Identification of a Novel Transferable cis Element in the Promoter of an Estrogen-Responsive Gene that Modulates Sensitivity to Hormone and Antihormone

Monica M. Montano, W. Lee Kraus1 and Benita S. Katzenellenbogen

Department of Molecular and Integrative Physiology (M.M.M., W.L.K., B.S.K.) Department of Cell and Structural Biology (B.S.K.) University of Illinois, Urbana, Illinois 61801


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The estrogen receptor (ER) is a ligand-regulated transcription factor that acts at the promoters of estrogen-regulated genes to modulate their expression. In the present study, we examined three estrogen-regulated promoters, namely the rat progesterone receptor gene distal (PRD) and proximal (PRP) promoters and the human pS2 gene promoter, and observed marked differences in their sensitivity to stimulation by estrogen and repression of estrogen-stimulated transcription by antiestrogen (AE)-occupied ER. ER-containing MCF-7 human breast cancer cells were transfected with reporter gene constructs containing estrogen response elements upstream of the three gene promoters. In this system, PRP and PRD showed similar dose-response curves for stimulation by estradiol whereas pS2 was activated by even lower concentrations of estradiol. By contrast, PRD was much less sensitive to repression of estrogen-stimulated activity by all AEs studied, relative to the PRP and the pS2 promoters. Using deletion and mutational analysis, we have identified a transferable cis element at -131 to -94 bp in PRD that is involved in modulating the sensitivity of this promoter to both estrogens and AEs. The element reduced the magnitude of estrogen-stimulated activity, enhanced the ability of AEs to repress estrogen-stimulated activity, and elicited similiar effects when transferred to the promoter of another estrogen-responsive gene. Thus, removal of this region from PRD further accentuated the insensitivity of this promoter to AE while enhancing its sensitivity (both EC50 and fold induction) to estrogen. Gel mobility shift assays showed that proteins from nuclear extracts of MCF-7 cells interact with this element and that the binding of these proteins is inversely correlated with the transcriptional effectiveness of the ER. The findings demonstrate that a specific cis element from the promoter of an estrogen-responsive gene can alter the transcriptional activity of hormone and antihormone-occupied receptor bound at its response element near the promoter. Such ligand response modulatory elements, and changes in the levels and activity of factors that bind to such elements, may underlie the different sensitivities of steroid hormone-regulated genes to both hormones and antihormones.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroid hormones, such as estrogen, modulate gene expression via intracellular receptors that belong to a large superfamily of hormone-regulated transcription factors. In the case of the estrogen receptor (ER), the binding of estrogen initiates a process of receptor activation that includes the high-affinity binding of ER to specific DNA sequences, termed estrogen response elements (EREs). The interaction of ER with EREs results in the modulation of specific gene expression, through which the physiological actions of estrogens are manifested (for reviews, see Refs. 1–5). The regulatory actions of estrogens on gene expression, which are generally stimulatory, can be inhibited by potent synthetic ER antagonists (6) termed antiestrogens (AEs).

The promoters of many known estrogen-regulated genes are complex, with binding sites for other transcription factors in addition to ER. Positive and negative interactions between ER and these transcription factors, which may be promoter- or cell-specific, provide an important step at which ER function may be regulated (reviewed in Refs. 1, 4, and 5). A number of studies from this laboratory and others have demonstrated the significance of promoter and cell context in modulating responses to both estrogens and AEs (7, 8, 9).

In the present study, we observed marked differences in the sensitivities of three estrogen-regulated promoters to repression by AEs, suggesting the involvement of promoter-specific factors capable of modulating the activity of the ER. Using several approaches, we have identified a transferable cis element in the rat progesterone receptor (PR) gene distal promoter that is involved in modulating promoter sensitivity to both estrogens and AEs. Gel mobility shift assays have been used to show that proteins from nuclear extracts of MCF-7 human breast cancer cells interact with this ligand response modulatory element (LRME) and that the binding of these proteins is inversely correlated with the transcriptional effectiveness of ER. Our results demonstrate that cis-acting elements in the promotor region of estrogen-responsive genes can alter the transcriptional activity of estrogen- and AE-occupied ER bound at its response element near a promoter. Such ligand response modulatory elements may be broadly applicable in the actions of many nuclear receptors in which gene-specific modulation of hormonal induction is known, but the underlying basis is poorly understood.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Examination of the Differential Sensitivity of Several Estrogen-Stimulated Promoters to Repression by AEs
As shown in Fig. 1Go, we analyzed three estrogen-regulated promoters [the PR gene distal and proximal promoters (PRD and PRP) and the promoter of the human pS2 gene; pS2] for their relative sensitivity to the stimulatory actions of estrogen and the repressive actions of AEs. We previously cloned the 5'-flanking region of the rat PR gene and demonstrated the presence of two promoters, a distal promoter (-131 to +65; PRD) and a proximal promoter (+461 to +675; PRP) (10), and we have shown that these promoters are functionally distinct with respect to activation by ER-dependent pathways (11). The PR distal and proximal promoters control production of the B and A isoforms of the PR (ca. 120- and 90-kDa, respectively), and the pS2 promoter regulates production of a growth factor-like secreted protein whose function is not completely known (12).



