Identification of a Third Autonomous Activation Domain within the Human Estrogen Receptor

John D. Norris, Daju Fan, Sandra A. Kerner and Donald P. McDonnell

Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Using a genetic selection system established in the yeast Saccharomyces cerevisiae, we have isolated, by random mutagenesis of the human estrogen receptor (ER), six mutants that display constitutive transcriptional activity. All of the mutants identified contained single base insertions or deletions leading to frameshift mutations, resulting in receptor truncations within the hormone-binding domain between amino acids (aa) 324–351. Interestingly, an ER mutant (aa 1–282) was transcriptionally inactive in yeast, suggesting that a domain important for transcriptional activity lies between aa 282 and 351 within human ER. Deletions representative of the mutants isolated in the yeast system were created in mammalian expression vectors and examined for transcriptional activity in animal cells to determine the physiological relevance of this domain. Receptors truncated at aa 282 were either weakly active or inactive; however, an ER deletion at aa 351 was approximately 50% as active as wild type ER (induced with estrogen). Furthermore, a chimeric receptor consisting of the DNA binding domain of GAL4 fused to aa 282–351 of the human ER was transcriptionally active on a GAL4 reporter. We conclude, therefore, that an autonomous activation domain (referred to as AF2a), functional in both yeast and mammalian cells, lies between aa 282–351 of the human ER.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human estrogen receptor (ER) is a member of the steroid receptor family of ligand-activated nuclear transcription factors (1). In the absence of hormone, ER resides in a transcriptionally inactive form within the nucleus of target cells (2). Interaction with its cognate ligand results in a conformational change within the receptor, allowing the displacement of associated heat shock proteins and permitting receptor homodimerization (3, 4). In this activated state, ER can interact with target gene promoters directly, by binding to specific estrogen response elements (5), or indirectly through its association with other DNA-bound proteins (6, 7, 8, 9, 10). The biological result of these interactions can lead to either up-regulation or down-regulation of target genes, depending on the cell and promoter context (11).

We and others have demonstrated that ER contains at least two distinct activation functions (AFs) that determine its transcriptional activity in target cells (11, 12). These ER activation sequences located within the amino (AF-1) and carboxyl (AF-2) terminus of the receptor function in a cell- and promoter-specific manner to manifest the activity of bound ligands (13). Specifically, it has been shown that estradiol acts as an ER agonist in contexts where either or both AFs are required. In contexts where AF-1 alone is required, tamoxifen and other triphenyl-ethylene-derived antiestrogens function as partial agonists, whereas in contexts where AF-2 alone is required, tamoxifen behaves as an ER antagonist (11, 13, 14, 15). The pure antiestrogen ICI 182,780 inhibits the activity of AF-1 and AF-2 and may also be involved in receptor degradation (11, 16, 17). However, it has become apparent recently that the ability to differentially regulate AF-1 and AF-2 cannot account for all of the biological effects of ER ligands. Specifically, we have shown that a novel ER antagonist GW5638 does not activate either AF-1 or AF-2, yet functions as a full agonist in the bone and cardiovascular systems (T. M. Willson, J. D. Norris, B. L. Wagner, I. Asplin, P. Baer, H. R. Brown, S. A. Jones, B. Henke, H. Sauls, S. Wolfe, D. Morris, and D. P. McDonnell, submitted). This would suggest that ER can operate in a nonclassic manner in these tissues or alternately that additional sequences within ER contribute to its transcriptional activity. Previous studies from our laboratory prompted us to pursue the latter hypothesis. Specifically, we observed that in the context of wild type ER (wtER), AF-2 was not required for AF-1 function. However, a construct containing the N-terminal AF-1 alone was not functional as a transcriptional activator in yeast or mammalian cells (11, 19). We hypothesized, therefore, that sequences in addition to AF-1 and AF-2 may be involved in ER transcriptional activity. Because similar findings were obtained in both yeast and mammalian cells, we considered that a genetic screen in yeast would be appropriate to examine this activity. Using a genetic selection system developed in the yeast Saccharomyces cerevisiae, we have identified a series of ER mutants that lead to the definition of a third activation sequence which functions in an autonomous manner. The discovery of an additional activation domain that functions in animal cells may help to explain the tissue-specific agonist activity of compounds that previously have been shown to inhibit AF-1 and AF-2.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of ER Mutants That Function Independently of AF-2
We have developed a genetic selection system in the yeast S. cerevisiae to screen for mutations within ER that manifest transcriptional activity in the absence of a functional AF-2. To accomplish this, we created an ER-dependent survival system in which the yeast HIS3 gene was placed under control of a chimeric estrogen response element (ERE)/GAL1 promoter (Fig. 1Go). This construct permits 17ß-estradiol-dependent growth of the ER containing his3 yeast strain YPH500 ER (20, 21). To permit a quantitative estimation of ER responsiveness, an additional reporter containing the Escherichia coli ß-galactosidase gene under the control of the ERE/CYC1 promoter was also introduced. This tester strain, containing both reporters, was then used to screen for ER mutants that displayed AF-2-independent transcriptional activity.



