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and Activating Protein-1 Mediate Estrogen Responsiveness of the Progesterone Receptor Gene in MCF-7 Breast Cancer Cells
Department of Molecular and Integrative Physiology, University of Illinois, Urbana, Illinois 61801
Address all correspondence and requests for reprints to: Ann M. Nardulli, Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, Illinois 61801. E-mail: anardull{at}life.uiuc.edu.
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Introduction |
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and ß with estrogen response elements (EREs) as the initiating event involved in estrogen-regulated gene expression (7, 8). However, it has become apparent that, in addition to mediating its effects through the ERE, the ER can interact with other DNA-bound transcription factors to influence transcription activation. For example, the ER cooperates with the activating protein-1 (AP-1) proteins Fos and Jun to confer estrogen responsiveness to simple, heterologous promoters (9, 10, 11) and to the ovalbumin (12), c-fos (13), collagenase (9), and IGF-I (14) genes. Likewise, the ER mediates estrogen’s effects through Sp1 recognition sites in a number a genes including the progesterone receptor (PR) gene (15, 16, 17, 18, 19, 20, 21). Previous studies have demonstrated that transcription of the PR gene is induced by E2 in MCF-7 human breast cancer cells. PR mRNA and protein levels increase 2- to 10-fold reaching maximal levels after 72 h of E2 treatment (22, 23, 24). Like ER, two distinct PR forms, the 120-kDa PR-B and the 94-kDa PR-A, are expressed in a tissue-specific manner (25, 26, 27). Kastner et al. (28) have hypothesized that two discrete promoters, A and B, are responsible for the production of PR-A and PR-B, respectively. In spite of the fact that both promoter A (+464 to +1105) and promoter B (-711 to +31) confer estrogen responsiveness to a heterologous promoter (28, 29), neither promoter contains an ERE.
We previously demonstrated that an ERE half-site and two adjacent Sp1 sites in the human PR gene were involved in E2-mediated activation of the PR gene (15). However, the ability of this half ERE/Sp1 site to mediate transcription activation was modest suggesting that other regions of the PR gene must also be involved in mediating estrogen’s effects. In this study, we have identified a putative AP-1 site at +90 in the PR gene that binds Fos and Jun in vitro and functions as an estrogen-inducible enhancer in transient transfection assays. The ER binds to an ERE half-site adjacent to the +90 AP-1, enhances binding of Fos and Jun to oligos containing the +90 AP-1 sequence, and is required for estrogen-mediated transactivation. More importantly, ER, Fos, and Jun are present at this AP-1 site in the endogenous PR gene in MCF-7 cells.
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Materials and Methods |
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strain of Escherichia coli, sequenced, and purified on two cesium chloride gradients.
Cell culture
The osteosarcoma cell line U2-OS was maintained in MEM with 10 µg/liter phenol red and 15% heat-inactivated fetal calf serum. Media were changed to phenol red-supplemented MEM with 5% charcoal dextran-stripped calf serum 3 d before transfection and then transferred to phenol red-free MEM supplemented with 5% charcoal dextran-stripped calf serum 2 d before transfections. MCF-7 cells were maintained as previously described (15).