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Figure 1. Differential Sensitivity of Three Promoters to the Inhibitory Actions of AEs

MCF-7 cells were transfected with CAT reporter constructs containing two consensus EREs upstream of the pS2 promoter [-90 to +10; (ERE)2-pS2-CAT], the proximal promoter of the rat PR gene [-131 to +65; (ERE)2-PRP-CAT], or the distal promoter of the rat PR gene [+461 to + 675; (ERE)2-PRD-CAT], and a ß-galactosidase expression plasmid, used as an internal control to correct for transfection efficiency, as described in Materials and Methods. The cells were then treated for 24 h with the estrogen E2 (10-9 M), or the AE ICI 164,384 (ICI, 5 x 10-7 M) or LY 117018 (LY, 10-6 M) alone or in the combinations as indicated. Cell extracts were prepared and analyzed for CAT activity as described in Materials and Methods. The activity for each construct was expressed as a percent of the activity observed with E2 treatment alone, which is set at 100%. Each bar represents the mean of three or more separate determinations + SEM. The numbers above the E2 bar show the fold induction observed with E2 alone for each of the three promoter constructs.

 
For the studies in Fig. 1Go, MCF-7 human breast cancer cells, which contain high levels of endogenous ER, were transfected with chloramphenicol acetyltransferase (CAT) reporter constructs containing two consensus EREs upstream of the pS2, PRP or PRD promoters. Extracts from the cells were analyzed for CAT activity after treatment with the lowest maximally stimulatory concentration of estradiol (E2; 10-9 M) in the absence or presence of a 500- or 1000-fold excess of the AEs, ICI 164,384 (ICI) or LY 117018 (LY), respectively. As shown in Fig. 1Go, E2 stimulated large (i.e. 50- to 130-fold) increases in the activity of the three promoter-reporter gene constructs. The AEs (ICI and LY) alone evoked essentially no activity, and they were able to repress greater than 90% of the E2-stimulated CAT activity from either the pS2 or PRP promoter-containing reporters. Of note, the AEs were substantially less effective at repressing E2-stimulated activity from the PRD-containing reporter (Fig. 1Go; and further investigated in Fig. 3Go below).



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Figure 3. Dose-Dependent Stimulatory Effects of E2 (Panel A) and Dose-Dependent Inhibitory Effects of ICI on E2 (10-9 M)-Stimulated CAT Activity (Panel B) Using the Three Estrogen-Responsive Promoter-Reporter Constructs

Constructs are defined in Figs. 1Go and 2Go. The truncated PRD is denoted PRD, B/N and lacks the -131 to -94 XmnI/BsmI fragment of the PRD promoter. Each value represents the mean of three or more separate determinations ± SEM.

 
Characterization of a Region of the PRD Promoter that Modulates Sensitivity to Estrogen and AE: Deletion and Mutational Analyses
We further analyzed PRD to identify region(s) involved in modulating the sensitivity of the promoter to the suppressive effects of AE on E2-stimulated activity. Reporter constructs containing two consensus EREs upstream of the full-length PRD, or deletion mutants of PRD, were analyzed for stimulation by E2 and repression by ICI. The results are shown in Fig. 2AGo. Deletion of the -131 to -94 XmnI/BsmI fragment from PRD (to generate a truncated promoter denoted PRD, B/N, since this truncated promoter contains the region -94 to +65, which spans from the BsmI to the NheI restriction sites) resulted in approximately 2-fold higher induction by E2 than was observed with the full-length promoter (Fig. 2AGo; compare line 2 with line 1). Surprisingly, we also observed a 2-fold decrease in the sensitivity of the deleted promoter to the inhibitory actions of ICI relative to the full-length promoter (Fig. 2AGo). This indicates, as shown in Fig. 3Go also (see below), that the -131 to -94 region plays a role in modulating the sensitivity of PRD to both estrogens and AEs and that in its absence (as in PRD,B/N), PRD gene responsiveness to estrogen and AE is even more discordant than that of PRP and pS2.



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Figure 2. Identification of a Region of PRD Involved in ER-Ligand Sensitivity

Panel A, Estrogen stimulation and AE (ICI) repression were examined using (ERE)2-PRD-CAT or CAT reporter constructs containing two EREs upstream of PRD deletion mutants (lines 1–5), or CAT reporter constructs containing the five natural ERE-containing estrogen-responsive regions of the PR gene linked together and placed upstream of PRD (line 6), which were transfected into MCF-7 cells as described in the legend of Fig. 1Go. Differential responsiveness of the reporter constructs to E2 (10-9 M) and to repression of E2-stimulated activity by ICI (5 x 10-7 M) was monitored. The magnitude of transactivation of the full-length PRD in response to E2 was set at 100%. Percent repression with ICI indicates the percent inhibition of E2-stimulated activity observed for each reporter construct upon cotreatment with 10-9 M E2 and 5 x 10-7 M ICI. Each value represents the mean of three or more separate determinations ± SEM. Panel B, Mutagenesis of the region from -131 to -80 encompassing the XmnI/BsmI fragment of PRD results in differential responsiveness to E2 (10-9 M) and repression of E2-stimulated activity by ICI (5 x 10-7M). Shown is the nucleotide sequence of the -131 to -80 region of the PRD promoter. The mutated nucleotides are indicated by the boxed regions in mutants 1 to 6. The CCAAT motif, Sp1-binding site, and putative binding site for CTF/NFI are also indicated. CAT reporter constructs containing two EREs upstream of the mutated PRD were examined for E2 responsiveness and for suppression of E2-mediated transactivation by ICI as described in the legend of Fig. 1Go. The magnitude of transactivation of wild type PRD in response to E2 was set at 100%. Percent repression with ICI indicates the percent inhibition of E2-stimulated activity observed for each reporter construct upon cotreatment with 10-9 M E2 and 5 x 10-7 M ICI. Each value represents the mean of three or more separate determinations ± SEM.