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Figure 1. Identification of ER Mutants That Demonstrate Transcriptional Activity in the Absence of a Functional AF-2

A library of ER mutants was created using the E. coli mutator strain XL-1 red. Resultant DNA was transformed into the yeast strain YPH500, and colonies that grew in the presence of raloxifene (keoxifene) were selected for further analysis as described in Materials and Methods.

 
Libraries of ER mutants were created by random mutagenesis and transformed into the yeast tester strain. To identify mutants that grew in the absence of a functional AF-2, we added raloxifene (an AF-2 antagonist) to the selection media (Fig. 1Go). Although ER antagonists do not effectively inhibit agonist-bound receptor in yeast and most exhibit partial agonist activity, we were able to use raloxifene in these screens to activate ER as it does not appear to function as an agonist in yeast. In addition, this compound was shown to be a full AF-2 antagonist in mammalian cells. Aminotriazole (a competitive inhibitor of the HIS3 enzyme) at a concentration of 1 mM was also added to increase the stringency of the screen. Yeast colonies that grew under these conditions within 5 days were selected for further analysis. Two successive rounds of primary screening, representing 100,000 individual transformants, were performed. A secondary screen was performed under the same conditions resulting in the selection of 23 individual colonies that demonstrated ER-dependent growth in the presence of raloxifene. The transcriptional activities of the resultant mutants were constitutive and thus unaffected by the addition of ligand. The receptor expression plasmids from each of these colonies were recovered, and the ER coding region was sequenced.

Characterization of the Mutants Identified in Yeast
DNA sequence analysis of the 23 mutants identified represented six different alleles generated by frameshift mutations at the boundary of the D (hinge) and E (ligand-binding) regions of the ER-coding sequence (Table 1Go). All of the mutations contained a single base insertion or deletion within an 80-bp region. Interestingly, ER62 in which a single C residue was deleted was isolated 13 times, and ER 65, which contained an inserted C residue, was found four times in our libraries. The six different alleles predicted receptor truncations with translation terminating at stop codons in the shifted frame yielding proteins of 339, 342, and 374 amino acids. Each of the six mutants were then remade in the yeast expression vector so that stop codons were introduced after the last authentic amino acid of ER (Table 1Go) to avoid any inaccuracies caused by the extraneous sequences. We confirmed that these mutations in the ER-DNA coding sequence gave rise to proteins of the predicted molecular mass by performing Western immunoblot analysis of protein extracts from representative isolated yeast colonies (Fig. 2AGo). The wtER migrates as an intact species at 65 kDa, whereas all of the mutants migrated at their predicted molecular masses (38–42 kDa). The additional minor bands are likely to be proteolytic fragments derived from the larger receptor species. Additionally, we observed that the expression level of wtER and that of the mutants were approximately the same in this strain.


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Table 1. AF-2 Independent ER Mutants Result from Carboxyl-Terminal Deletions

 


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Figure 2. Characterization of the Biochemical Properties of the AF-2-Independent Mutations

A, Expression vectors for each of the mutants M324-M351, wtER, and ERN282g were transformed separately into BJ5457, a protease-deficient strain of S. cerevisiae. Extracts were prepared from the resultant strains and examined by Western immunoblot analysis using anti-ER antibody H226. Extracts were also prepared from an empty expression vector as control. B, Expression vectors for each of the mutants (M324-M351), ERN282g, and wtER were transformed separately into YPH500 together with the ERE-CYC1-ß-GAL reporter plasmid. The transcriptional activity of wtER was assayed in both the presence and absence of 10-6 M 17ß-estradiol. The data shown for each of the mutants represent constitutive activity since these proteins are unable to bind ligand. Experiments were performed several times in triplicate and representative data are shown. The percent error in all yeast transcriptional assays was less than 20%.