Genomic Southern analysis
MCF-7 cells were exposed to ethanol vehicle or 10 nM E2 for 24 h, harvested, and washed twice with cold PBS. Pelleted cells were resuspended in hypotonic buffer (10 mM Tris, pH 7.5; 15 mM NaCl; 60 mM KCl; 1 mM EDTA; 0.2% Nonidet P-40; 0.15 mM spermine; 0.5 mM spermidine; 5% sucrose; and 1 mM phenylmethylsulfonylfluoride), homogenized, layered onto a sucrose cushion (10 mM Tris, pH 7.5; 15 mM NaCl; 60 mM KCl; 1 mM EDTA; 0.15 mM spermine; 0.5 mM spermidine; 10% sucrose; and 1 mM phenylmethylsulfonylfluoride), and centrifuged at 1600 x g for 10 min. The nuclear pellet was resuspended in wash buffer (10 mM HEPES, pH 7.5; 15 mM NaCl; 60 mM KCl; 0.15 mM spermine; and 0.5 mM spermidine) and centrifuged at 400 x g at 4 C. The pelleted nuclei were resuspended in wash buffer and treated with 0, 40, 80, 120, 200, or 240 U deoxyribonuclease (DNase) I/ml (Roche Molecular Biochemicals, Indianapolis, IN) in DNase dilution buffer (10 mM HEPES, pH 7.5; 50 mM NaCl; 20 mM MgCl2; 10 mM CaCl2; and 0.1 mg/ml BSA) for 3 min at 4 C. Lysis solution (0.1 M EDTA, pH 8.0; 1% sodium dodecyl sulfate (SDS); 0.4 mg proteinase K/ml) was added to the mixture and incubated for 2 h at 55 C. DNA was incubated with ribonuclease A, phenol chloroform extracted, ethanol precipitated, resuspended in TE (10 mM Tris, pH 7.5; 1 mM EDTA) and stored at -20 C. Ten micrograms of purified genomic DNA were digested with BamHI (2 U/µg DNA) overnight at 37 C. The DNA was phenol chloroform extracted, precipitated, and fractionated on a 1.5% TBE agarose gel. The gel was rinsed with distilled water, soaked in denaturing solution (0.5 M NaOH, 1.5 M NaCl) at room temperature, rinsed with distilled water and soaked in neutralization buffer (1.5 M NaCl; 0.5 M Tris, pH 7.2; 0.001 M EDTA) at room temperature for 30 min. The DNA was transferred to a Duralon-UV membrane (Stratagene, La Jolla, CA) in 20x SSC, and UV cross-linked. The membrane was prehybridized with 10 ml of Rapid-hyb buffer (Amersham Pharmacia Biotech, Piscataway, NJ) for 2 h at 65 C. A random primed, 300 bp 32P-labeled PCR product (-714 to -436) was amplified using the pBL3 B promoter (kindly provided by Pierre Chambon, Strasbourg, France) as a template. The membrane was incubated with the 32P-labeled DNA overnight at 65 C and washed twice with 2x SSC and 0.5% SDS and once with 0.2x SSC and 0.5% SDS at room temperature for 15 min. Radioactive bands were visualized by autoradiography.
Transient cotransfections
A total of 4 x 105 U2-OS cells were plated in each well of a 24-well plate the day before transfection. Transfections were carried out using lipofectin (Life Technologies, Inc., Grand Island, NY) as described previously (31) with 7.5 (see Figs. 2A
and 6) or 3.0 (Fig. 2B) µg of the indicated reporter plasmid and 150 ng of the ß-galactosidase vector CMVß-gal (Promega Corp., Madison, WI). One hundred nanograms of the human ER expression vector CMV5hER (32) were added as indicated. Cells were maintained in media containing ethanol vehicle or 10 nM E2 for 24 h. ß-Galactosidase activity was determined at 37 C as previously described (33) and used to normalize the amount of CAT activity in each sample. CAT assays were carried out as described (31).
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Preparation of MCF-7 nuclear extracts and purified Fos and Jun
MCF-7 cells were exposed to 10 nM E2 for 72 h, harvested, and pelleted. Pelleted cells were resuspended in 400 µl of TEG (50 mM Tris, pH 8.5; 7.5 mM EDTA; 10% glycerol), homogenized, and centrifuged at 10,200 x g for 10 sec. The nuclei were resuspended in TEG containing 0.5 mM KCl and incubated at 4 C for 20 min. The nuclear extract was centrifuged at 150,000 x g for 30 min, aliquoted, assayed for protein concentration, and stored at -80 C. The expression and purification of Fos and Jun have been described (36, 37, 38, 39).