 
Further deletion of the PRD promoter to -67 resulted in a substantial loss of estrogen-inducible activity (line 3). Deletion from the 3'-end of PRD (the +25 to +65 region) similarly reduced promoter activity (line 4 vs. line 1), and further deletion to +1 almost fully destroyed PRD activity (line 5), as expected. We also tested PRD in the context of the natural estrogen-responsive sequences of the PR gene (line 6). With the ERE-like sequences contained in the five estrogen-responsive fragments of the rat PR gene linked together and placed upstream of PRD [5E-PRD-CAT (11)], we observed good stimulation by E2 [50% of the magnitude observed with (ERE)2-PRD-CAT, line 1] and the same poor sensitivity to repression by ICI (Fig. 2AGo, line 6, only 60% repression) as seen with 2ERE-PRD-CAT (line 1). This indicates that the unusual resistance of PRD to AE inhibition is a function of the 5'-flanking region to -131 and not the nature of the estrogen-response element regions.

Mutagenesis of the -131 to -84 region of PRD (Fig. 2BGo) identified a nucleotide sequence that appears to be involved in conferring differential sensitivity to stimulation by estrogen and repression by AEs. Introduction of mutations at -115 to -110 of the PR gene distal promoter (Mut3) increased the magnitude of the response to E2 (2-fold) and decreased the ability of ICI to suppress E2-mediated transactivation with respect to the wild type promoter construct, reproducing what was observed upon deletion of the -131 to -94 region of PRD (Fig. 2AGo, line 2). Mutations in the nucleotide sequence corresponding to the putative CTF/NF-1 site (Mut 4 and Mut 5) decreased E2-mediated transactivation from PRD to 40–50% of the wild type promoter, suggesting an involvement of CTF/NF-1 nuclear factors in estrogen regulation of PRD, possibly similar to that observed previously for the vitellogenin B1 gene (13). Mutations at other sites within the -131 to -84 region (Mut 1, 2, and 6) had relatively little effect on the response to E2 or ICI.

Analysis of the Relationship between Estrogen Stimulation of the Three Promoters and the Sensitivity of the Three Promoters to AE Repression
We performed E2 and AE (ICI) dose-response experiments using the estrogen-responsive reporter constructs containing the three different promoters (PRD, PRP, and pS2). We also assessed whether the decreased sensitivity of the -131 to -94 deleted PRD (i.e. the PRD,B/N promoter) to AE was attributable to the greater sensitivity of PRD, B/N to stimulation by E2 relative to PRD.

The PRP and PRD gene promoter constructs showed similar dose-response curves for stimulation by E2 (Fig. 3AGo), with half-maximal stimulation at approximately 3 x 10-11 M E2. The pS2 promoter-containing construct [(ERE)2-pS2-CAT] showed a 2- to 3-fold greater maximal CAT activity, and half-maximal activity required 5- to 10-fold less E2 (~5 x 10-12 M E2, Fig. 3AGo). The pS2 and PRP promoter constructs were equally and highly sensitive to suppression of E2-stimulated CAT activity by the AE ICI, whereas the PRD promoter was much less sensitive to ICI suppression over the entire concentration range tested (Fig. 3BGo), consistent with the data shown in Fig. 1Go, in which only a single concentration of ICI was used to inhibit E2 activation. Thus, promoters that have similar sensitivities to the stimulatory actions of estrogens can have very different sensitivities to the inhibitory actions of AEs.

Shown in Fig. 3Go, panels A and B, is our observation that deletion of the -131 to -94 portion of the PRD promoter [to give (ERE)2-PRD,B/N-CAT] resulted in a 3-fold increase in the magnitude of CAT activity in response to E2 (Fig. 3AGo) and also resulted in a reduced sensitivity to suppression by ICI relative to that shown by the intact PRD promoter construct [(ERE)2-PRD-CAT] (Fig. 3BGo). In addition, the truncated promoter construct [(ERE)2-PRD,B/N-CAT] required 10-fold less E2 (i.e. ~3 x 10-12 M E2) for half-maximal activity compared with the PRD-containing reporter [(ERE)2-PRD-CAT] (Fig. 3AGo).

It is of note that although the PRD,B/N and the pS2 promoter constructs required similar E2 concentrations for half-maximal activity, they had very different dose-response curves for suppression by ICI (Fig. 3BGo). These results illustrate the lack of correlation between the estrogen and AE sensitivities of a particular promoter and suggest that the decreased sensitivity of PRD,B/N to repression by ICI relative to PRD was not related to its increased sensitivity to the stimulatory actions of E2.

Our initial expectation in deletion and mutagenesis studies in the PRD promoter was that we would identify a region conferring the resistance that this promoter shows to AE antagonism. We failed to find such a region as far as we were able to study through deletions and mutations. Although this aspect merits further study, we have found that further deletions (Fig. 2Go, lines 3–5) reduced estrogen responsiveness of the promoter, complicating such an approach. On the other hand, in the PRD gene, we have made the unusual observation that a small region in PRD has a strong modulatory effect on estrogen and AE responsiveness, and its presence confers higher estrogen sensitivity and higher AE repression, even though overall the PRD is less estrogen and AE responsive than other genes such as pS2 and PRD. Therefore, we investigated this modulatory element further.

