Sp1 Binding Sites and An Estrogen Response Element Half-Site Are Involved in Regulation of the Human Progesterone Receptor A Promoter

Larry N. Petz and Ann M. Nardulli

Department of Molecular and Integrative Physiology University of Illinois Urbana, Illinois 61801


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


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen is a hormone of critical importance in the development and maintenance of reproductive tissues and also plays an important role in cardiovascular and bone physiology. Estrogen’s effects are mediated through its interaction with the intracellular estrogen receptor (ER). Numerous studies have demonstrated that the two ERs, {alpha} and ß, mediate their effects by binding to specific DNA sequences, estrogen response elements (EREs), thereby initiating changes in transcription of target genes (1 2 ).

It has become apparent that, in addition to binding directly to an ERE, the ER may also modulate transcription indirectly by interacting with other DNA-bound proteins. For example, ER interaction with AP1-bound fos and jun proteins confers estrogen responsiveness to the ovalbumin (3 ), c-fos (4 ), collagenase (5 ), and insulin-like growth factor I (6 ) genes. In addition, a growing body of evidence suggests that the ER may influence binding of Sp1 to its recognition site and thereby confer estrogen responsiveness to the creatine kinase B (7 ), c-myc (8 ), retinoic acid receptor {alpha} (9 ), heat shock protein 27 (10 11 ), cathepsin D genes (12 ), and uteroglobin (13 ) genes.

The progesterone receptor (PR) gene is under estrogen control in normal reproductive tissues (14 15 ) and in MCF-7 human breast cancer cells (16 17 ). MCF-7 PR mRNA and protein increase and reach maximal levels after 3 days of 17ß-estradiol (E2) treatment (16 17 18 ). Like ER, two distinct PR forms are differentially expressed in a tissue-specific manner (19 20 21 22 23 ). PR-B is a 120-kDa protein containing a 164 amino acid amino-terminal region that is not present in the 94-kDa PR-A. Two discrete promoters, A and B, which are thought to be responsible for the production of PR-A and PR-B, respectively, have also been defined (24 ). The activities of these two promoters are increased by estrogen treatment of transiently transfected Hela cells. Interestingly, no consensus EREs have been identified in either promoter A (+464 to +1105) or promoter B (-711 to +31). Promoter A does, however, contain an ERE half-site located upstream of two Sp1 sites (24 ). The presence of these adjacent binding sites suggests that the ER might be able to influence PR expression directly by binding to the ERE half-site, indirectly by interacting with proteins bound to the putative Sp1 sites, or a combination of these two methods. To determine whether the ERE half-site and the two Sp1 sites present in the human PR-A promoter might impart estrogen responsiveness to the PR gene, a series of in vivo and in vitro experiments were carried out.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In Vivo Footprinting of the PR Gene
A number of studies have suggested that an Sp1 site alone or in combination with an imperfect ERE or ERE half-site may be involved in conferring estrogen responsiveness to target genes (7 8 9 10 11 12 13 ). To determine whether the ERE half-site and two potential Sp1 sites residing in the endogenous human PR gene (+571 to +595, Ref. 24 ) might be involved in estrogen-regulated transactivation, in vivo deoxyribonuclease I (DNase I) footprinting was carried out using MCF-7 cells. The region of the PR-A promoter containing the consensus ERE half-site and two potential Sp1 sites is shown in Fig. 1Go and will hereafter be referred to as the half-ERE/Sp1 binding site.



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Figure 1. Sequence of the Half-ERE/Sp1 Binding Site

The sequence of the half-ERE/Sp1 binding site in the PR-A promoter originally reported by Kastner et al. (24 ) is shown. The locations of the ERE half-site and the proximal and distal Sp1 sites (Sp1P and Sp1D, respectively) are indicated.

 
To carry out in vivo footprinting assays, MCF-7 cells were treated with ethanol vehicle or with E2 for 2 or 72 h and then exposed to DNase I. The cells were rapidly lysed, DNA was isolated, and ligation-mediated PCR (LMPCR) procedures were carried out (25 ). Naked genomic DNA, which had been treated in vitro with DNase I, served as a reference in identifying sequences that were susceptible to cleavage in the absence of proteins (Fig. 2Go, Vt). When cells were treated with E2 for 2 h, the protection of the proximal Sp1 site (Sp1P), the distal Sp1 binding site (Sp1D), and the ERE half-site was greater than seen in cells that had not been exposed to hormone. After 72 h of E2 treatment, a time when PR mRNA and protein reach maximal levels (16 17 18 26 27 ), the protection of the half-ERE/Sp1 binding site was sustained. E2 treatment also elicited protection of regions flanking the half-ERE/Sp1 binding site. Thus, we were able to detect distinct differences in protection of the half-ERE/Sp1binding site on the coding strand of the endogenous PR gene after E2 treatment. Despite numerous attempts, we were unable to obtain a footprint of the noncoding PR DNA strand in this region. The failure of these LMPCR reactions may have been due to formation of an extensive stem loop structure ({Delta}G = -11.5 kcal/mol) extending from +674 to +733 (24 ) that limited primer annealing or interfered with the ability of polymerase to proceed through this region. Nonetheless, our in vivo footprinting of the coding strand demonstrated that the half-ERE/Sp1 binding site residing in the endogenous PR gene was differentially protected in ethanol- and E2-treated MCF-7 cells and suggested that the ERE half-site as well as the proximal and distal Sp1 sites might be involved in regulation of the endogenous PR gene in MCF-7 cells.