Chromatin immunoprecipitation (ChIP) assays
MCF-7 cells were exposed to ethanol vehicle or 100 nM E2 for 24 h and ChIP assays were carried out essentially as described in Upstate Biotechnology, Inc. Tech Note 50105 (Upstate Biotechnology, Inc., Waltham, MA). The ER-specific antibody sc-8002 or sc-8005 (Santa Cruz Biotechnology), Jun-specific antibody, sc-45, or Fos-specific antibody, sc-7202, was used for immunoprecipitation of protein-DNA complexes. PCR primers (5'-GGCTTTGGGCGGGGCCTCCCTA-3' and 5'-TCTGCTGGCTCCGTACTGCGG-3') flanking the +90 AP-1 site produced 234-bp DNA fragments. As a negative control, primers that annealed from -711 to -693 and from -458 to -436 of the PR gene were used to produce a 275-bp fragment. This region of the PR gene does not contain an identifiable ERE, Sp1, or AP-1 site.
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Results |
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, lanes 2–6 and 8–12). Interestingly, both of these DNase I-cleaved products were present in the absence and presence of E2, suggesting that these regions were accessible to DNase I in the absence and in the presence of hormone. Nuclei that had not been exposed to DNase I (lanes 1 and 7) produced a prominent 1.5-kb band, which resulted from BamHI cleavage of the genomic DNA. Another minor, lower molecular weight band, which resulted from a cross-reacting sequence (data not shown), was sometimes visible.
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The +90 AP-1 site confers estrogen responsiveness to a heterologous promoter
Interestingly, the putative +90 AP-1 site is present in the +31 to +464 region of the PR gene between promoters A (+464 to +1105) and B (-711 to +31) defined by Kastner et al. (28). This region has not previously been implicated in conferring estrogen responsiveness to the PR gene. To determine whether this putative AP-1 site could confer estrogen responsiveness, CAT reporter plasmids containing a TATA sequence alone (TATA-CAT) or in combination with the +90 AP-1 site (+90 AP-1 TATA-CAT) were tested for their abilities to function as transcriptional enhancers. U2-OS cells were transfected with a CAT reporter plasmid and a ß-galactosidase expression vector in the absence or in the presence of a human ER expression vector. When the ER expression vector was used (+ER), exposure of transfected cells to E2 resulted in a significant increase in CAT activity when the reporter plasmid contained the +90 AP-1 site (Fig. 2A
, +90 AP-1 TATA-CAT). Although CAT activity was distinctly different in vehicle- and E2-treated cells when the reporter plasmid contained the +90 AP-1 site, hormone treatment did not affect CAT activity when the reporter plasmid contained the TATA sequence alone (TATA-CAT). The estrogen-induced increase in CAT activity was dependent on the presence of ER because no difference in CAT activity was observed when the ER expression vector was not included (-ER). These findings demonstrate that the +90 AP-1 site confers estrogen responsiveness to a heterologous promoter and may assist in mediating estrogen’s effects on the endogenous PR gene.
The importance of the +90 AP-1 site was also assessed in transient transfection assays in the context of a 1.5-kb region of the PR gene (-711 to +817). When this 1.5-kb region of the PR gene was included in the TATA-CAT reporter plasmid, transcription was increased 72-fold in the presence of E2 (Fig. 2B, -711/+817 TATA-CAT). When the +90 AP-1 site in the 1.5-kb region of the PR gene was mutated from TGAGTGA to CGAGCTG (-711/+817 mut +90 TATA-CAT), the E2-induced increase in CAT activity was dramatically reduced. The residual E2-induced CAT activity was most likely due to the participation of other cis elements in the -711/+817 region of the PR gene such as an ERE half-site and adjacent Sp1 sites, which have previously been implicated in ER-mediated transactivation (15). This E2-dependent transcription was observed only when an ER expression vector was used in transfection assays (data not shown). These combined transfection studies document the importance of the +90 AP-1 site in mediating estrogen responsiveness of the PR gene.