Evaluation of the -131 to -94 Region of the PRD Promoter as a Transferable cis Element
To determine whether the -131 to -94 XmnI/BsmI fragment of PRD had the properties of a cis element, one or two copies of the fragment were cloned 50 bp upstream of the EREs in both the (ERE)2-PRD-CAT and (ERE)2-PRD,B/N-CAT reporter constructs (Fig. 4Go). In the context of the full- length PRD, which contains the -131 to -94 region in its natural location, the presence of additional XmnI/BsmI (i.e. -131 to -94) fragments resulted in no substantial change in magnitude of E2-stimulated activity or ICI repression (Fig. 4Go, lines 1–3). Deletion of the -131 to -94 fragment from PRD caused a doubling of the level of induction by E2 and a reduction in the magnitude of repression by ICI (Fig. 4Go, line 4), as noted earlier in Figs. 2Go and 3Go. The addition of one XmnI/BsmI fragment 50 bp upstream of the two EREs in (ERE)2-PRD,B/N-CAT reduced E2-inducibility by 50% with no change in AE repression (Fig. 4Go, line 5 vs. line 4). Two XmnI/BsmI fragments had the same effect on the level of E2 induction and gave a greater (67%) repression by the AE ICI (Fig. 4Go, line 7), as seen with the intact PRD (Fig. 4Go, line 1). The effect was not seen with the mutated (Mut 3) form of the XmnI/BsmI fragment (Fig. 4Go, line 6). Therefore, the effect of the -131 to -94 fragment was to reduce E2 stimulation and increase ICI repression.



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Figure 4. The -131 to -94 Region of PRD Acts as a Transferable cis Element

CAT reporter constructs containing two EREs upstream of either PRD (-131 to +65 bp) or the truncated PRD (-94 to +65 bp) denoted PRD,B/N [lines 1–7] or pS2 (-90 to +10 bp) [lines 8–10] with one or two copies of the -131 to -94 XmnI/BsmI fragment of PRD cloned 50 bp upstream of the EREs, were examined for E2 responsiveness and for repression of E2-stimulated CAT activity by ICI in MCF-7 cells as described in the legend of Fig. 1Go. For line 6 and line 10, one copy of the -131 to -94 XmnI/BsmI fragment containing the mutated nucleotides in mut3 (see Fig. 2BGo) was cloned upstream of ERE-containing PRD,B/N or pS2, respectively. The magnitude of transactivation of wild type PRD or wild type pS2 (without an XmnI/BsmI fragment cloned upstream of the ERE) was set at 100%. The percent repression with ICI indicates the percent inhibition of E2-stimulated activity observed for each construct upon cotreatment with 10-9 M E2 and 5 x 10-7 M ICI. Each value represents the mean of three or more separate determinations ± SEM.

 
The activity of the -131 to -94 fragment was also observed in the context of the promoter of another estrogen-responsive gene. When the fragment was cloned upstream of the EREs in the (ERE)2-pS2-CAT reporter construct, E2-stimulated CAT activity was reduced to approximately 30% when compared with (ERE)2-pS2-CAT lacking the fragment (Fig. 4Go, lines 8–9). ICI suppression, which in (ERE)2-pS2 itself was greater than 90%, remained strong and unaffected, as expected (since ICI repression was complete and could not be increased further). This effect on E2 stimulation was specific for the intact -131 to -94 XmnI/BsmI fragment and was not observed when the XmnI/BsmI fragment contained the Mut3 mutations (Fig. 4Go, line 10). Thus, the -131 to -94 XmnI/BsmI fragment satisfies two criteria of a regulatory cis element in that it is positionally independent and is transferable, being active in the context of a heterologous promoter, in this case, the promoter of another estrogen-responsive gene.

Analysis of the Effects of the -131 to -94 Region of the PRD Promoter on Another Mediator of ER Action
The preceding studies examined the effects of the -131 to -94 region of PRD on the actions of a typical positive regulator of ER function, namely E2, and a typical fully negative regulator of ER function, namely the AE ICI. Since the ligand trans-hydroxytamoxifen (TOT) is a partial agonist/antagonist that can exhibit agonistic activity in certain promoter contexts (5, 6, 7, 8, 9), we wanted to determine whether the -131 to -94 region of PRD could also modulate the agonistic effect of TOT.