 
We then measured the transcriptional activity of the ER mutants on the ERE-ß-galactosidase reporter plasmid and compared their activity to that of the wtER (induced with 17ß-estradiol) containing both AFs and ERN282g (19) containing only AF-1. As shown in Fig. 2BGo, wtER functions as an efficient hormone-dependent transcription factor whereas ERN282g is transcriptionally inactive under identical conditions. However, all six classes of ER truncations displayed transcriptional activity that was approximate to or greater than wtER. Thus, it appears that in yeast, sequences contributing to the transcriptional activity of ER lie between aa 282 and 351. These data are in agreement with the work of Pierrat et al. (22) who also found that truncations within this region of ER resulted in receptor mutants that displayed constitutive activity in a yeast system. They referred to this sequence as AF2a, a nomenclature that we have adopted.

In Vitro DNA-Binding Affinity of wtER and ER Mutants
Before concluding that the region within ER between aa 282 and 351 was directly involved in transcriptional activity, we examined whether this region was important for DNA binding. One possible explanation for the inability of ERN282g to activate transcription, as opposed to the ability of isolated truncation mutants to activate transcription, would be that the region between aa 282 and 351 enhanced the ability of the mutant to bind DNA, allowing for transcriptional activation by AF-1. This was examined using in vitro gel shift analysis. Yeast were transformed with expression vectors for wtER, ERN282g, or M351 (one of the most active mutants). Nuclear extracts were prepared from these yeast, and the ability of the expressed wtER or ER mutant to interact with a radiolabeled vitellogenin probe was measured. The results of this analysis are shown in Fig. 3Go. Extracts containing wtER, but not control extract, were able to interact with the vitellogenin probe. The specificity of this association was confirmed by showing that the resultant band was not affected by the addition of a 100-fold molar excess of unlabeled progesterone response element (PRE). Additionally, it was shown that the resultant complex could be supershifted by an anti-ER antibody (H226), indicating that the complex contained ER. Under identical conditions, extracts containing either ERN282g or M351 were not capable of high-affinity DNA interactions with the labeled ERE, although ERN282g did display some affinity for DNA. This may be due to the lack of more C-terminal sequences that contain a strong dimerization domain (23). However, upon the addition of anti-ER antibody, dimerization and high-affinity interactions were established. Stabilization of DNA/ER interactions by antibody are consistent with the work of others (17). Contrary to what was expected, it appeared that ERN282g interacted with DNA in a more efficient manner than did M351, yet they demonstrate dissimilar transcriptional activities. Thus, it appears in this system that DNA-binding affinities do not correlate well with transcriptional activity. Moreover, it indicates that the dramatic difference between the transcriptional activities between ERN282g and M351 does not appear to be the result of an increased affinity for target DNA. These data therefore suggest that the region between aa 282 and 351 is important for efficient ER transcriptional activity.



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Figure 3. Characterization of the in Vitro DNA-Binding Properties of wtER, ER-M351, and ERN282g

Recombinant proteins corresponding to wtER, ERN282g, and M351 were obtained after transformation in yeast (BJ5457) and isolation of protein extracts. The ability of these proteins to interact with a radiolabeled vitellogenin ERE was determined by electrophoretic mobility shift assay. Extracts from nontransformed yeast were used as control. The specificities of the DNA/ER complexes were tested using 100-fold molar excess of a progesterone response element (PRE), and the presence of ER within the complexes was ascertained using anti-ER antibody H226 (Ab) to supershift the complex.

 
The Sequence between aa 282 and 351 Contributes to ER Transcriptional Activity in Mammalian Cells
We next sought to determine whether the activation sequence identified in the yeast system also functions in a mammalian cell line. To accomplish this, representative mutants isolated in yeast were subcloned into the mammalian expression vector pRST7ER (20). Transient transfections were performed in the human liver cell line HepG2. Cells were cotransfected with both a receptor expression plasmid and a reporter plasmid containing three EREs placed upstream of an enhancerless luciferase reporter vector containing only a TATA sequence element. The results of this analysis are shown in Fig. 4Go. As expected, wtER functioned as a potent transcriptional activator in the presence of 17ß-estradiol. Mutants terminating at aa 282, 325, or 335 were either weakly active or inactive compared with wtER in the absence of hormone. However, mutants terminating at aa 343 and 351 showed considerable transcriptional activity with the mutant terminating at aa 351 possessing more than 50% activity of wtER induced with ligand. These results support the finding in yeast that an efficient transcriptional domain lies between aa 282 and 351 (putative AF2a) in the human ER.