Proteins present in E2-treated MCF-7 nuclear extracts bind to the AP-1 site
To determine whether Fos and Jun might help mediate estrogen’s effects on PR gene expression in MCF-7 cells, gel mobility shift assays were carried out with 32P-labeled oligos containing the +90 AP-1 site and nuclear extracts from E2-treated MCF-7 cells, which contain both Fos and Jun (data not shown). Proteins present in the MCF-7 nuclear extracts bound to the AP-1 site (Fig. 3, lane 2, 
). One protein-DNA complex was disrupted by the Fos-specific antibody sc-52, which recognizes c-Fos, but does not cross-react with Fos B, Fra-1, or Fra-2 (lane 3). This protein-DNA complex was also disrupted by the Jun-specific antibody sc-45, which recognizes c-Jun, but does not cross-react with Jun B or Jun D (lane 4). These data indicate that Fos and Jun present in MCF-7 nuclear extracts bind to the +90 AP-1 site. The abilities of both Fos and Jun antibodies to disrupt the protein-DNA complex suggests that these proteins bind to the AP-1 site as a heterodimer. The ER-specific antibody H151 (lane 5) did not affect formation or migration of the protein-DNA complex, suggesting that the ER was not present. The failure of ER to participate in protein-DNA complex formation may result from the low levels of ER in our binding reactions compared with the levels of ER in intact MCF-7 nuclei (15).
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, lane 2, ). As increasing concentrations of Fos and Jun were added to the binding reaction (lanes 3–6), the intensity of the gel-shifted band increased. The protein-DNA complex was diminished by inclusion of either the Fos- specific antibody sc-52 (lane 7) or the Jun-specific antibody sc-45 (lane 8). These findings demonstrate that purified Fos and Jun were capable of forming a stable heterodimeric complex with the +90 AP-1 site. Parallel experiments performed with Fos or Jun alone indicated that neither Fos nor Jun alone bound to the AP-1 site (data not shown).
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, Fos/Jun, lane 1, 
). When the concentration of purified Fos and Jun was decreased to 5 nM, a faint gel shift band was observed (lane 2). Addition of increasing concentrations of purified E2-occupied ER to 5 nM purified Fos and Jun resulted in a dose-dependent increase in complex formation (Fos/Jun , lanes 3–5), indicating that ER enhanced the binding of Fos and Jun to the +90 AP-1 site. Addition of purified ER to the binding reaction also resulted in the appearance of a second, more rapidly migrating protein-DNA complex (ER , lanes 3–5), which was supershifted by an ER-specific antibody (lane 6, ER). Other nonspecific complexes were sometimes formed with increasing amounts of ER; however, the migration of these complexes was unaffected by addition of an ER-specific antibody.
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, ER , lanes 1–4). When parallel experiments were carried out with oligos containing the +90 AP-1 site and mutations in the adjacent putative ERE half-site (TAGAC), hereafter referred to as mut ERE +90 AP-1 site, no complex was observed (lanes 5–8), indicating that the ER bound the wild-type, but not the mutant ERE half-site.
It seemed possible that ER binding to the ERE half-site might influence binding of Fos and Jun to the adjacent AP-1 site. To determine whether this was the case, 32P-labeled oligos containing the mut ERE +90 AP-1 site were incubated with 50 nM Fos and Jun and fractionated on a nondenaturing acrylamide gel. A single faint, protein-DNA complex was formed (Fig. 5C, lane 1, Fos/Jun, ), which was significantly attenuated when compared with Fos/Jun binding to the wild-type + 90 AP-1 site (Fig. 5A, lane 1). When the concentration of purified Fos and Jun was decreased to 5 nM, no gel shift band was formed (Fig. 5C, lane 2). Addition of increasing concentrations of ER to 5 nM Fos and Jun failed to enhance Fos and Jun binding (lanes 3–5), indicating that mutation of the ERE half-site dramatically decreased the ability of Fos and Jun to bind to the +90 AP-1 site (compare panels A and C, lanes 3–5). It should be noted that the gels shown in panels A and C were carried out in parallel with the same binding reactions and were run on the same gel. Thus, differences in binding of Fos, Jun, and ER cannot be attributed to interexperimental variation and must be due to changes in the ERE half-site sequence. These results suggest that the integrity of the imperfect ERE half-site is not only required for ER binding but is also needed for effective binding of Fos and Jun to the adjacent +90 AP-1 site. This could reflect the ability of ER to stabilize Fos/Jun binding to the adjacent +90 AP-1 site or the ability of the sequence flanking the +90 AP-1 site to influence Fos/Jun binding. Previous studies have demonstrated that the DNA sequence flanking an AP-1 site can influence Fos and Jun binding (12).