As shown in Fig. 5Go, we examined the ability of the -131 to -94 region of PRD to reduce the agonistic actions of TOT on different promoters, similar to the way it reduced the agonistic actions of E2 as described above. Reporter constructs lacking the -131 to -94 XmnI/BsmI fragment from PRD [i.e. (ERE)2-PRD,B/N-CAT and (ERE)2-pS2-CAT] showed dose-dependent increases in CAT activity in response to treatment with TOT (Fig. 5Go, panels A and B) that were greater in magnitude than that of constructs containing the -131 to -94 XmnI/BsmI fragment [(ERE)2-PRD-CAT, Fig. 5AGo]. In addition, deletion of the -131 to -94 fragment from PRD resulted in a promoter construct in which the EC50 was lowered about 10-fold, from 10-10 M for (ERE)2-PRD-CAT to approximately 10-11 M for (ERE)2-PRD,B/N-CAT. Also, addition of the XmnI/BsmI fragment to PRD,B/N (to give X/B-(ERE)2-PRD,B/N) reduced TOT stimulation back to that of the intact PRD construct (Fig. 5AGo). The same effect was seen with a different promoter (the pS2 gene promoter, Fig. 5BGo), in which addition of the XmnI/BsmI fragment [i.e. X/B-(ERE)2-pS2-CAT] resulted in a great reduction in the response to TOT, whereas addition of a mutated XmnI/BsmI fragment [X/Bmut3(ERE)2-pS2-CAT] resulted in virtually no change in the response to TOT (Fig. 5BGo). Thus, the -131 to -94 region of PRD reduced the ability of ER to respond positively to at least two different types of stimulatory signals, namely E2 and TOT acting as an estrogen agonist.



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Figure 5. The Effects of the -131 to -94 Region of PRD on the Estrogen Agonist Activity of TOT

The (ERE)2-PRD-CAT, (ERE)2-PRD,B/N-CAT, (ERE)2-PRP-CAT, or (ERE)2-pS2-CAT reporters were examined for responsiveness to the stimulatory (agonistic) actions of TOT (panels A and B) using MCF-7 cells as described in Materials and Methods. The activity for each construct is expressed as a percent of the maximal stimulation observed with 10-9 M E2. Each value represents the mean of three or more separate determinations ± SEM.

 
Interaction of MCF-7 Cell Factors with the -131 to -94 Region of PRD
The identification of a region of PRD (i.e. the -131 to -94 region) that can alter the sensitivity of the promoter to the stimulatory actions of estrogens and the inhibitory actions of AEs suggested the presence of a specific trans-acting factor(s) that could interact with this region. One major band (indicated in Fig. 6BGo) was detected in gel mobility shift assays using extracts from MCF-7 cells and a radiolabeled double-stranded oligomer containing the -131 to -94 sequence (shown in Fig. 6AGo). The band was competed by a 50-fold or 25-fold excess amount of unlabeled oligomer (Fig. 6BGo, lanes 7 and 11 vs. no competitor, lanes 6 and 10), but not by an excess of unlabeled -131/-94 oligonucleotide with a 6-bp mutation from -115 to -110 (mut3) (Fig. 6BGo, lane 8), indicating that the protein-DNA interaction producing the band was specific. Since a portion of the -131 to -94 sequence shows some homology to an NF-1 binding site (see Fig. 2BGo), an unlabeled double-stranded oligonucleotide containing an NF-1 binding site was also tested for its ability to compete for binding to the -131 to -94 oligomer. However, the complex was not competed by the NF-1 oligomer (not shown).



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Figure 6. Analysis of Protein Interactions with the -131 to -94 Region of PRD

A, The sequence of the coding strand of the -131 to -94 region of PRD and of the double- stranded oligomer used in the gel mobility shift assay. B, Gel mobility shift assays were performed using a double-stranded oligomer containing the -131 to -94 sequence of PRD and extracts from MCF-7 cells as described in Materials and Methods. With extracts treated with E2 (10-8 M, lane 1), or ICI (10-7 M, lane 2), or TOT (10-7 M, lane 3) for 15 min on ice; with extract + 200-fold excess unlabeled GRE (lane 4); with extract + 200-fold excess unlabeled mutated ERE (lane 5); extract alone (lane 6); with extract + 50-fold excess unlabeled -131 to -94 double stranded oligo (lane 7); with extract + 50-fold excess unlabeled -131 to -94 double-stranded oligo with a 6-bp mutation from -115 to -110 (mut3, lane 8); with extract + 50-fold excess unlabeled ERE (lane 9); extract alone (lane 10); with extract + 25-fold excess unlabeled -131 to -94 double-stranded oligo (lane 11); with extract + 25-fold excess unlabeled ERE (lane 12); no extract (lane 13). The positions of the shifted complex and the free probe are indicated. The autoradiograph is representative of three separate experiments.

 
Interestingly, although the -131 to -94 sequence shares no homology with an ERE, the band was competed by a 25-fold excess of unlabeled ERE (Fig. 6BGo, lane 12) and more fully by a 50-fold excess of unlabeled ERE (lane 9), but was not competed by 200-fold excess mutated ERE (lane 5) or consensus glucocorticoid response element (GRE) (lane 4). The band was not supershifted in the presence of an anti-ER antibody (not shown), suggesting that ER was not present in the complex. These results suggest that a protein in the complex is also capable of interacting with ERE or with ER when it is bound to its response element in a manner that disrupts the binding of the labeled oligo. Because ER is present in the MCF-7 cell nuclear extract, this protein may be titrated away from the complex upon the addition of excess competing ERE.

