Extreme Position Dependence of a Canonical Hormone Response Element

Steven K. Nordeen, Carol A. Ogden, Laima Taraseviciene and Benjamin A. Lieberman

Department of Pathology and Program in Molecular Biology University of Colorado Health Sciences Center Denver, Colorado 80262


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hormone response elements (HREs) are considered enhancers, activating transcription in a relatively position- and orientation-independent fashion. Upon binding to an HRE, steroid receptors presumably contact coactivators and/or proteins associated with the transcription initiation complex. As a receptor target site is moved further from a fixed position such as the TATA box, not only will the spatial separation of the receptor with respect to its interaction partners change, so will the orientation due to the rotation of the DNA helix. Additional constraints may be imposed by the assembly of DNA into chromatin. Therefore, we have endeavored to test rigorously the assertion that HRE action is position independent. We have constructed a series of 42 chloramphenicol acetyltransferase expression vectors that contain a single progesterone/glucocorticoid receptor-binding site separated from a TATA box by 4 to 286 bp. The enhancer activity of the HRE was assessed after transient transfection of progesterone receptor-expressing fibroblasts. We find that the position of the HRE has a dramatic influence on induction by progestins. When closely juxtaposed to the TATA box, the HRE was unable to support a hormone response, perhaps due to direct steric hindrance with the transcription initiation complex. Full activity was gained by moving the HRE 10 bp further from the TATA sequence. As the HRE was moved incrementally further, activity remained near maximal over the next 26 bp. HRE activity then declined over the subsequent 26 bp and remained low for another 2.5 helical turns. Surprisingly, a narrow window of HRE activity occurred at an HRE-TATA box separation of 90–100 bp. Little or no hormone-induced transcriptional activity was observed when the HRE was positioned further from the TATA box. The addition of a second HRE or a basal (nuclear factor-1) element failed to relieve this constraint. A similar series of experiments was carried out in a mammary carcinoma cell line that expressed high levels of both glucocorticoid and progestin receptors. Data in these cells indicate that glucocorticoids and progestins supported a similar HRE position-activity profile, but this pattern of HRE activity was quite distinct from that seen in fibroblasts. This may be indicative of cell type-specific interactions between steroid receptors and adapter/coactivator proteins or cell type-specific activities such as acetylases or deacetylases participating in the steroid response.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Enhancers are defined operationally as transcriptional control elements that increase gene expression in a heterologous context and independently of orientation and position. The hormone response element (HRE) of the mouse mammary tumor virus (MMTV) promoter was among the first transcriptional control regions shown to meet these operational criteria (1). While it is clear that activity of enhancers may decrease over some distance, activity is generally retained when an enhancer is positioned at sites ranging over several hundreds of bases from the promoter. Indeed, glucocorticoid response elements of the tyrosine amino transferase and rat uteroglobin genes are found 2–3 kb from the start of transcription of the natural gene (2, 3).

There are a number of constraints that have the potential to favor or proscribe the activity of a transcriptional control element at different positions. Models often depict the DNA or chromatin template folding back so that transcription factors binding the enhancer can interact with proteins at the initiation complex. Such cartoons fail to consider the inherent stiffness of DNA. The helical nature of DNA also compounds the difficulty of devising models that account for position-independent interaction of transcription factors especially between separation distances of less than the persistance length of DNA (4) (~150 bp). Packaging of DNA into chromatin can deform the path of the DNA template to juxtapose factors but also introduces its own set of constraints with regard to position independence.

Part of the reason that enhancers are relatively position independent may be that they are generally aggregates of individual elements (enhansons, Ref.5) that together determine the overall properties of an enhancer. The multipartite structure may disguise position constraints if, at a given position, some enhansons are in a favorable location even though others may not be. The functional synergism between enhansons and between enhansons and elements of the basal promoter may also serve to circumvent positional constraints. The mechanistic basis of synergism between transcriptional control elements is poorly understood. In this study we have sought to evaluate whether a single steroid receptor-binding site, in the context of a promoter lacking control elements other than a TATA box, can activate transcription in a position-independent manner. Our data indicate that the activation potential of an HRE with a simple promoter exhibits remarkable position dependence. The pattern of this activity is, however, not readily reconciled in terms of known structural constraints of DNA or its packaging as chromatin.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Vectors for Assessing Position Effects on the Activity of an HRE
To assess the position dependence of an HRE in a systematic fashion, we engineered a series of reporter vectors with a promoter comprised simply of a TATA box and an HRE (Fig. 1AGo). The HRE is comprised of an optimized 15-bp sequence binding a glucocorticoid or progesterone receptor (PR) dimer. This straightforward constitution obviates complicating factors introduced due to the interplay and synergistic behavior between multiple receptor recognition sites or between receptor-binding sites and other transcription control elements. A polylinker sequence separates the TATA box and HRE so that vectors with different spacing between the two elements could be readily generated.