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Figure 2. In Vivo DNase I Footprinting of the Endogenous PR Gene in MCF-7 Cells

MCF-7 cells were maintained in serum-free medium for 5 days, treated with ethanol control (0 h E2) or 1 nM E2 for 2 or 72 h, and then exposed to DNase I. Genomic DNA was isolated and used in in vivo footprinting. Naked genomic DNA, which had been treated in vitro with either DMS (G) or DNase I (Vt), was included as a reference. The locations of the proximal Sp1 site (Sp1P), distal Sp1 site (Sp1D), and ERE half-site are indicated.

 
Estrogen Enhances Transcription of a Reporter Plasmid Containing the Half-ERE/Sp1 Binding Site
To determine whether the half-ERE/Sp1 binding site could confer estrogen responsiveness to a heterologous promoter, transient cotransfection experiments were carried out with a human ER expression vector and a chloramphenicol acetyltransferase (CAT) reporter plasmid containing either a TATA box alone (TATA CAT) or in combination with the half-ERE/Sp1 binding site (ERE/Sp1-TATA CAT). Exposure of transiently cotransfected CHO cells to E2 resulted in a 1.7-fold increase in CAT activity when the reporter plasmid contained the half-ERE/Sp1 binding site (Fig. 3Go, ERE/Sp1-TATA CAT). This difference was statistically significant. In contrast, no statistical difference in activity was observed with E2 treatment when the parental TATA CAT reporter plasmid was used. These findings suggest that the half-ERE/Sp1 binding site is involved in estrogen-mediated activation of the PR-A promoter.



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Figure 3. Estrogen-Enhanced Activity of a Plasmid Containing the Half-ERE/Sp1 Binding Site

CHO cells were transfected with TATA CAT or ERE/Sp1-TATA CAT reporter plasmid, hER expression plasmid, ß- galactosidase expression plasmid, and pTZ nonspecific DNA using the calcium phosphate coprecipitation method as described in Materials and Methods. Cells were treated with ethanol vehicle or 10 nM E2. Data represent the average of 9 independent experiments. Values are presented as the mean ± SEM. *, P < 0.02 for E2 vs. ethanol control by Student’s t test when ERE/Sp1-TATA CAT was used.

 
Proteins Present in MCF-7 Nuclear Extracts Bind to the Half-ERE/Sp1 Binding Site in Vitro
Our in vivo footprinting and transient transfection experiments provided evidence for the involvement of the half-ERE/Sp1 binding site in mediating estrogen’s effects on the PR-A promoter. However, these studies did not allow us to identify proteins that interact with this DNA sequence. To begin to identify proteins that bind to this site, gel mobility shift assays were carried out with MCF-7 nuclear extracts. When 32P-labeled oligos, each containing the half-ERE/Sp1 binding site, were combined with nuclear extracts prepared from E2-treated MCF-7 cells, one major protein-DNA complex was formed (Fig. 4Go, lane 1). Since we anticipated that ER and Sp1 might bind to this region, antibodies to these proteins were included in separate binding reactions. The major protein-DNA complex was supershifted by the Sp1-specific antibody 1C6, which binds to Sp1, but does not cross-react with Sp2–4 (lane 2), suggesting that Sp1 was present in substantial amounts in our MCF-7 nuclear extracts and that it bound efficiently to the half-ERE/Sp1 binding site. In contrast, the major protein-DNA complex was not affected by the ER-specific antibody H222 (lane 3).



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Figure 4. Binding of MCF-7 Sp1 Protein to the Half-ERE/Sp1 Binding Site

32P-labeled oligos containing the half-ERE/Sp1 binding site were incubated with nuclear extracts from E2-treated MCF-7 cells. The ER-specific antibody H222 (ER Ab) or the Sp1-specific antibody IC6 (Sp1 Ab) was added to the binding reaction as indicated. The 32P-labeled oligos were fractionated on a nondenaturing gel and visualized by autoradiography.

 
Although our gel mobility shift experiments suggested that Sp1 was involved in regulation of the PR gene expression, they did not provide evidence that the ER was involved in formation of a protein-DNA complex. However, gel mobility shift experiments require the formation of stable protein-DNA complexes, which must be maintained during extended periods of electrophoresis. To determine whether a more transient or lower affinity interaction might occur between MCF-7 nuclear proteins and the ERE half-site and/or either one or both of the Sp1 binding sites, in vitro DNase I footprinting was carried out. DNA fragments (181 bp), each containing the half-ERE/Sp1 binding site and additional PR flanking sequence, were 32P-labeled on one end, incubated with increasing amounts of MCF-7 nuclear extract, and exposed to limited DNase I cleavage (Fig. 5Go, lanes 3–5 and 8–10). When DNA fragments that had been 32P-labeled on the coding strand were used, the proximal and distal Sp1 sites were partially protected by proteins present in the MCF-7 nuclear extracts (lanes 3–5). Quantitative analysis of the coding strand revealed slightly greater protection of the proximal Sp1 site than the distal Sp1 site. Although the ERE half-site was not protected, nucleotides within and immediately flanking the ERE half-site were hypersensitive to DNase I cleavage upon addition of increasing concentrations of nuclear proteins (lanes 3–5). When the noncoding DNA strand was labeled and used in in vitro footprinting experiments with MCF-7 nuclear extracts, the proximal Sp1 site was more extensively protected than the distal Sp1 site (lanes 8–10). As seen with the coding strand, hypersensitive sites were observed within and adjacent to the ERE half-site on the noncoding strand. Control lanes containing DNA fragments that had been exposed to dimethylsulfate (DMS) (lanes 1 and 6) or DNase I (lanes 2 and 7) in the absence of protein were included for reference. The enhanced protection of the Sp1 sites observed in our in vitro footprints in the presence of MCF-7 nuclear extracts was similar to the increased protection of the Sp1 sites in the endogenous gene upon E2 treatment of MCF-7 cells. The ERE half-site was not protected in our in vitro footprints as seen in the in vivo footprints, but rather displayed hypersensitivity to DNase I cleavage on both strands. Since DNase I hypersensitivity can result from binding of a protein to the major groove of the DNA helix, making the minor groove more accessible to DNase I cleavage (28 ), the hypersensitivity observed within and adjacent to the ERE could result from binding of a protein to the major groove in the region of the ERE.



