintained in Iscove’s modified Dulbecco’s media (IMDM) (Life Technologies Inc., Gaithersburg, MD) supplemented with 10% FBS (Atlanta Biologicals, Norcross, GA) and 1% penicillin and streptomycin (Life Technologies Inc.). Cells used for transfection were grown in IMDM without phenol red supplemented with 10% charcoal-dextran treated FBS and antibiotics for 24 h before transfection.

Sequences of EREs
EREc38 5'-CCAGGTCAGAGTGACCTGAGCTAAAATAACACATTCAG-3' 1/2EREc38 5'-CCA-GGTCAGAGCATTTCGAGCTAAAATAACACATTCAG-3'

Plasmids
Single or multiple tandem copies of EREc38 and 1/2EREc38 were isolated by KpnI and SacI double digestion from pGEM7Zf(+) plasmid constructs (described in Ref.19) and cloned directly into the upstream multiple cloning site of pGL3-Promoter vector (Promega, Madison, WI) to place the closest ERE 83 nucleotides upstream of the SV40 promoter driving the luciferase reporter gene. The center-to-center spacing of the consensus EREs and the distance between adjacent half-EREs in these constructs is 38 nucleotides. The constructs containing EREc38 were called pGL3–1EREc38, 2EREc38, 3EREc38, and 4EREc38 (Fig. 3AGo). The constructs containing 1/2EREc38 were called pGL3–1/2EREc38, 2(1/2)EREc38, and 3(1/2)EREc38.

EREc38 double-stranded oligomer with a NheI cohesive end was synthesized and ligated end-to-end and cloned upstream of the luciferase-coding region at the NheI site. The NheI site was located at 28 nucleotides from the SV40 promoter, and the ERE center-to-center spacing was 23 nucleotides. The constructs containing one or two EREs at the NheI site, with no EREs downstream of the luciferase gene, were called pUP1 and pUP2 (Fig. 4AGo). Double-stranded EREc38 with a SalI cohesive end was synthesized (Genosys, The Woodlands, TX), ligated end-to-end using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN), and cloned into the SalI site located downstream of the luciferase-coding region of pGL3-Promoter vector. The resulting constructs containing one and two copies of the ERE, at 2400 nucleotides downstream of the promoter, were called pLL1 and pLL2, respectively. The center-to-center spacing between the dimeric EREs of pLL2 is 23 nucleotides (Fig. 5AGo).The construct containing one ERE upstream and one ERE downstream was called pUP1-down1. The construct containing one ERE upstream and two EREs downstream was called pUP1-down2 (Fig. 6AGo).

CAT reporter constructs were made by removing the two EREs from the pATC2 vector (provided by Dr. David Shapiro, University of Illinois, Urbana, IL), and inserting the XbaI/HindIII fragments containing single or multiple tandem copies of EREc38 from pGEM7Zf(+) plasmid. These constructs were called pATC-1EREc38, 2EREc38, 3EREc38, and 4EREc38. The closest ERE in each construct was located at 52 nucleotides from the minimal promoter, which contained the Xenopus vitellogenin B1 TATA box. The ERE center-to-center distance in these constructs was 38 nucleotides. The orientation and nucleotide sequence of the ERE(s) in each reporter construct were verified by dideoxynucleotide sequencing (Sequenase version 2.0, USB). Large-scale purification of plasmids was performed using an Endo-free Qiagen maxiprep kit (Chatsworth, CA).

Transfection
Cells (2.5 x 105) were plated in each well of a 12-well plate in IMDM without phenol red supplemented with 10% stripped serum and 1% antibiotics. After 24 h, at 50% confluency, the cells were transfected using a liposome-mediated transfection protocol (44). Either 0.3 µg of pCMVßGal (CLONTECH, Palo Alto, CA) and 1.5 µg of CAT reporter construct, or 0.6 µg of pCMVßGal and 0.6 µg of luciferase reporter DNA, were transfected into each well using a DNA to liposome ratio of 1 µg:10 nmol. The DNA and liposomes were mixed in Opti-MEM I without insulin, estrogen, and other growth factors (Life Technologies). Three hours after transfection, 1 nM 17ß-estradiol (Sigma, St. Louis, MO), 100 nM 4-OHT (Research Biochemicals International, Natick, MA) or an equal volume of ethanol was added to the wells in duplicate. The cells were maintained in medium containing 1% stripped FBS post-transfection. The cells were lysed after 24 h in 200 µl of 1x reporter lysis buffer (Promega), and the extracts were assayed for activity.

CAT Assays
Sixty microliters of extracts were assayed in a total volume of 125 µl containing 0.25 µCi of [3H] chloramphenicol (DuPont NEN, Boston, MA) diluted in 0.25 M Tris-HCl, pH 8.0, and 5 µl n-butyryl Coenzyme A, 5 mg/ml (Sigma), at 37 C for 90 min. The reaction was stopped with 300 µl of mixed xylenes (J.T Baker, Inc., Phillipsburg, NJ) and mixed on a vortexer for 30 sec. The upper xylene phase was extracted twice with 0.25 M Tris-HCl, pH 8.0, and 100 µl of each sample were counted in Ecoscint A (National Diagnostics, Atlanta, GA).

ß-Galactosidase Assays
Fifty microliters of extracts were assayed in a total volume of 300 µl containing 1x reporter lysis buffer and 2x assay buffer (protocol from Promega). In parallel, several serial dilutions of the ß-galactosidase (Promega) were assayed for activity to obtain a standard curve. The samples were incubated at 37 C until yellow color developed. The reaction was stopped with 500 µl of 1 M sodium carbonate. The activity was plotted as a function of the absorbance at 420 nm to obtain enzyme activity in the extracts.

Luciferase Assays
Twenty microliters of extracts were used for luciferase assay (Promega) with 100 µl of reconstituted substrate. The activity was determined using a luminometer (Turner Designs, Sunnyvale, CA, model 20E) for 15 sec for each sample.

Luciferase and CAT activities of each transfection were normalized using ß-galactosidase activity, and the ratio of normalized activity with and without estradiol was calculated. A value of 1.0 indicates the basal normalized activity without any added hormone, and values greater than 1 represent the fold of induction for each reporter construct. Each experiment was performed at least three times to obtain mean and SEM values.


    ACKNOWLEDGMENTS
 
The authors wish to thank Dr. David Shapiro, University of Illinois, for the gift of the pATC2 vector; Dr. Barry Stripp, University of Rochester, for the use of the luminometer to measure luciferase activity; Jianghong Zhou and Julia Rozenblit for all their assistance; and Dr. Mark Driscoll for his critical review of this manuscript.


    FOOTNOTES
 
Address requests for reprints to: Robert A. Bambara, Department of Biochemistry and Biophysics and the Cancer Center, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642.

This work was supported by NIH Grant HD-24459, in part by NIH Grant CA-16660 (to R.H.), and in part by NIEHS IP20 ES06832 and Veterans Administration Center for the Study of Environmental Hazards to Reproductive Health Grant 0006, Department of Veterans Affairs Medical Center, Louisville, KY (to C.M.K.).

Received for publication June 12, 1997. Revision received September 16, 1997. Accepted for publication September 23, 1997.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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