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Figure 1. HRE-TATA Spacing Vectors

A, Schematic of the series of HRE-TATA vectors. Different spacings of the HRE and the TATA box were generated by digesting and religating the polylinker sequence as described in Table 1Go. B, HRE-44 is the vector from which all other vectors of this series are derived. The number indicates the number of base pairs separating the 5'-T of the TATA box and the 3'-most position of the HRE (the first C of the XhoI site). C, HRE 165 is derived from HRE 44 by the insertion of a 121-bp polylinker sequence. Vectors with numbers greater than 56 are derived directly or indirectly from HRE 165 (Table 1Go).

 
The progenitors of all the vectors in this series are shown in Fig. 1Go. An optimal glucocorticoid/progestin response element sequence was cloned into the XhoI site of the vector pE1bcat that contains a 14-bp sequence encompassing the TATA box of the adenovirus E1b gene upstream of the chloramphenicol acetyltransferase (CAT) reporter gene (6) (Fig. 1AGo). This created HRE-44 (Fig. 1BGo), a hormone-responsive vector in which 44 bp separates the 3'-most base of the HRE and the 5'T of the TATA sequence. A polylinker sequence was removed from the vector pBend2 (7) with XhoI and ligated into the SalI site of HRE-44, creating HRE-165 (Fig. 1CGo) and HRE-286 (insertion of two copies of the 121-bp polylinker). All other vectors were derived from manipulation of HRE-44 and HRE-165 as outlined in Table 1Go. In all, a series of 42 vectors was constructed with HRE-TATA spacing from 4–286 bp.


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Table 1. HRE-TATA Spacing Vectors

 
HRE Activity Assay
Each HRE vector of the entire series was transfected into 4F cells. 4F cells are Ltk - fibroblasts engineered to express the B isoform of human PR. 4F cells express about 0.8 pmol receptors per mg protein (8). These cells were chosen because they transfect well and because the high expression of PR gave a robust hormone response with HRE-44, the progenitor of the entire vector series. Although these cells express GR, they do so at low levels and support only a small induction of CAT activity in response to glucocorticoid with HRE-44. With such a large series of vectors, it is not possible to assess all in a single experiment. To normalize between experiments HRE-44 was assessed in all experiments. The induction obtained with HRE-44 was set at 100%, and induction data for other plasmids were calculated relative to that standard within each experiment. The HRE activity data for the entire set of plasmids are plotted in Fig. 2Go relative to HRE-44. Each point represents the average of data from 3 to 11 independent experiments (median 5) where that spacing was assayed. It is predominantly the poorly inducible to uninducible HRE-plasmids at the larger HRE-TATA separations that were assayed only three times. Some plasmids consistently exhibit greater inducibility than HRE-44 and many exhibit less. When the HRE is closely juxtaposed to the TATA box (HRE-4), the promoter is uninducible. It is likely that steric interference prevents the simultaneous occupancy of the HRE and TATA box by receptor and the basal transcription complex. However, moving the HRE an additional 8 bp further from the TATA box permits recovery of some activity, while increasing the separation another 2 bp (HRE-14) results in a plasmid that supports an induction consistently greater than for HRE-44. The HRE activity remains consistently greater than the HRE-44 standard for all plasmids from HRE-14 to HRE-40. From HRE-40 through HRE-66 the HRE inducibility declines steadily to baseline. Inducibility remains negligible as the HRE-TATA separation is increased to about 88 bp. There is a sharp peak of inducibility between HRE-88 and HRE-100. Little or no HRE activity is exhibited by plasmids HRE-103 through HRE-169. One even larger spacing tested, HRE-286, also exhibited no inducibility.