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Figure 4. The AF2a Sequence Identified in Yeast Is Also Functional in Mammalian Cells

Plasmids expressing wtER (pRST7-ER) and ER mutants (indicated in the figure) were cotransfected into the human liver cell line HepG2 along with a reporter construct consisting of three EREs placed upstream of a TATA sequence element initiating transcription of the luciferase gene. Transcriptional activities of wtER are shown in the presence and absence of 10-7 M 17ß-estradiol. Transfections were normalized for efficiency using a constitutive ß-galactosidase reporter plasmid (pCMV-ß-GAL) (20). Representative assays are shown in which triplicate transfections were performed; error bars represent the SEM.

 
Amino Acid 282–351 Exhibits Autonomous Transcriptional Activity in Mammalian Cells
Having demonstrated that aa 282–351 of the human ER is important for the transcriptional activity of truncation mutants in both yeast and mammalian cells, we next wanted to determine whether this domain could act independently of AF-1 (N-terminal domain of ER). To define this sequence within ER as an activation domain (or AF), we felt that it was necessary to demonstrate that it can activate transcription in an autonomous fashion. To accomplish this, we fused aa 282–351 of the human ER to the DNA-binding domain (DBD) of the yeast transcription factor GAL4. This chimeric protein was then assayed for transcriptional activity in a transient transfection system using a reporter plasmid containing five GAL4 DNA-binding sites placed upstream of a TATA sequence element initiating transcription of the luciferase gene. For comparative purposes, the AF-1 sequence between ER aa 52–149 (24) and the hormone binding domain (AF-2) consisting of ER aa 312–595 were also fused to the DBD of GAL4. This analysis was performed using several cell lines to determine whether there was any cell specificity within the defined parameters of this experiment. In all cell lines tested (Fig. 5Go, A, B, and C), the fusion containing the putative AF-2a sequence was transcriptionally active in an autonomous manner. As expected, the ER-hormone-binding domain (HBD) fusion protein (containing AF-2) functioned in a ligand-dependent manner in all cells (Fig. 5Go, A, B, and C). AF2a appeared to be most active in the HepG2 and Hela cell lines where it induced expression of the reporter 36- and 25-fold, respectively (Fig. 5Go, A and B). The GAL4 DBD is included as control and shows no transcriptional activity on its own. Interestingly, in HepG2 cells where tamoxifen functions as a partial agonist (11, 15), ER-AF1 is clearly the dominant AF, and in Hela cells where tamoxifen acts as a complete ER antagonist, ER-AF-2 is dominant (Fig. 5Go, A and B). AF-1 also appears to be the dominant AF in CV-1 cells; however, tamoxifen shows no agonist activity in this cell line (14). The transcriptional activity of AF2a in CV-1 cells was much weaker than that of the other cell lines tested (Fig. 5CGo). In light of these results, it is interesting to speculate that in order for tamoxifen to display agonist activity through AF-1, a fully active AF2a is required. Regardless, we have shown for the first time that the region marking the boundary between the D (hinge) and E (HBD) domains of ER act as a bona fide transcriptional activation domain in animal cells.



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Figure 5. Amino Acids 282–351 Representing the ER AF2a Sequence Function in an Autonomous Fashion in Mammalian Cells