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Figure 5. In Vitro Footprinting of the Half-ERE/Sp1 Binding Site with MCF-7 Nuclear Extracts

DNA fragments (181 bp) containing the half-ERE/Sp1 binding site were end-labeled on either the coding or noncoding strand and incubated with increasing concentrations of nuclear extract from E2-treated MCF-7 cells (lanes 3–5 and 8–10). The binding reactions were subjected to limited DNase I digestion, and the cleaved DNA fragments were fractionated on a denaturing gel. DNA fragments, which had been treated in vitro with either DMS (lanes 1 and 6) or DNase I (lanes 2 and 7), were included as references. The locations of the proximal Sp1 site (Sp1P), distal Sp1 site (Sp1D), and ERE half-site are indicated.

 
Purified Sp1 Binds to the Half-ERE/Sp1 Binding Site
Our antibody supershift experiments indicated that native Sp1 present in MCF-7 nuclear extracts was binding to the half-ERE/Sp1 binding site. However, the MCF-7 extracts used in these assays contained a complex combination of nuclear proteins. To determine whether Sp1 protein alone was capable of binding to the half-ERE/Sp1 binding site or whether other proteins present in MCF-7 nuclear extracts were required for Sp1 binding, gel mobility shift experiments were carried out with purified Sp1 protein. 32P-labeled oligos, each containing the half-ERE/Sp1 binding site, were incubated with increasing concentrations of purified Sp1 protein and fractionated on a nondenaturing acrylamide gel (Fig. 6Go, lanes 2–5). At the lowest Sp1 concentration used (1 ng), a single gel-shifted band was observed (<-1, lane 2). As increasing concentrations of Sp1 were added to the binding reaction, there was a dose-dependent increase in a second, higher mol wt complex (<-2, lanes 3–5). These experiments demonstrate that purified Sp1 was capable of forming a stable complex with the half-ERE/Sp1 binding site. Additional gel shift assays demonstrated that the more rapidly migrating Sp1-DNA complex had the same mobility as the complex formed with MCF-7 nuclear extracts (data not shown).



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Figure 6. Gel Mobility Shift Assay of half-ERE/Sp1 Binding Site-Containing Oligos and Purified Sp1 Protein

32P-labeled oligos containing the half-ERE/Sp1 binding site were incubated alone (lane 1) or with increasing concentrations of purified Sp1 protein (lanes 2–5) and fractionated on a nondenaturing gel. The locations of the more rapidly (<-1) and more slowly (<-2) migrating Sp1/DNA complexes are indicated.

 
It seemed likely that the formation of the higher order complex in our gel shift experiments represented the simultaneous binding of two Sp1 proteins to the two Sp1 sites and the more rapidly migrating complex represented Sp1 binding to one of the two Sp1 sites. To determine whether Sp1 was binding to one or both of the Sp1 sites and whether it displayed any preference in binding to the proximal or the distal Sp1 site, in vitro footprinting experiments were carried out. DNA fragments (181 bp), each containing the half-ERE/Sp1 binding site, were 32P-labeled on the coding strand and incubated with increasing concentrations of purified Sp1 protein. When 12.5 ng of purified Sp1 were included in the binding reaction, the proximal and distal Sp1 sites were protected (Fig. 7Go, lanes 3). Addition of 25 and 37.5 ng of purified Sp1 protein further enhanced protection of the two Sp1 sites (lanes 4 and 5). When DNA fragments labeled on the noncoding strand were incubated with increasing amounts of purified Sp1, the proximal Sp1 site was more protected than the distal Sp1 site (lanes 8–10). This preference for the proximal Sp1 site was also evident in the in vitro footprints of the noncoding strand in the presence of MCF-7 nuclear extracts (Fig. 5Go). Control lanes containing DNA fragments that had been exposed to DMS (Fig. 7Go, lanes 1 and 6) or DNase I (lanes 2 and 7) in the absence of proteins were included for reference. These data, combined with our gel mobility shift assays, support the idea that Sp1 binds first to the proximal Sp1 site and then to the distal Sp1 site.



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Figure 7. In Vitro Footprinting of the Half-ERE/Sp1 Binding Site with Purified Sp1

DNA fragments (181 bp) containing the half-ERE/Sp1 binding site and flanking regions were end-labeled on either the coding or noncoding strand and incubated with increasing concentrations of purified Sp1 protein (lanes 3–5 and 8–10). The binding reactions were subjected to limited DNase I digestion, and the cleaved DNA fragments were fractionated on a denaturing gel. DNA fragments that had been treated in vitro with either DMS (lanes 1 and 6) or DNase I (lanes 2 and 7) were included as references. The locations of the proximal Sp1 site (Sp1P), distal Sp1 site (Sp1D), and ERE half-site are indicated.