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Figure 2. Position Dependence of the HRE in Response to the Progestin R5020 in 4F Cells

In a large series of experiments each of 42 HRE vectors representing 38 different HRE-TATA box separations of 4–286 bp was transfected into 4F cells, and the inducibility was assessed by activity of the CAT reporter. Inducibility of HRE 44 was assessed in each experiment, and all inductions were normalized to HRE 44, which was assigned an induction of 100%. HRE-44 is indicated by the solid circle. The average CAT activity in 4F cells transfected with HRE-44 was 4 pmol product/mg protein/min without hormone, a level indistinguishable from assay background, and 1300 pmol product/mg protein/min with hormone treatment. Thus, the average magnitude of induction of HRE-44 by R5020 is more than 300-fold above background. The other plasmids were assessed in 3 to 11 independent experiments (median 5) and tested in duplicate transfections within an experiment. It is generally the plasmids with the larger HRE-TATA separations where little or no induction is observed that were repeated in only three experiments. Several vectors, HRE-24, HRE-40, HRE-94, and HRE-165, were constructed in two ways to test whether flanking polylinker sequences were influencing inducibility. In each case, both vectors gave similar results and data points represent the average of the two. Data points at or below the horizontal dashed line are effectively uninducible as compared with the activity of HRE-less pE1bCAT in hormone-treated cells.

 
It is clear from these data that the activity of a single HRE is highly position sensitive. There is, however, no hint of a helical periodicity. One can operationally define three domains each defined by six or seven HRE plasmids and each spanning about two and one-half helical turns. The closest to the TATA box is a region where HRE activity is high. HRE activity declines steadily in the second domain and is negligible in the third. Adjacent to this domain, there is a narrow window where significant HRE function is recovered before again falling to baseline at all greater HRE-TATA separations. This pattern is not readily explicable in terms of the structure of a nucleosome or the helical structure of DNA, as will be discussed.

HRE Position-Activity Profile: Progestins vs. Glucocorticoids
We wanted to compare the activity profile of the same series of vectors in response to glucocorticoids to test whether the two receptors may exhibit distinct position preference for their shared target element. 4F cells are glucocorticoid responsive. However, they contain low levels of glucocorticoid receptors (GRs) and show only a barely detectable response with HRE 44 and several of the other vectors that respond well to progestin treatment in 4F cells. Therefore, we have performed another large series of transfection experiments with T47DA/1–2 cells, a derivative of T47D mammary carcinoma cells that have been engineered to express high levels of GR along with the high expression of endogenous PR. We have used these cells extensively to compare glucocorticoid and progestin action (9, 10). Here we have compared the induction of the series of HRE-spacing vectors by the synthetic glucocorticoid, dexamethasone, and the synthetic progestin, R5020. For every inducible vector, the absolute value of CAT activity is higher with dexamethasone than R5020 despite the fact that there is somewhat less GR than PR in these cells. We previously observed that transiently transfected mouse mammary tumor virus promoter also exhibits a greater response to glucocorticoids in these cells (9). Figure 3Go compares the glucocorticoid and progestin induction for the entire series of HRE-TATA-spacing vectors. The activity of each spacing vector is normalized to the induction of HRE 44 with that hormone. The overall HRE activity-position profile is similar for both receptors showing only some minor quantitative differences that are less remarkable than the extent of similarity. Indeed, the two patterns are, for the most part, superimposable, suggesting that the two receptors act or are acted upon by similar mechanisms to regulate transcription in T47D/A1–2 cells.



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Figure 3. Position Dependence of the HRE in Response to Progestin or Glucocorticoid in T47DA/1–2 Cells

Plasmids from the HRE library were transfected into T47D/A1–2 cells, and CAT activity was assessed after treatment with R5020, dexamethasone, or vehicle. HRE-44 was assessed in every experiment. All R5020 inductions were normalized to the R5020 induction of HRE-44, and all dexamethasone inductions were normalized to the dexamethasone induction of HRE-44. Hormone inductions of HRE-44 were assigned an induction value of 100% (solid symbols). The average CAT activity in T47D/A1–2 cells transfected with HRE-44 was 3 pmol product/mg protein/min without hormone, a level indistinguishable from assay background. Activity after R5020 treatment averaged 27 pmol/mg protein/min and, after dexamethasone treatment, 66 pmol/mg protein/min. These activities represent a magnitude of induction 9-fold and 22-fold above background. Data points for the remaining plasmids represent the average of two to six independent experiments with each transfection done in duplicate in an experiment. In general, it is the uninducible plasmids at the larger HRE-TATA separations for which only two independent experiments have been done.