Vectors expressing fusion proteins consisting of the DNA binding domain (DBD) of GAL4 and ER fragments representing AF-1 (aa 51–149), AF-2 (aa 312–595), and AF2a (aa 282–351) were cotransfected into (A) HepG2, (B) Hela, or (C) CV-1 cells along with a reporter construct consisting of five GAL4 DNA-binding sites placed upstream of a tata sequence element initiating transcription of the luciferase gene. Addition of 17ß-estradiol was used to activate the GAL4 fusion containing the HBD of ER. An expression vector containing only the GAL4 DBD was included as control. Induction values were calculated by dividing the activity of individual ER-AF fusions by the activity of the GAL4-DBD and are indicated in the figure within the corresponding bar. Representative assays are shown in which triplicate transfections were performed; error bars represent the SEM.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Several constitutive human ER mutants were obtained using a phenotypic screen in yeast. Although the primary goal of the screen was to isolate ER mutants that displayed AF-2-independent or raloxifene-dependent activation, only mutants corresponding to a coding sequence terminating at the boundary between the D and E regions of the receptor were detected. Perhaps the method of mutagenesis or the possibility that such raloxifene-dependent mutants are not viable resulted in the selection of mutants that displayed hormone-independent activity. Similar results were obtained by Pierrat et al. (22) using a yeast-screening system. Interestingly, mutations truncated at aa 282 appear to have little or no activity in yeast unless the yeast transcriptional repressor complex SSN6/TUP1 (19) is disrupted, indicating that AF-1 (N terminus) alone is not sufficient for transactivation. However, extension of the receptor to aa 351 results in a more efficient transcription factor that no longer requires AF-2. The reason for these differences in ER transcriptional activities cannot be explained by differences in DNA binding or protein expression levels, as their affinities for DNA and expression levels both appear to be roughly equivalent. This would suggest that an efficient transcriptional activation sequence lies between aa 282–351 (AF2a) and that perhaps in yeast it is required in order for full activation of the N-terminal AF-1. The fact that full transcriptional activation (equivalent to that obtained by ligand-induced wtER) is obtained by these truncated mutants in yeast would also suggest that AF-1 and AF2a are the dominant AFs in this biological system.

Although yeast has proven to be a valuable tool in the dissection of the functional domains of the nuclear receptors, including ER (4, 25), any data collected there must be tempered by the fact that this is only a model system as yeast do not contain any proteins homologous to these receptors. Therefore, we extended our studies of the AF2a region to ascertain whether the region identified in yeast could function also in mammalian cells. We were initially surprised to find that the AF2a identified in the yeast screen functioned analogously in the animal models; previous studies by Pierrat et al. (22) and Webster et al. (26) were unable to show activity in mammalian cells. Webster fused the protein sequence coded by ER exon 4 (aa 254–366) to a GAL4 DBD and did not show any autonomous function. It is possible that the contrast between our data and previous studies could be accounted for by differences in reporter constructs or cell lines as we and others have determined that both of these parameters play a major role in determining the transcriptional activity of any given AF (11, 13, 27). It is also likely that minor amino acid sequence variations in the region selected for analysis could account for the variance in the data obtained by a particular study. Nevertheless, we were able to demonstrate that aa 282–351 were not only important for the activity of truncated mutants (inactivity of ERN282g as opposed to activation by 351T), but that this amino acid sequence, when fused to a GAL4 DBD, could function as an autonomous activation domain. We did detect some differences in the results regarding transcriptional activation in yeast and mammalian cells (Figs. 2AGo and 4Go). Mutants terminating at aa 324 and 335 demonstrate transcriptional activation comparable to wtER in yeast, yet are inactive when analyzed in the HepG2 cell line. The contrast in transcriptional activity between these two biological systems may be the result of interactions with different coactivators.

Thus far, no proteins interacting with the N-terminal (AF-1) domain of the human ER have been identified. The general transcription factor TAFII30, however, has been shown to associate with the hinge region of ER in vitro (28), and homologous regions of related nuclear transcription factors have been implicated in the binding of nuclear corepressors such as N-CoR (29). Moreover, a transactivation domain within the glucocorticoid receptor called {tau}2 is located within a homologous region equivalent to the AF2a sequence isolated in ER (30). Interestingly, a codon 351 mutant (Asp>Tyr) isolated from a human breast tumor line demonstrated increased agonistic activity of a fixed-ring tamoxifen analog (31). Thus, this often overlooked region of the receptor appears to be more important in transcriptional regulation than previously thought.