The effect of ligand treatments on the formation of the DNA-protein complex was also examined (Fig. 6BGo). There was no difference in complex formation using nontreated (lanes 6 and 10) vs. ICI-treated cell extracts (Fig. 6Go, lane 2). Notably, using cell extracts treated with E2, there was a marked decrease in the intensity of the shifted complex (lane 1). Cell extracts treated with TOT (lane 3) showed a gel shift pattern similar to, but slightly less strong than, that observed with ICI treatment. Therefore, differential binding of factors to the XmnI/BsmI fragment may occur in the presence of estrogens vs. AEs, and disappearance of the DNA-protein complex is correlated with the presence of transcriptionally productive, E2-liganded ER.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Differential Sensitivity of Estrogen-Stimulated Promoters to the Inhibitory Actions of AEs
The experiments described herein have demonstrated the differential sensitivity of a number of estrogen-regulated promoters, namely PRD, PRP and the pS2 promoter, to the actions of estrogens and AEs. In general, there was no correlation between the sensitivity of a given promoter to stimulation by estrogens when compared with its sensitivity to inhibition by AEs. For example, although the PRD and the pS2 promoters required relatively comparable levels of E2 for half-maximal stimulation, they showed markedly different dose-response profiles to the inhibitory actions of AEs. These findings suggest that the magnitude of estrogen responsiveness of a particular promoter is intrinsic to the nature of the promoter and that promoter responsiveness to the actions of estrogen- and AE-occupied ERs are separable. Furthermore, they implicate the involvement of inhibitory cis elements and promoter-specific factors acting to modulate the response of each promoter to different ER-ligand complexes. These findings are consistent with earlier reports in which it has been noted that reporter constructs containing EREs upstream of different promoters are differentially activated by estrogen in transient transfection assays, even when other experimental variables remain constant (10, 14), and with increasing evidence for promoter-specific actions of estrogens and AEs (5, 7, 8, 9, 14, 15).

Identification of an Inhibitory cis Element that Modulates the Sensitivity of Promoters to Estrogen- and AE-Occupied ER
Our search for ligand response modulatory elements began with our observation that the PRD promoter showed reduced sensitivity to suppression by the AE-ER complex relative to the two other promoters, PRP and pS2, examined. Deletion and mutational analyses led to the identification of a region in PRD that, in fact, made the PRD and a different estrogen-responsive (pS2) promoter more sensitive to inhibition by AE. Although these studies have allowed us to identify this novel element, which directs the AE sensitivity that PRD does have, it is evident that the reduced AE sensitivity of PRD overall must derive from activities from other portions of the promoter, which may normally act in concert with this element. Further analysis of PRD would be of interest but may be complicated by our observation that more extensive deletions in the 5'-flanking region reduced activity altogether.

The identified element in PRD had the following properties: 1) it reduced the magnitude and sensitivity of estrogen-stimulated activity, 2) it enhanced the ability of AEs to repress estrogen-stimulated activity, and 3) it elicited similiar effects when transferred to the promoter of another estrogen-responsive gene. This LRME appears to have no clear homology to previously identified cis elements. Gel mobility shift assays showed that a cellular factor or factors were capable of binding to the element. Although we know very little about the nature of these factors, changes in the level or activity of these trans-acting factors would be predicted to play important roles in the gene-selective actions of hormone- and antihormone-receptor complexes.

Relevant to our findings are reports from the Simons’ laboratory (16, 17) of a cis-acting glucocorticoid modulatory element that, like the element we identify here in the PR gene, alters the sensitivity of the tyrosine aminotransferase (TAT) gene to glucocorticoid and to mixed agonist/antagonist antiglucocorticoids. The element differs, however, from the one we have identified in that it is located much further away from the promoter (3646 bp upstream of the start of TAT gene transcription). In addition, our LRME reduces the magnitude of E2 stimulation or TOT agonism and increases the EC50 for E2 stimulation or TOT agonism, while the glucocorticoid-modulatory element enhances the sensitivity of the TAT gene to glucocorticoid (lower EC50, i.e. left-shifted dose-response curve) and confers greater agonistic activity with partial agonist/antagonist antiglucocorticoids. However, the magnitudes of the shift in the dose-response curves (~10-fold) and the maximum activity levels (~2- to 3-fold) effected by the glucocorticoid-modulatory element and our LRME are very similar.

Of note, PRD is a TATA-less promoter. However, the reduced AE sensitivity and the activity of the -131/-94 element is not exclusive to TATA-less promoters. For example PRP, which is also TATA-less, shows strong sensitivity to AEs. Furthermore, the -131/-94 element can be transferred to the pS2 promoter, which is TATA-containing, and elicits the same activity.

Implications for Gene-Specific Regulation by Estrogens and AEs
Our results suggest that the sensitivity of a given promoter to the stimulatory actions of estrogens is not necessarily correlated with its sensitivity to the inhibitory actions of AEs. Furthermore, we have demonstrated that the presence of a modulatory cis element in the promoter region of a gene can dramatically influence the response of that promoter to agonist- and antagonist-occupied receptor. Ligand response-modulatory elements, such as we have identified in the PRD promoter, may participate in regulating the activity of different estrogen-responsive genes by altering the pharmacology of estrogen and AE ligands that regulate these genes. They may thus be important in selectively modulating the properties of gene induction by estrogen agonists and antagonists and may underlie the known differences in dose-response curves for estrogen induction of different genes (5).

A BLAST search for this 38-bp cis element sequence in other genes revealed a related sequence (26-bp sequence, 84% identity) in the vinculin gene. Interestingly, vinculin, which encodes an actin-binding cytoskeletal protein, is also known to be under estrogen regulation (18). Thus, this sequence may potentially influence estrogen and AE sensitivity of several genes.