 
HRE Position-Activity Profile: Cell Type Dependence
It is readily apparent that the HRE position-activity profile for progestin induction differs between 4F fibroblasts and T47D/A1–2 mammary carcinoma cells. R5020 induction data from Figs. 2Go and 3Go have been replotted on the same graph for direct comparison (Fig. 4Go). Both cell types exhibit a narrow activity peak at about HRE-94 but from there the profiles are quite different. In T47D/A1–2 cells there is a very narrow peak at HRE-82 that is completely absent in 4F cells. Note that the same plasmid preparation of HRE-82 was used in both series of experiments. In T47D/A1–2 cells there is a peak of activity at HRE-48 and modest inducibility from HRE-14 through HRE-30 in contrast to the broad pattern of activity of HRE-14 through HRE-40 in 4F cells. The differences in the HRE position-activity profile in the two cell lines suggest that protein-protein interactions between receptor and other components of the transcription apparatus differ between cell types.



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Figure 4. HRE Position Dependence: 4F vs. T47D/A1–2 Cells

R5020 induction data from Figs. 2Go and 3Go have been replotted to compare directly the HRE position-activity profile in the two cell lines.

 
Multiple Elements and HRE Action at a Distance
The data we have presented indicate that an isolated HRE in the context of a simple promoter composed of only a TATA box does not act in a position- independent manner. Indeed, HRE activity is extremely position dependent for both GR and PR action. Thus, in this circumstance, an HRE does not fulfill the operational criteria of an enhancer. One possibility suggested by these data is that position independence may be conferred by the interaction of multiple elements acting in a synergistic fashion just as position-dependent SV40 enhansons interact to create a position-independent enhancer (5). We tested whether the responsiveness of a poorly inducible HRE vector could be restored by introduction of a second HRE or another transcription control element at a position where the second element cannot itself promote transcription. An 18-bp oligonucleotide with an HRE sequence was cloned in both orientations at the SpeI site of HRE-165 (Fig. 1CGo). This HRE is separated from the TATA box by 127 bp in orientation 1 or 130 bp in orientation 2 and from the distal HRE by 41 and 38 bp, respectively. The two HRE constructs were transfected into 4F fibroblasts, and the induction of CAT activity by R5020 was compared with HRE-165, HRE127, or HRE-44. As before, induction of the latter averaged more than 300-fold and was assigned a value of 100%. Consistent with the data of Fig. 2Go, little induction of HRE-165 and HRE-127 was seen (3.4% ± 2.7 and 10.8% ± 8.5, Fig. 5Go). The two HRE constructs were somewhat inducible (25.0% ± 10.8 and 27.8% ± 8.8 compared with the HRE-44 single HRE construct). Thus, a pair of HREs can support more induction than each does alone, but the modest synergism seen suggests the existence of additional constraints and/or the need for additional mechanisms to relieve spatial limitations of HRE action. It should be noted that the two HRE constructs, although less inducible than HRE44, still exhibit 80-fold inductions in these circumstances. This suggests that an alternative way to approach these data is to ask not why the more distant elements give small inductions but rather why HRE-44 and closer elements are so effective.



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Figure 5. An Additional HRE or Basal Element Fails to Relieve Spatial Constraints on HRE Action

4F cells were transfected with plasmids containing a second HRE or a basal transcription element (NF-1). In the 2HRE vectors, both individual HREs are at positions that permit little or no inducibility. The activity of the HRE-NF-1 vector, like the HRE vectors, was at or below background in the absence of hormone (R5020). The results are representative of three or four experiments. The mean induction relative to HRE-44 is plotted ± 1 SE.