The ERE half-site helps confer estrogen responsiveness
To determine whether this putative ERE half-site was involved in conferring estrogen responsiveness, CAT reporter plasmids containing the +90 AP-1 site and either a wild-type (+90 AP-1 TATA-CAT) or mutated (mut ERE +90 AP-1 TATA-CAT) ERE half-site were used in transient transfection assays. U2-OS cells were transfected with a CAT reporter plasmid and a ß-galactosidase expression vector in the absence or in the presence of a human ER expression vector. When the ER expression vector was used (+ER), exposure of transfected cells to E2 resulted in a 2.9- or 2.4-fold increase in CAT activity when the reporter plasmid contained the +90 AP-1 site with a wild-type or a mutated ERE half-site, respectively (Fig. 6). The levels of transcription observed with the wild-type and mutant ERE half-sites were statistically different (P < 0.04), indicating that the ERE half-site is important for effective activation of this estrogen-responsive reporter plasmid. The increase in CAT activity was dependent on the presence of ER because no difference in CAT activity was observed when the ER expression vector was not included (-ER).
ER, Fos, and Jun are present at the +90 AP-1 site in native chromatin
Our transient cotransfection assays documented that the +90 AP-1 site could confer estrogen responsiveness to a heterologous promoter, that this site played an important role in conferring estrogen responsiveness to a 1.5-kb region of the PR gene, and that the ER was required for E2-mediated transactivation. Furthermore, our gel mobility shift assays demonstrated that Fos and Jun bound to the + 90 AP-1 site and that the ER bound directly to an ERE half-site and enhanced the binding of Fos and Jun in vitro. To determine the physiological relevance of these findings in an in vivo setting, we examined the association of ER, Fos, and Jun with the +90 AP-1 site in the endogenous PR gene in the absence and in the presence of hormone using ChIP assays. MCF-7 cells were treated with ethanol vehicle or E2 for 24 h, exposed to formaldehyde to cross-link proteins and DNA, and sonicated to fragment the chromatin. An ER-, Fos-, or Jun-specific antibody was used to immunoprecipitate the protein-DNA complexes. The immunoprecipitated DNA was used as a template to generate 234-bp DNA fragments containing the +90 AP-1 site. A discrete amplified product was obtained when MCF-7 cells were treated with E2 and an ER-, Fos-, or Jun-specific antibody was used for immunoprecipitation (Fig. 7, +90 AP-1, lanes 6, 8, 10). Likewise, genomic DNA that had not been subjected to immunoprecipitation was readily amplified (+90 AP-1, Input, lanes 1 and 2). In contrast, no amplified product was obtained when cells were maintained in a hormone free environment and immunoprecipitated with an ER-, Fos-, or Jun-specific antibody (+90 AP-1, lanes 5, 7, 9). Similarly, no DNA product was obtained when no antibody (-Ab) was used for immunoprecipitation regardless of hormone exposure (+90 AP-1, lanes 3 and 4). To ensure that the antibodies used precipitated only ER-, Jun-, or Fos-containing protein-DNA complexes, we determined whether a region of the PR gene that contains neither an ERE nor an AP-1 site (-711 to -436) could be immunoprecipitated and amplified. In fact, no amplified product was observed with any of the antibodies used (-711/-436, lanes 5–10) even though the -711 to -436 region of the PR gene was readily amplified when genomic DNA was used as a control (-711/-436, Input, lanes 1 and 2). Similarly, no DNA product was obtained when no antibody (-Ab) was used for immunoprecipitation regardless of hormone exposure (-711/-436, lanes 3 and 4). These data demonstrate that the ER, Fos, and Jun were associated with the +90 AP-1 site in the endogenous PR gene and that the association of these proteins with this region was dependent upon exposure of the MCF-7 cells to E2.