 
ER Enhances Sp1 Binding to the Half-ERE/Sp1 Binding Site
Our in vitro binding assays suggested that Sp1 was involved in regulating the PR gene, but left some question about the involvement of ER in this process. From previous studies examining ER-mediated transcription activation, it seemed possible that ER could increase transcription either directly by binding to the ERE half-site or indirectly by enhancing Sp1 binding (7 8 9 10 11 12 29 30 ). To determine whether ER affected protein-DNA complex formation, gel mobility shift assays were carried out. When a 32P-labeled oligo containing the half-ERE/Sp1 binding site was incubated with 3 ng of purified Sp1, a single gel-shifted band was formed (Fig. 8AGo, lane 1, Sp1->). When the amount of purified Sp1 protein was decreased to 0.25 ng, a faint gel-shifted band was barely visible (lanes 2 and 8). Addition of 150, 350, or 700 fmol of purified, unoccupied (lanes 3–7), or E2-occupied (lanes 9–13) receptor to 0.25 ng purified Sp1 protein elicited a dose-dependent increase in Sp1 binding. This ER-enhanced Sp1 binding was not due to an increase in protein concentration since all reactions contained the same amount of total protein. Incremental addition of ER also resulted in a dose-dependent increase in a more rapidly migrating protein-DNA complex. The ER-specific antibody H151 supershifted this more rapidly migrating complex (ER->) but did not affect the Sp1-DNA complex (lanes 6 and 12). The Sp1-specific antibody IC6 supershifted the Sp1-DNA complex but did not affect the more rapidly migrating ER-DNA complex (lanes 7 and 13). These findings demonstrate that ER enhances Sp1 binding and that both ER and Sp1 can bind directly to the half-ERE/Sp1 binding site.



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Figure 8. ER-Enhanced Binding of Sp1 to the Half-ERE/Sp1 Binding Site

32P-labeled oligos containing the half-ERE/Sp1 binding site were incubated with purified Sp1, ER, or Sp1 and ER and fractionated on nondenaturing gels. A, Binding reactions contained either 3 ng of Sp1 (lane 1) or 0.25 ng of Sp1 alone (lanes 2 and 8) or in combination with 150 (lanes 3 and 9), 350 (lanes 4 and 10), or 700 (lanes 5–7 and 11–13) fmol of unoccupied (-E2) or E2-occupied (+E2) ER. B, Binding reactions contained 350 (lane 1) or 700 (lanes 2–4) fmol of ER, 3 ng of Sp1 (lane 5), or 1 ng of Sp1 (lanes 6–9) and 350 (lane 6) or 700 (lanes 7–9) fmol of ER. The ER-specific antibody H151 (ER Ab) or the Sp1-specific antibody IC6 (Sp1 Ab) was added as indicated. The locations of the ER/DNA (<-ER), Sp1/DNA (<-Sp1), and ER/Sp1/DNA (<-ER/Sp1) complexes are indicated.

 
In addition to the major ER-DNA and Sp1-DNA complexes, three minor, higher order protein-DNA complexes, which we thought might reflect the formation of a trimeric ER-Sp1-DNA complex, were consistently observed in our gel shifts (Fig. 8AGo, lanes 5 and 11). To determine whether this was the case, 32P-labeled oligos containing the half-ERE/Sp1 binding site were incubated with ER in the absence and in the presence of Sp1. When ER, but not Sp1, was included in the binding reaction (Fig. 8BGo, lanes 1–4), the major ER-DNA complex was formed (<-ER) as well as two minor, higher order complexes, which most likely contained ER and Sf-9 proteins that copurified with the receptor. When 1 ng Sp1 and 350 or 700 fmol ER were included in the binding reaction (lanes 6–9), the major ER (<-ER) and Sp1 (<-Sp1) complexes and three minor, higher order complexes were formed. The one unique, higher order protein-DNA complex (lanes 6 and 7,<-ER/Sp1) formed in the presence of both ER and Sp1 was supershifted by ER- and Sp1-specific antibodies, demonstrating that both ER and Sp1 were present. A lane containing Sp1 alone was included as a reference (lane 5). These combined experiments suggest that ER and Sp1 can form a trimeric complex with DNA at the half-ERE/Sp1 binding site in the PR-A promoter.

To determine how ER affected Sp1 protection of the half-ERE/Sp1 binding site, in vitro DNase I footprinting experiments were carried out with purified ER and Sp1 proteins. When 15 ng of purified Sp1 were incubated with the 32P-labeled coding strand, the proximal and distal Sp1 sites were protected (Fig. 9Go, lane 3). Addition of 15 ng Sp1 and 0.33–1.3 pmol of purified ER incrementally enhanced the protection of both the proximal and distal Sp1 sites (lanes 4–6). As suggested from the gel mobility shift assays, the consensus ERE half-site was protected in the presence of higher ER concentrations (lane 6). When DNA fragments labeled with 32P on the noncoding strand were incubated with 15 ng of purified Sp1 and increasing concentrations of purified ER, enhanced protection of both the proximal and distal Sp1 sites and the half-ERE was observed (lanes 9–12). As seen in the in vitro footprints with MCF-7 nuclear extracts and with purified Sp1, the proximal Sp1 site on the noncoding strand was more extensively protected than the distal Sp1 site. The ERE half-site was partially protected on the noncoding strand. Control lanes containing DNA fragments that had been exposed to DMS (lanes 1 and 7) or DNase I (lanes 2 and 8) in the absence of proteins were included for reference.



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Figure 9. In Vitro DNase I Footprinting of the Half-ERE/Sp1 Binding Site with Purified Sp1 and ER

DNA fragments containing the half-ERE/Sp1 binding site and flanking regions were end-labeled on either the coding or noncoding strand and incubated with 15 ng of purified Sp1 protein (lanes 3–6 and 9–12) and 0.33, 0.66, or 1.3 pmol of purified ER (lanes 4–6 and 10–12). The binding reactions were subjected to limited DNase I digestion, and the cleaved DNA fragments were fractionated on a denaturing gel. DNA fragments that had been treated in vitro with either DMS (lanes 1 and 7) or DNase I (lanes 2 and 8) were included as references. The locations of the proximal Sp1 site (Sp1P), distal Sp1 site (Sp1D), and ERE half-site are indicated.