 
We also tested whether the presence of a target sequence for a basal transcription factor could functionally synergize with a single HRE to relieve spatial constraints on hormone inducibility. We chose to introduce a nuclear factor-1 site into the HindIII site of HRE-165 since a nuclear factor-1 site is present in the strongly inducible mouse mammary tumor virus promoter. However, the nuclear factor-1 site did not promote R5020 inducibility in the context of the HRE-165 promoter (Fig. 5Go). The presence of the nuclear factor-1 site also failed to stimulate a detectable glucocorticoid induction in response to dexamethasone in 4F cells (data not shown). These results highlight how little we really understand about the spatial constraints on factor-factor interaction and the mechanisms that account for the relative lack of such constraints on HRE action in the context of natural control elements.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
HREs have been classified as enhancers based on data indicating steroid-dependent transcriptional activation to be relatively insensitive to position, orientation, or the basal promoter employed. This is consistent with observations that the HREs of natural promoters are found at varying distances from the promoter. Glucocorticoid response elements have been identified more than 2 kb distant from the transcription initiation site (2, 3). However, natural steroid response elements are generally composed of multiple receptor binding sites that may be located over some distance and that may act synergistically. Also, steroid receptors may work in conjunction with other factors such that these additional promoter elements may have significant influence on the hormone response. Such assemblages in the tyrosine amino transferase gene and the phosphoenolpyruvate carboxykinase gene have been termed glucocorticoid-respons(iv)e units (11, 12, 13). Thus, natural response elements and response units may achieve position independence as the result of the contribution of multiple elements to activity.

The present studies were designed to systematically test whether a single HRE in the context of a basal promoter composed only of a TATA box is capable of functioning in a position-independent manner. In PR expressing 4F fibroblasts, the HRE exhibited a complex pattern of distance dependence as shown in Fig. 2Go. There was no evidence of a 10-bp periodicity that would implicate a constraint imposed by the helical nature of DNA. However, we find that the distance-activity pattern depicted in Fig. 2Go is also not easily accommodated in terms of nucleosome structure. It should be noted that the beginning of the broad plateau of high HRE activity (HRE-14 to HRE-40) is positioned about 80 bp (or one wrap of the DNA around a nucleosome) from the sharp spike of activity exhibited by HRE 94a, HRE-94b, and HRE-98. However, arguing against a role for nucleosomes in this pattern are the reports that although transiently transfected DNA is packaged into chromatin, the nucleosomes are not arrayed in the same specific pattern as seen with stable transfections (15). Yet, it is conceivable that binding of a steroid receptor could determine the local positioning of nucleosomes around the promoter. This could favorably position the TATA box so as to permit access by the TFIID complex. Arguing against such simple models accounting for the position dependence of the HRE are the progestin induction data in T47D/A1–2 cells. The HRE position-activity profile obtained in this line is quite different than that of 4F cells (Fig. 4Go). In both cases, however, a peak of activity is seen at an HRE-TATA spacing of 94 bp, and no induction is seen at spacings greater than 100 bp. It is not obvious what determines this limit nor the cell-specific pattern of induction at lesser spacing of the HRE from the TATA box. Nonetheless, these data would argue that it is the interaction of the receptor with factors expressed in a cell type-specific manner rather than general factors that determine hormone responsiveness of this simple promoter. Such cell-specific factors could be involved in the tissue-specific expression of agonist activity by steroid antagonists.

We had anticipated that glucocorticoid response might show a different HRE position-activity profile than the progesterone response. Counter to these expectations, GR and PR mediated a similar pattern of response throughout the library of HRE position vectors. Thus, with this template the activation domains of the two receptors may interact in a similar manner with other components of the transcription apparatus. There clearly must exist other mechanisms to distinguish glucocorticoid and progestin action since the two hormones may have very different physiological activities in tissues such as the mammary epithelium that express both receptors. In other work we have shown that a stably integrated mouse mammary tumor virus promoter is differentially induced by glucocorticoids in a mammary carcinoma cell line (10) in a locus-specific fashion (J. R. Lambert and S. K. Nordeen, submitted), implying that the packaging of promoters as stable chromatin and the influence of the surrounding chromatin may play a commanding role in governing hormone inducibility.

In summary, we have used a strategy that employs simplified promoter constructs to make interpretation of the results as straightforward as possible. Of course, there are caveats to any reductionist approach. As with natural promoters, it is not possible to guarantee that any DNA sequence is completely inert. Thus, we cannot rule out that the polylinker sequence is structured in some unusual fashion that could affect hormone action or that factors, possibly cell type specific, may bind to the polylinker DNA, thereby enhancing or suppressing hormone action. We have considered this where possible. For example, certain constructs, such as the active HRE-94 spacing, were made in more than one way to minimize the possibility that a chance juxtaposition fortuitously created a factor-binding site or an unusual DNA structure. Over the entire set of HRE-TATA vectors, various portions of the polylinker sequence are present in different vectors, suggesting that no single sequence could easily account for the complex spacing-activity patterns observed or the cell type differences of this pattern.