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Discussion |
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Role of Fos and Jun in regulating PR gene expression
The AP-1 family is comprised of a number of proteins including Fos and Jun (42, 43). Fos and Jun heterodimerize and bind to AP-1 sites in target genes (44, 45, 46). Although Fos/Fos homodimers do not bind to the consensus AP-1 site, Jun/Jun homodimers can bind to and activate transcription through AP-1 sites (47). These Jun/Jun homodimers, however, are typically less potent in activating transcription than Fos/Jun heterodimers. Purified Fos and Jun, but neither Fos nor Jun alone, formed detectable complexes with the +90 AP-1 site. Furthermore, Fos- and Jun-specific antibodies disrupted the protein-DNA complexes formed when MCF-7 nuclear extracts were combined with the +90 AP-1 site. The fact that Fos/Jun heterodimers present in MCF-7 nuclear extracts interact with this AP-1 site in vitro suggests that these two proteins play a role in regulating expression of the human PR gene in MCF-7 cells in vivo. In fact, the ability of Fos- and Jun-specific antibodies to immunoprecipitate this region of the PR gene containing the + 90 AP-1 site demonstrates that these proteins are involved in regulating expression of the endogenous gene in MCF-7 cells and that their association with this region is dependent upon E2 treatment.
Estrogen-regulated expression of the PR gene
One way that E2 might alter expression of an estrogen-responsive gene is through direct binding of ER to an ERE. Although no EREs are present in the human PR gene, our gel mobility shift assays demonstrated that the ER was capable of binding to an imperfect ERE half-site adjacent to the +90 AP-1 site and that the integrity of this ERE half-site was required for effective binding of Fos and Jun and for efficient gene expression. We have also demonstrated that transcription of a promoter containing the PR +90 AP-1 site was enhanced by E2 treatment only in the presence of ER, suggesting that both hormone and the receptor are required for estrogen responsiveness of the PR gene. Thus, the ER and the ERE half-site play important roles in activation of the human PR gene.
Another way that E2 might regulate expression of an estrogen-responsive gene is through ER-enhanced binding of a transcription factor to its cognate recognition site. In fact, ER effectively enhanced the binding of Fos and Jun to the + 90 AP-1 site but only when the wild-type ERE half-site was present. More importantly, E2 treatment of MCF-7 cells, which is required for activation of the PR gene, promoted the recruitment of ER, Fos, and Jun to the +90 AP-1 site in the endogenous PR gene. The association of ER with this site in vivo could be mediated by direct interaction with the imperfect ERE half-site, by interaction with the amino terminus of Jun (10) and/or simultaneous interaction of coactivators with the ER and AP-1 proteins (11). Our studies suggest that estrogen’s effects on PR gene expression are derived from the ability of the receptor itself to play a role in forming an active transcription complex with the Fos/Jun heterodimer at the +90 AP-1 site. This is, to our knowledge, the first demonstration that an AP-1 site residing in the human PR gene plays a role in mediating estrogen-regulated gene expression.
In this study, we have provided evidence that the + 90 AP-1 site helps to confer estrogen responsiveness to the PR gene, but we believe that other sites are also involved in estrogen-regulated expression of the PR gene. In addition to influencing binding and activity of the +90 AP-1 site in the PR gene, the ER enhances Sp1 binding to its recognition site and binds directly to an adjacent ERE half-site in the PR promoter A (15). These combined studies suggest that the ER has direct and indirect effects on formation of an active transcription complex and that multiple transcription factors including ER, Fos, Jun, and Sp1 must act in concert to confer estrogen responsiveness to the PR gene in MCF-7 human breast cancer cells.
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Acknowledgments |
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Footnotes |
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Abbreviations: AP-1, Activating protein-1; CAT, chloramphenicol acetyl transferase; ChIP, chromatin immunoprecipitation; DNase, deoxyribonuclease; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; PR, progesterone receptor; SDS, sodium dodecyl sulfate.
Received April 3, 2002.
Accepted for publication September 4, 2002.
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and ß with estrogen response elements. Mol Cell Endocrinol 181:151–163[CrossRef][Medline]
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