 
Sp1 Does Not Enhance ER Binding to the ERE Half-Site
Our binding assays indicated that ER greatly enhanced binding of Sp1 to the half-ERE/Sp1 binding site. To determine whether Sp1 influenced ER binding, gel mobility shift assays were carried out. When 32P-labeled oligos containing the half-ERE/Sp1 binding site were incubated with 150 fmol of purified ER, a single major gel shifted band was formed (Fig. 10Go, lane 1, <-ER). When the amount of purified ER protein was decreased to 50 fmol, a fainter gel shifted band was produced (lanes 2–6,<-ER). Addition of 0.25, 0.5, 1.5, or 3 ng of purified Sp1 protein (lanes 3–6, <-Sp1) elicited a dose-dependent increase in Sp1 binding. In contrast, incremental addition of Sp1 slightly decreased ER binding to the half-ERE/Sp1 binding site. These combined findings demonstrate that although ER greatly enhanced Sp1 binding, Sp1 did not enhance ER binding to the half-ERE/Sp1 binding site.



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Figure 10. Effect of Sp1 on ER Binding to the Half-ERE/Sp1 Binding Site

32P-labeled oligos containing the half-ERE/Sp1 binding site were incubated with 150 fmol (lane 1) or 50 fmol of purified ER (lanes 2–6) and 0.25, 0.5, 1.5, or 3 ng of purified Sp1 (lanes 3–6) and fractionated on nondenaturing gels. The locations of the ER-DNA (<-ER) and Sp1-DNA (<-Sp1) complexes are indicated.

 
Purified Sp1 and ER Bind Differentially to Wild- Type and Mutant Half-ERE/Sp1 Binding Sites
The in vitro footprinting experiments reproducibly suggested a preference of Sp1 for the proximal Sp1 site. To determine how each of the Sp1 sites and the ERE half-site contributed to protein-DNA complex formation, each of the individual elements was mutated and tested in gel mobility shift assays. Complementary oligos containing the wild-type half-ERE/Sp1 binding site (wt) or mutations in both Sp1 sites (mP/D), the distal Sp1 site (mD), the proximal Sp1 site (mP), or the ERE half-site (mE) were synthesized, annealed, and labeled with 32P. The labeled oligos were combined with purified Sp1 (Fig. 11Go, lanes 1–5) or purified Sp1 and ER (lanes 6–10) and fractionated on nondenaturing gels. Sp1 or Sp1 and ER bound effectively to the wt half-ERE/Sp1 binding site (lanes 1 and 6). As anticipated, Sp1 did not bind to the oligo containing mutations in both Sp1 sites, in the absence (lane 2) or in the presence of ER (lane 7). Sp1 alone or in combination with ER bound to oligos containing a mutation in one of the two Sp1 sites, but more protein-DNA complex was formed when the oligo contained an intact proximal Sp1 site (mD; compare lanes 3 and 8 with lanes 4 and 9). PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) analysis of four gel shift experiments demonstrated that there was a 30.8% (±7 SEM) increase in complex formation with the proximal Sp1 site compared with the distal Sp1 site. These findings corroborate the preferential binding of Sp1 to the proximal Sp1 site observed in the in vitro footprinting studies. The ability of ER to bind to oligos containing an intact ERE half-site (lanes 6–9), but not to an oligo containing a mutated ERE half-site (lane 10), further supports the idea that ER can bind to the ERE half-site. When the ERE half-site was mutated, increased Sp1-DNA complex formation was observed (lanes 5 and 10). The reason for this apparent increase in Sp1 binding is unclear, but it was a reproducible finding.



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Figure 11. Interaction of Purified Sp1 and ER with wt and Mutant Half-ERE/Sp1 Binding Sites

32P-labeled oligos containing the wild-type half-ERE/Sp1 binding site (wt), or mutations in both Sp1 binding sites (mP/D), the distal Sp1 binding site (mD), the proximal Sp1 binding site (mP), or the ERE half-site (mE) were incubated with 3 ng of purified Sp1 (lanes 1–5) or 3 ng of purified Sp1 and 0.33 pmol of purified ER (lanes 6–10). The 32P-labeled oligos were fractionated on a nondenaturing gel and visualized by autoradiography. The locations of the ER-DNA (<-ER) and Sp1-DNA (<-Sp1) complexes are indicated.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Sequence comparison of the PR gene from different species has been used to identify cis elements that are involved in estrogen-regulated transactivation. The rabbit PR gene contains an imperfect ERE, which overlaps with the translation start site and is capable of conferring estrogen responsiveness to a heterologous promoter in transient transfection assays (31 ). Although a similar sequence is present in the chicken PR gene (32 ), no homologous sequence has been identified in the human PR gene (24 ). A number of studies have suggested that ER and Sp1 may be involved in conferring estrogen responsiveness to the creatine kinase B (7 ), c-myc (8 ), retinoic acid receptor {alpha} (9 ), heat shock protein 27 (10 11 ), cathepsin D (12 ), and uteroglobin (13 ) genes. The identification of an ERE half-site adjacent to two Sp1 sites in the human PR gene (24 ) led us to investigate whether this region might be involved in conferring estrogen-responsiveness to the human PR gene. We initiated our studies by examining the endogenous PR gene in MCF-7 cells. Unlike transient transfection assays, which examine the ability of ER to activate transcription of synthetic promoters in supercoiled plasmids, our in vivo DNase I footprinting experiments allowed us to examine the endogenous PR gene as it exists in native chromatin and assess whether the half-ERE/Sp1 binding site might play a physiological role in gene expression. E2 treatment of MCF-7 cells did elicit more extensive protection of the half-ERE/Sp1 binding site than was observed in the absence of hormone. The enhanced protection of the half-ERE/Sp1 binding site seen after 72 h of hormone treatment, a time when PR mRNA and protein reach maximal levels (16 17 18 26 27 ), suggests that sustained protein-DNA interactions are required for maximal production of PR mRNA and protein. Furthermore, the ability of the half-ERE/Sp1 binding site to enhance transcription of a CAT reporter plasmid in the presence of E2 suggests that this region is involved in estrogen responsiveness of the PR-A promoter.