Our findings add to the growing list of modulators of ER activity. Modulators at almost every step in the process of transcriptional activation by ER have been identified: the type of ligand, receptor phosphorylation (19, 20, 21), the sequence of the estrogen response element (Refs. 22 and 23 for reviews), coactivator proteins (such as TIF-1, SRC-1, SPT-6, and others) (24, 25, 26, 27, 28, 29), some other nuclear hormone receptors (30, 31, 32, 33), and chromatin structure (34, 35). The identification of so many potential modulators of ER activity suggests that transcriptional activation by ER is not a simple process and that there are many checkpoints in the process suitable for regulation.

The modulatory cis element that we have identified, which is capable of increasing the sensitivity of a promoter to the inhibitory actions of AEs, is especially interesting in light of the therapeutic uses of AEs in the treatment of breast cancer. A detailed understanding of the mechanisms by which this element and the factors that bind to it alter responsiveness to AEs may assist ultimately in the development of more effective therapeutic agents. In addition, since the ER is a member of a large superfamily of structurally and functionally related ligand-activated transcription factors, it is likely that similar cis elements, as identified previously in the glucocorticoid-regulated TAT gene (16, 17), will be found to modulate the sensitivity of genes regulated by other steroid receptors, and thyroid and retinoic acid receptors, to their agonist and antagonist ligands.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reagents and Radioisotopes
Cell culture media and antibiotics were purchased from GIBCO (Grand Island, NY). Calf serum was from Hyclone Laboratories (Logan, UT) and FCS from Sigma Chemical Company (St. Louis, MO). 35S- and 32P-radiolabeled nucleotides and [dichloroacetyl-1,2-14C]-chloramphenicol (50–60 Ci/mmol) were from Dupont/NEN Research Products (Boston, MA). Custom oligonucleotides were purchased from National Biosciences Inc. (Plymouth, MN). DNA restriction and modifying enzymes were from New England Biolabs (Beverly, MA), GIBCO/Bethesda Research Laboratory (Gaithersburg, MD), and U.S. Biochemicals (Cleveland, OH). DNA sequencing reagents were from U.S Biochemicals. E2 was from Sigma. The AEs ICI 164,384 and TOT were kindly provided by Alan Wakeling (Zeneca Pharmaceuticals, Macclesfield, U.K.). The AE LY 117,018 was kindly provided by Eli Lilly & Co. (Indianapolis, IN). All general reagents were of molecular biology grade and were purchased from Sigma Chemical Co., U.S. Biochemicals, or Fisher Scientific (Houston, TX).

Plasmid Construction and Mutagenesis
All cloning was done using standard techniques (36, 37). When necessary to make termini compatible, 3'- and 5'-overhangs generated by restriction digestion were blunted with T4 DNA polymerase and the Klenow fragment of Escherichia coli DNA polymerase, respectively. The insertion of double-stranded oligonucleotides and the deletion of DNA fragments were confirmed by dideoxy chain termination DNA sequencing. Other manipulations were confirmed by restriction digest analyses.

The construction of pTZ-TK-CAT, PRD-CAT, PRP-CAT, and (ERE)2-PRP-CAT (10), and 5E-PRD-CAT (11), as well as that of pS2-CAT (38), has been described previously. PRD,B/N-CAT was constructed by releasing and blunting the BsmI/NheI fragment from the rat PR genomic clone EE(3.1)3Z (10) and cloning it into SalI/BglII-digested pTZ-TK-CAT. (ERE)2-PRD, B/N-CAT and (ERE/HindIII)2-PRD-CAT were made by annealing the single-stranded oligomers 5'-AATTAGTCAGGTCACAGTGACCTGATC-3' and 5'-AATTGATCAGGT-CACTGTGACCTGACT-3' and cloning two copies of the resultant double-stranded oligomer into the HindIII sites of PRD,B/N-CAT and PRD-CAT, respectively. (ERE)2-pS2-CAT was made by annealing the single-stranded oligomers 5'-GATCCAAAGTCAGGTCACAGTGACCTGATCAAAGA-3' and 5'-GATCTCTTTGATCAGGTCACTGTGACCTGACTT-TG-3' and cloning two copies of the resultant double-stranded oligomer into the BamHI site of pS2-CAT. (ERE/HindIII)2-pS2-CAT was made by replacing the BamHI/NcoI fragment from (ERE/HindIII)2-PRD-CAT with the BamHI/NcoI fragment from pS2-CAT.

(X/B)N-(ERE)2-PRD-CAT and (X/B)N-(ERE)2-PRD, B/N-CAT were made by annealing the single-stranded oligomer 5'-TTTTCTTCTCGAAGTCTGATGTTCCAGGTGGAATGCC-3' with its complement and cloning one or two copies of the resultant double-stranded oligomer into EagI-digested and blunted (ERE/HindIII)2-PRD-CAT and (ERE/HindIII)2-PRD, B/N-CAT, respectively. (X/B)2-(ERE)2-pS2-CAT was made by replacing the BamHI/NcoI fragment from (ERE)2-PRD-CAT with the BamHI/NcoI fragment from pS2-CAT.