Introduction of a second HRE or a nuclear factor-1 site into the poorly inducible HRE-165 construct results at best in limited relief of spatial constraints on hormone induction. Again, in this reductionist approach it is difficult to guarantee that functional synergism would not have been seen if the constructs were formulated differently. A GRE2tkCAT construct exhibited considerably more inducibility than a single GRE construct (16), but this is a more complicated circumstance since the basal thymidine kinase promoter contains at least three elements, two GC boxes and a CCAAT box. Sathya et al. (17), in a paper published after submission of our work, analyzed the functional consequences of multiple estrogen response elements (EREs) and indicated that a single ERE 52 bp upstream of the TATA box of a minimal promoter gave a borderline induction (1.2-fold). This was improved only marginally by the addition of a second ERE (1.9-fold induction). Addition of a third or fourth ERE did lead to a synergistic increase in inducibility (16- and 38-fold, respectively). These authors went on to show that the presence of other basal elements in the promoter, spacing between the EREs, and position of the EREs with respect to the promoter all influenced the functional synergism of EREs (17). Together, this work and ours suggest that the synergistic interactions between multiple HREs or between HREs and other transcription control elements must not be completely promiscuous and are, therefore, subject to constraints of their own. These data highlight the paucity of our understanding of how steroid receptors and other transcription factors can communicate with the basal transcription apparatus in many different promoter contexts and over a remarkable variety of configurations. No understanding of mechanisms of steroid receptor action will be complete without a better conception of how receptors deal with these cir-cumstances.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
A hormone-responsive reporter gene vector bearing a single HRE was constructed by introducing a synthetic, optimized HRE into the XhoI site of plasmid pE1bCAT (6). The promoter of the E1bCAT consists of a 14-bp DNA sequence spanning the TATA box of the adenovirus E1b gene embedded in a polylinker sequence upstream of the CAT reporter gene. The 3'-most base of the HRE is separated by 44 bp from the first T of the TATA box. This vector is termed HRE-44 (Fig. 1Go). A 121-bp XhoI fragment containing a polylinker sequence from pBend2 was cloned at the SalI site of HRE-44 between the HRE and the TATA box. The separation of the HRE and TATA box was increased to 165 bp (HRE-165). A clone that contains two copies of the polylinker was also isolated (HRE-286). All of the remaining plasmids in the HRE series were derived from HRE-44 or HRE-165 and their products by cutting with the indicated restriction enzyme(s), blunting the ends where necessary with Klenow polymerase or Mung Bean nuclease, and religation (Table 1Go). The sequence of all HRE plasmids was verified, and the instances of deviation from the predicted joint are indicated in Table 1Go.

The 2HRE-1 and 2HRE-2 plasmids were constructed by introducing the oligonucleotide; 5'-CTAGTGGTACAAACTGTT-3'; 3'-ACCATGTTTGACAAGATC-5' into the SpeI site of HRE-165. The plasmid 2HRE-1 has one copy of the oligonucleotide in the orientation shown. 2HRE-2 has one copy of the oligonucleotide in the opposite orientation. HRE-nuclear factor-1 (NF-1) was constructed by introducing the oligonucleotide 5'-AGCTTGGCTTGAAGCCA-3'; 3'-ACCGAACTTCGGTTCGA-5' into the HindIII site of HRE-165.

Transfection and Reporter Assays
The development of PR-expressing 4F fibroblasts and GR-expressing T47D/A1–2 mammary carcinoma cells has been described (8, 9, 18). Both lines are cultured in modified Eagles medium supplemented with 5% FBS, glutamine, and penicillin + streptomycin. Additionally, 4F medium contains HAT (hypoxanthine-aminopterin-thymidine), and T47D/A1–2 medium contains Geneticin. Geneticin is not included when stock cultures are split for transfection experiments as GR expression has been found to be stable for some time in the absence of continued selection. Both cell lines were transiently transfected using a diethylaminoethyl (DEAE)-dextran protocol.