A Role for Sp1 in Regulating Expression of the PR Gene
Sp1 was originally described as a trans-acting factor that bound to a GC box (5'-GGGCGG-3') and activated transcription of the SV40 promoter (33 34 ). Subsequent comparison of numerous Sp1 binding sites led to the identification of a higher affinity, consensus Sp1 site, 5'-GGGGCGGGGC-3' (35 ), and the discovery that sequences that varied from this consensus sequence displayed decreased affinities for Sp1. While both of the Sp1 sites in the human PR half-ERE/Sp1 binding site contain the GC box motif, only the proximal Sp1 site contains the 10-bp consensus Sp1 sequence (Fig. 1Go). The increased affinity of Sp1 for the 10-bp proximal Sp1 site, when compared with the distal Sp1 site, was repeatedly observed in our in vitro footprinting assays and was most obvious on the noncoding strand (Figs. 5Go, 7Go, and 9Go). Gel mobility shift assays carried out with oligos containing mutations in the proximal or distal Sp1 site confirmed Sp1’s preference for the proximal Sp1 site (Fig. 11Go). Interestingly, the centers of the two GC boxes present in the half-ERE/Sp1 binding site are separated by 10 bp or one turn of the DNA helix (Sp1D +580 to +585, Sp1P +590 to +595). The periodicity of these elements could either favor interaction between adjacent DNA-bound proteins resulting in cooperative binding or sterically hinder binding of two Sp1 proteins. Our gel mobility shift and in vitro DNase I footprinting assays provided evidence for additive, not cooperative, binding of Sp1 to these sites and indicate that Sp1 binds first to the proximal Sp1 site and then to the distal Sp1 site.

A Role for ER in Regulating Expression of the PR Gene
One way that estrogen might affect PR gene expression is through direct binding of the receptor to the ERE half-site. The ERE was protected in our in vitro footprinting experiments with ER and Sp1, but not with Sp1 alone, demonstrating that the ER did bind to the ERE half-site. Likewise, gel mobility shift experiments carried out with purified ER alone or in combination with Sp1 indicated that the ER bound surprisingly well to the ERE half-site and formed a stable protein-DNA complex that was capable of withstanding the extensive periods of electrophoresis required for gel mobility shift experiments. Furthermore, the ERE half-site was protected in our in vivo footprinting experiments after treatment of MCF-7 cells with E2, suggesting that this element is involved in regulation of the endogenous gene. Although we were unable to detect protection of the ERE half-site in our in vitro binding assays using MCF-7 nuclear extracts, the level of ER in these extracts (0.42 fmol/µg protein) was significantly lower than the level present in an intact cell nucleus. Assuming a nuclear radius of 6 µm and 150,000 ER sites per cell (36 ), the ER concentration in an MCF-7 nucleus would be 273 nM. These ER concentrations are significantly higher than the 7–57 nM concentrations used in our in vitro binding assays and would most likely favor ER binding to the ERE half-site. The 10 bp separating the ERE half-site and the distal Sp1 binding site would place the ER on the same side of the DNA helix as the DNA-bound Sp1 proteins and could help to foster protein-protein interactions.

Estrogen could also modulate PR gene expression through ER-enhanced Sp1 binding. ER effectively enhanced Sp1 binding to the two Sp1 sites in the PR-A promoter and formed a trimeric complex with Sp1 and DNA in our in vitro binding assays. Direct ER-Sp1 interaction has also been documented in immunoprecipitation and glutathione-S-transferase (GST) pulldown experiments (11 29 ).

We have considered only ER{alpha} in our studies since MCF-7 cells express high levels of ER{alpha}, but do not express ERß (36 37 ). Although we have not ruled out the possibility that another nuclear protein might bind to the ERE half-site, the high levels of nuclear ER, the differential protection of the ERE half-site in the presence and absence of E2, and the demonstrated ability of ER to bind to the ERE half-site in vitro suggest that it is most likely the ER that interacts with this site in vivo and helps to regulate transcription of the PR-A promoter.

Regulation of the PR-A Promoter in MCF-7 Cells
Estrogen treatment of transfected cells resulted in a modest, but reproducible 1.7-fold increase in transcription of a plasmid containing the ERE/Sp1 binding site. Since estrogen treatment of MCF-7 cells results in a 2- to 10-fold increase in PR mRNA levels (16 17 18 ), it seems likely that the ERE/Sp1 binding site may play a significant role in mediating the estrogen responsiveness of this gene. However, the ERE/Sp1 site represents a small part of the complex PR promoter, which contains multiple regulatory elements. Unlike promoters that contain a palindromic ERE, transcription of the estrogen-regulated PR gene may require ER action at multiple cis elements. Preliminary experiments from our laboratory suggest that additional Sp1 and AP1 sites in the PR promoter may also be involved in estrogen-regulated gene expression (L. Petz and A. Nardulli, unpublished data). Thus, the cooperative action of the multiple sites within the PR promoter may be required for effective estrogen-regulated transcription.