Six reporter constructs, each containing 6-bp mutations introduced sequentially from the -131 to -84 region of the rat PR gene distal promoter, were constructed by site-directed mutagenesis (39) with modifications (40). The EcoRI fragment of (ERE/HindIII)2-PRD-CAT was first inserted into the EcoRI site of Bluescript II SK+ (Stratagene, La Jolla, CA) to make (ERE/HindIII)2-PRD-BSK+. Mutagenic oligonucleotides were then annealed to single-stranded DNA generated using the f1 origin of replication in Bluescript II SK+. The mutagenic oligonucleotides used in six separate mutagenesis reactions were: 5'-ATCAGACTTCGATTCTGCAGTCGACTCTAGAG-3' 5'-CCTGGAACATCACCATGGACGAAGAAAATCGA-3' 5'-GCATTCCACCTGAAGATATCGACTTCGAGAAG-3' 5'-TGGAGTTGGCATGGATCCAAGAACATCAGACT-3' 5'-TCCAAAACTGGACAAGATCTTCCACCTGGAAC-3' 5'-TGGCGAGATCCATTCATATGGTTGGCATTCCA-3'

To make each of the six (ERE/HindIII)2-PRD,mut-CAT reporter constructs, the EcoRI/EcoRI fragment of (ERE/HindIII)2-PRD-CAT was then replaced with the mutated EcoRI/EcoRI frag-ment of (ERE/HindIII)2-PRD,mut-BSK+. To simplify the reporter construct nomenclature used, we will refer to (ERE/HindIII)2-PRD-CAT, (ERE/HindIII)2-PRD, B/N-CAT, (ERE/HindIII)2-PRD, mut CAT, and (ERE/HindIII)2-pS2-CAT as (ERE)2-PRD-CAT, (ERE)2-PRD, B/N-CAT, (ERE)2-PRD, mut CAT, and (ERE)2-pS2-CAT, respectively.

The plasmid pCMVß, which constitutively expresses ß-galactosidase, was obtained from Clonetech (Palo Alto, CA) and was used as an internal control for transfection efficiency in all experiments. The plasmid pTZ19, used as a carrier DNA, was provided by Dr. Byron Kemper of the University of Illinois.

DNA Preparation
Plasmid DNA for transfections was prepared on CsCl gradients as previously described (8, 37) or with a plasmid preparation kit (Qiagen, Chatsworth, CA).

Cell Culture and Transfections
MCF-7 cells (K1 subline, see Ref.41) were maintained in MEM plus phenol red supplemented with 5% calf serum. For transfection experiments, the cells were switched to MEM plus phenol red supplemented with 5% charcoal-dextran-treated calf serum for 2 days, and then to MEM without phenol red plus 5% charcoal-dextran-treated calf serum for 6 days before plating for transfection. All media included penicillin (100 U/ml) and streptomycin (100 µg/ml). For transfections, the cells were plated at a density of 3.5 x 106 per 100-mm diameter dish and were given fresh medium about 30 h after plating. The cells were transfected by the calcium phosphate coprecipitation method (42) 16 h later with 15 µg of CAT reporter plasmid plus 400 ng of pCMVß. The cells remained in contact with the precipitates for 6 h and were then subjected to a 3-min shock (25% glycerol in culture medium), which was followed by a rinse with HBSS. Treatments were added in fresh medium after the rinse.

ß-Galactosidase and CAT Assays
All cells were harvested 24 h after hormone treatment. Extracts were prepared in 200 µl of 250 mM Tris-HCl (pH 7.5) using three freeze-thaw cycles. ß-Galactosidase activity, which was measured to normalize for transfection efficiency, and CAT activity were assayed as previously described (43).

Gel Shift Assays
Whole cell extracts from MCF-7 cells for use in the gel shift assays were prepared by freeze-thaw lysis as described previously for transfected COS-1 cells (44). The single-stranded oligomer 5'-TTTTCTTCTCGAAGTCTGATGTTCCAGGTGGAATGCC-3', which represents the -131 to -94 region of the rat PR gene, was annealed with its complement. The resultant double-stranded oligomer was gel purified on a nondenaturing 10% polyacrylamide gel run in 0.5x Tris-borate-EDTA. The ability of extract protein(s) to bind to the -131 to -94 fragment was analyzed using standard gel mobility shift assays. Briefly, 2 µl (~5 µg) of MCF-7 whole cell extract was incubated with 1 ng of end-labeled -131/-94 oligomer, under conditions described previously (11). The specificity of binding was assessed by competition with excess unlabeled double-stranded -131/-94 oligomer or with excess unlabeled double-stranded -131/-94 oligonucleotide with a 6-bp mutation from -115 to -110 (mut3; single-stranded oligomer 5'-TTTTCTTCTCGAAGTCgatatcttCAGGTGGAATGCC-3'annealed to its complement) as well as with excess unlabeled double-stranded oligomers containing the consensus ERE, mutated ERE, or consensus GRE sequence. The nondenaturing gels used to analyze the protein-DNA complexes were run as described previously (11, 44).


    FOOTNOTES
 
Address requests for reprints to: Dr. Benita S. Katzenellenbogen, Department of Molecular and Integrative Physiology, University of Illinois, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, Illinois 61801.

This work was supported by NIH Grant CA-18119 and US Army Grant DAMD17–94-J-4205. M.M.M. is the recipient of a postdoctoral fellowship from the Susan G. Komen Foundation.

1 Present address: Department of Biology, University of California, San Diego, La Jolla, California 92093. Back

Received for publication October 14, 1996. Revision received December 17, 1996. Accepted for publication December 19, 1996.


    REFERENCES
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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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