4F cells were plated on 60-mm culture dishes at 1.4 x 106 cells per dish the day before transfection. For transfection, cells were exposed to 1 ml growth medium containing 200 µg/ml DEAE-dextran, 2 µg/ml test plasmid, 0.5 µg/ml of internal transfection control plasmid (pSV2luc), and 100 µM chloroquine for 2 h. Transfection medium was replaced by shock buffer (137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM glucose, 21 mM HEPES, pH 7.1) with 15% dimethylsulfoxide for 6 min before cultures were returned to growth medium. Two days after the start of transfection, R5020 (10 nM) or vehicle was added and the incubation was continued for 24 h before harvest. For both 4F cells and T47D/A1–2 cells all plasmids were transfected in duplicate dishes for each treatment.

T47D/A1–2 cells were plated on 60-mm culture dishes at 1.4 x 106 cells per dish the afternoon before transfection. For transfection, cells were exposed to 1 ml of growth medium containing 500 µg/ml DEAE-dextran, 2 µg/ml test plasmid, and 0.1 µg/ml of internal transfection control plasmid [cytomegalovirus(CMV)-ßgal). The transfection solution was replaced after 4 h with 1 ml shock buffer with 15% dimethylsulfoxide for 6 min. This was replaced by growth medium containing 100 µM chloroquine for 2 h before returning to growth medium. Two days after the start of transfection, hormone (R5020 10 nM or dexamethasone 100 nM) or vehicle was added and the incubation was continued for 24 h before harvest.

To harvest, cells were rinsed twice with wash buffer (40 mM Tris-HCl, 140 mM NaCl, 1 mM EDTA, pH 7.4) and then lysed with 500 µl lysis buffer (20 mM K2HPO4, 5 mM MgCl2, 0.5% Triton X-100, pH 7.8). Lysate was transferred to a microfuge tube, and insoluble debris was pelleted by centrifugation. Reporter gene assays were performed on the cleared lysate.

CAT activity was assessed by an enzymatic/organic extraction method described previously (19). Acetylation cocktail (200 µl) was incubated for 5 min at 37 C. To this was added 50 µl of lysate supernatant, and the incubation was continued for 4 h. The components of the cocktail were at the following concentrations after addition of lysate: Tris-Cl, pH 7.8, 100 mM; MgCl2, 6 mM; KCl, 100 mM; sodium acetate, 0.4 mM; ATP, 3 mM; Coenzyme A, 0.4 mM; chloramphenicol, 1 mM. Each reaction also contained 7.8 µCi [3H]acetate and 0.015 U acetyl CoA synthetase (Sigma, St. Louis, MO). The CAT assays were stopped by pipetting an aliquot (100 µl) into 1 ml benzene and immediately vortexed. The phases were separated by centrifugation. An aliquot (750 µl) of the upper phase was removed to a scintillation vial. The benzene was evaporated overnight in a fume hood to lower background (19), and scintillation fluor (Beckman Ready Safe, Beckman Instruments, Fullerton, CA) was added to the dry scintillation vials. Duplicate aliquots were assayed from each reaction.

For luciferase assays, lysate (15–50 µl) was added to 350 µl luciferase buffer (100 mM K2HPO4, 1 mM dithiothreitol, 5 mM ATP, 15 mM MgSO4, pH 7.8) The reaction was initiated by the injection of 100 µl 1 mM luciferin and light output integrated for 10 sec after a 2-sec delay using a Monolight 2001 luminometer (Analytical Luminescence Laboratories, Ann Arbor, MI). For ß-galactosidase, 2–5 µl of lysate were added to 100 µl of Galactolight reaction buffer containing the Galacton-Plus substrate (Tropix, Bedford, MA), and the mixture was incubated at room temperature for 1 h. Using the Monolight 2001 luminometer, readings were taken after the injection of 100 µl Galacto-light accelerator into the reaction mixture. CAT induction data calculated by normalizing for the internal transfection control were compared with induction data normalized to protein content of the lysate or to unnormalized induction data. Similar results were obtained by all three methods.


    ACKNOWLEDGMENTS
 
Cell culture medium was obtained through the Tissue Culture and Monoclonal Antibody Core of the University of Colorado Cancer Center. Some of the vector sequence confirmation was done by the Sequencing Core of the Cancer Center.


    FOOTNOTES
 
Address requests for reprints to: Steven K. Nordeen, Department of Pathology B216, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Denver, Colorado 80262.

This work was supported by NIH Grant DK-37061.

Received for publication June 11, 1997. Revision received December 9, 1997. Revision received February 10, 1998. Accepted for publication February 16, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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