Our studies support the idea that ER and Sp1 are involved in estrogen-regulated expression of the human PR-A promoter. The protection of nucleotides flanking the half-ERE/Sp1 binding site in our in vivo footprinting experiment suggests that other proteins are associated with the promoter and are involved in transcription activation. Interestingly, the E2-occupied ER, but not the unoccupied ER, interacts with a number of coactivator proteins, which participate in transcription activation and chromatin remodeling (38 39 40 41 42 43 44 45 46 ). The recruitment of these proteins to the DNA-bound, liganded receptor could account for protection of sequences flanking the half-ERE/Sp1 binding site and serve as the initiating event in the formation of an active transcription complex.

While models of DNA are typically drawn in a linear array, the packaging of DNA and protein into the nucleus of a cell requires tremendous compaction. This compaction could facilitate interaction between trans acting factors bound to more distant cis elements. Thus, the association of upstream activators, such as ER and Sp1, with factors bound to downstream elements could be fostered. In fact, both ER and Sp1 are known to directly associate with TFIID components. ER interacts with TATA binding protein (TBP), transcription factor IIB (TFIIB), and TBP-associated factor (TAF)II30 (47 48 49 ), and Sp1 interacts with TBP, TAFII130, and TAFII55 (50 51 52 53 ). The interaction of ER and Sp1 with TBP and its associated proteins could foster formation of a protein-protein network that helps to establish an active transcription complex. Furthermore, the E2-dependent recruitment of coactivators such as CBP/p300, which can function as a histone acetyltransferase (39 ), could help remodel chromatin in the PR-A promoter and enhance formation of an interconnected protein-protein and protein-DNA network involved in activation of the human PR gene.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
MCF-7 human breast cancer cells (54 ) were maintained in Eagle’s MEM containing 5% heat-inactivated calf serum. Cells were seeded in 10-cm plates and transferred to phenol red-free, serum-free improved MEM (55 ) 5 days before the experiments were conducted. Chinese Hamster Ovary (CHO) cells were maintained in DMEM/F12 supplemented with 5% charcoal dextran-stripped calf serum (27 ).

Oligonucleotides and Plasmid Constructions
The names and sequences of wild-type (wt) or mutant half-ERE/Sp1 binding sites are listed. Nucleotides that differ from the endogenous, wt half-ERE/Sp1 binding site are underlined.

ERE/Sp1 wt: 5'-GATCTAGGAGCTGACCAGCGCCGCCCTCCCCCGCCCCCGACCA-3'

and 5'-GATCTGGTCGGGGGCGGGGGAGGGCGGCGCTGGTCAGCTCCTA-3',

ERE/Sp1 mP/D: 5'-GATCTAGGAGCTGACCAGCGTTGTACTCCCTTGTACCCGACCA-3'

and 5'-GATCTGGTCGGGTACAAGGGAGTACAACGCTGGTCAGCTCCTA-3',

ERE/Sp1 mD: 5'-GATCTAGGAGCTGACCAGCGTTGTACTCCCCCGCCCCCGACCA-3'

and 5'-GATCTGGTCGGGGGCGGGGGAGTACAACGCTGGTCAGCTCCTA-3',

ERE/Sp1 mP: 5'-GATCTAGGAGCTGACCAGCGCCGCCCTCCCTTGTACCCGACCA-3'

and 5'-GATCTGGTCGGGTACAAGGGAGGGCGGCGCTGGTCAGCTCCTA-3',

ERE/Sp1 mE: 5'-GATCTAGGAGCTGATTAGCGCCGCCCTCCCCCGCCCCCGACCA-3'

and 5'-GATCTGGTCGGGGGCGGGGGAGGGCGGCGCTAATCAGCTCCTA-3'.

ERE/Sp1 wt oligos with BglII compatible ends were subcloned into the BglII-cut, dephosphorylated CAT reporter plasmid, TATA CAT (56 ), to create ERE/Sp1-TATA CAT. The ligated vector was transformed into the DH5{alpha} strain of Escherichia coli, sequenced, and purified on two cesium chloride gradients.

In Vitro and in Vivo Treatment of Genomic DNA
MCF-7 cells were exposed to ethanol vehicle or 1 nM E2 for 0, 2, or 72 h before DNase I treatment. Cells were permeabilized with 0.4% NP-40 and treated with 750 U DNase I/ml (Roche Molecular Biochemicals, Indianapolis, IN) for 3 min at 25 C. Isolation of genomic DNA was carried out as described by Mueller and Wold (25 ). The genomic DNA was purified, incubated with RNase A, resuspended in TE (10 mM Tris, pH 7.5, 1 mM EDTA) and stored at -20 C.

Naked genomic DNA was treated in vitro with DMS as described (25 ). In vitro DNase I-treated DNA was prepared by adjusting 100 µg of protein-free, RNase A-treated DNA to 175 µl with TE. DNA was incubated with 2.5 x 10-5 U DNase I for 5 min at 37 C. The reaction was stopped by the addition of 10 mM EDTA and processed as described for in vivo-treated genomic DNA.

In Vivo Footprinting
Ligation-mediated PCR (LMPCR) footprinting was carried out essentially as described by Mueller and Wold (25 57 ). Two micrograms of genomic DNA were subjected to LMPCR procedures using nested primers, which annealed to sequences upstream of the half-ERE/Sp1 binding site (+571 to +595) in the human PR gene. The primer sequences were: primer 1, 5'-TCCCCGAGTTAGGAGACGAGAT-3'; primer 2, 5'-CGCTCCCCACTTGCCGCTC-3'; and primer 3, 5'-GCTCCCCACTTGCCGCTCGCTG-3'. The annealing temperatures for the primers were 55 C, 62 C, and 69 C, respectively. The linker primers LMPCR 1 and LMPCR 2 described by Mueller and Wold (57 ) were also used, except that LMPCR 1 was modified by removing the two 5'-nucleotides to eliminate potential secondary structure.

In Vitro DNase I Footprinting
Primers, which annealed 88 bp upstream (primer 3) or 79 bp downstream (primer 4, 5'-TCGGGAATATAGGGGCAGAGGGAGGAGAA-3') of the half-ERE/Sp1 binding site, were subjected to 30 rounds of PCR amplification with 30 ng of the PR-(+464/+1105) CAT (24 ). Labeling of the coding and noncoding strands was carried out with 32P-labeled primer 3 or primer 4, respectively. The 181-bp singly end-labeled amplified fragments were fractionated on an acrylamide gel and isolated. End-labeled DNA fragments (100,000 cpm) containing the half-ERE/Sp1 binding site were incubated for 15 min at room temperature in a buffer containing 10% glycerol, 50 mM KCl, 15 mM Tris, pH 7.9, 0.2 mM EDTA, 1 mM MgCl2, 50 ng of poly dIdC, and 0.4 mM dithiothreitol (DTT) in a final volume of 50 µl with either 30–60 µg of MCF-7 nuclear extract, 12.5–37.5 ng of purified Sp1 protein (Promega Corp., Madison, WI), or 15 ng of purified Sp1 and 0.13–1.3 pmol of purified Flag-tagged ER, which had been expressed and purified as described by Kraus and Kadonaga (58 ). E2 (10 nM) was included in binding reactions containing the purified ER. BSA, ovalbumin, and KCl were added as needed to maintain constant protein and salt concentrations. When MCF-7 nuclear extracts were used, poly dI/dC was increased to 1 µg per reaction. RQ1 ribonuclease-free DNase I (1–2 U) (Promega Corp., Madison, WI) was added to each sample and incubated at room temperature for 0.75–8 min. The DNase I digestion was terminated by addition of stop solution (200 mM NaCl, 1% SDS, 30 mM EDTA, and 100 ng/µl tRNA). The DNA was phenol/chloroform extracted, precipitated, and resuspended in formamide loading buffer (59 ). Samples were fractionated on an 8% denaturing acrylamide gel. Radioactive bands were visualized by autoradiography and quantitated with a PhosphorImager and ImageQuant software (Molecular Dynamics, Inc.).

Gel Mobility Shift Assays
Gel mobility shift assays were carried out essentially as described previously (60 61 ). 32P-labeled (10,000 cpm) half-ERE/Sp1-containing wt or mutant oligos were incubated for 15 min at room temperature in a buffer containing 10% glycerol, 50 mM KCl, 15 mM Tris, pH 7.9, 0.2 mM EDTA, 1 mM MgCl2, 50 ng of poly dI/dC, and 0.4 mM DTT in a final volume of 20 µl with either 20 µg of MCF-7 nuclear extract, 0.25–3 ng of purified Sp1 protein, or 0.25 ng of purified Sp1 and 50–700 fmol of purified ER. E2 (10 nM) was included in all binding reactions containing ER unless otherwise indicated. BSA, ovalbumin, and KCl were added as needed to maintain constant protein and salt concentrations. When MCF-7 nuclear extracts were used, the nonspecific DNA for each reaction included 1 µg of salmon sperm DNA, and poly dI/dC was increased to 2 µg. For antibody supershift experiments, the Sp1-specific monoclonal antibody, 1C6 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or the ER-specific monoclonal antibody H222 or H151 (kindly provided by Drs. Geoffrey Greene, The University of Chicago, Chicago, IL and Dean Edwards, University of Colorado Health Science Center, Denver, CO, respectively) was added to the protein-DNA mixture and incubated for 10 min at room temperature. Low ionic strength gels and buffers were prepared as described previously (59 ). Radioactive bands were visualized by autoradiography and quantitated with a PhosphorImager and ImageQuant software (Molecular Dynamics, Inc.).

Transient Transfections
CHO cell transfections were performed using the calcium phosphate method (62 ). Crystals were formed in the presence of 3 µg of the indicated CAT reporter, 200 ng of the ß-galactosidase vector pCH110 (Pharmacia Biotech, Piscataway, NJ), 5 ng of the human ER{alpha} expression vector pCMVhER (63 ), and 4.8 µg of pTZ18U and incubated with CHO cells for 16 h followed by a 2 min 20% glycerol shock. Cells were maintained in media containing ethanol vehicle or 10 nM E2 for 24 h. Protein concentration was determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Inc. Hercules, CA) with BSA as a standard. Mixed-phase CAT assays were performed using 35 µg protein as previously described (64 ). The ß-galactosidase activity was determined at room temperature as previously described (65 ) and used to normalize the amount of CAT activity in each sample.


    ACKNOWLEDGMENTS
 
We are very grateful to Jennifer Wood for providing MCF-7 nuclear extracts and purified ER; W. Lee Kraus and James T. Kadonaga for providing the viral stock used in ER production; Geoffrey Greene and Dean Edwards for the ER antibodies; and Pierre Chambon for the PR-(+464/+1105) CAT. We also thank Jongsook Kim for providing technical expertise and Robin Dodson for helpful comments during the preparation of this manuscript.


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

This research was supported by United States Army Medical Research and Materiel Command Grant DAMD17–96-1–6267 and NIH Grants R29 HD-31299 and DK-53884 (to A.M.N.). Postdoctoral support for L. Petz was provided by USMRMC Grant DAMD17–97-1–7201 and NIH Reproductive Training Grant PHS 2T32 HD-0728–19.

Received for publication September 7, 1999. Revision received April 6, 2000. Accepted for publication April 7, 2000.


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