The transcriptional activities of AF-1 and AF-2 are greatly influenced by their cellular and promoter context. Estradiol acts as an ER agonist regardless of the cellular context, whereas antiestrogens such as tamoxifen behave as either complete antagonists or partial agonists, depending on the activity of AF-1 within a given environment (11, 13). Thus, different ligands demonstrate a differential requirement for AF-1 and AF-2. The recent cloning of proteins that interact with AF-2 (HBD) of the nuclear receptors has provided insight into the way in which activation sequences mediate their activities (32, 33, 34). For instance, it has been shown that in the presence of estradiol, ER can engage the receptor-interacting protein, mSUG1, whereas tamoxifen, an AF-2 antagonist, disrupts this interaction (35). However, the core AF-2 sequence postulated to mediate the transcriptional activity of the HBD (36) may not be the only activation sequence within this region. A recent study demonstrating that SRC-1 can interact with an ER mutant devoid of the core AF-2 sequence (37) and our own data showing that GRIP (glucocorticoid receptor interacting protein) can interact with mutants in which the core AF-2 sequence has been destroyed by point mutations (our unpublished observations) would indicate that these receptors may indeed contain many transcriptional activation sequences and may interact in different ways with receptor coactivators. Thus, it is likely that nuclear receptor transcriptional activity in a given cellular environment may ultimately depend on the relative expression of a multitude of coactivators and corepressors. These findings, coupled with our current study identifying a previously unrecognized AF (AF2a) within mammalian cells, could help to explain why compounds such as raloxifene (14) and GW 5638 (18), which do not permit AF-1 or AF-2 activity, function as full agonists in the bone and cardiovascular systems. It is possible that the cofactor environment within relevant tissues permit AF2a to function as a dominant activator. However, until the target cells and genes in these systems are identified, this hypothesis cannot be tested.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Enzymes and Chemicals
Restriction and modification enzymes were obtained from Promega (Madison, WI) Boehringer Mannheim (Indianapolis, IN), or New England Biolabs Inc. (Beverly, MA). Raloxifene was kindly provided by Dr. Eric Larsen (Pfizer Inc., Groton, CT). 17ß-Estradiol and 3-aminotriazole were purchased from Sigma (St. Louis, MO). Oxaliticase was purchased from Enzogenetics (Corvallis, OR). XL-1-Red cells were purchased from Stratagene (La Jolla, CA). PCR reagents were purchased from Perkin Elmer Cetus (Palo Alto, CA) or Promega. Anti-ER antibodies for mobility shift assays and Western blots were a gift from Dr. Geoffrey Greene (Ben May Institute, Chicago, IL).

Yeast Plasmids and Strains
Yeast strain BJ5457 (MAT{alpha} ura3–52 trp1 lys2–801 leu2{Delta}1 his3{Delta}200 pep4::HIS3 prb1{Delta}1.6R can1 GAL) was used to express wtER and ER mutants. All screening was performed in yeast strain YPH500 (MAT{alpha} ura3–52 lys2–801 ade2–101 trp1-{Delta}63 his3-{Delta}200 leu2-{Delta}1). YPH500 ER was obtained by transforming YPH500 with YEpE22 (21). Reporters pBM2389 (21) and YRpE2 (38) have been described previously.

Random, Site-Directed, and Oligo-Directed Mutagenesis
Random mutations within ER were generated according to the manufacturer’s instructions by transforming YEpE22 into the mutator E. coli strain XL1-Red. ER mutants 324c, 335c, 343g, and 351t were created by insertion of YEpE22 into m13mp19 followed by site-directed mutagenesis according to the manufacturer’s protocol (Bio-Rad, Hercules, CA). The resulting mutants were then subcloned into pRST7-ER (20). GAL4-ER fusions were generated using oligo-directed mutagenesis as previously described (15).

Identification of ER Mutants Using a Phenotypic Screen in Yeast
Randomly mutated YEpE22 plasmid DNA, along with YRpE2 (ERE-CYC-ß-galactosidase) and the survival plasmid pBM2389, were introduced into the yeast strain YPH500 by the lithium acetate protocol (20). Transformants were plated on synthetic defined media lacking leucine, tryptophan, histidine, or uracil. In addition, 1 mM 3-aminotriazole, 50 µM CuSO4, and 10-6 M raloxifene (dissolved in ethanol) were added to the media. Colonies appearing within 5 days were selected for subsequent analysis. More than 100,000 transformants were screened.

ß-Galactosidase Assays in Yeast
The transcriptional activities of ER and ER mutants in yeast were assayed on the ERE-CYC1-ß-galactosidase reporter as previously described (21). Briefly, mutants were grown overnight in the appropriate media ± hormone and assayed for ß-galactosidase activity.

Bandshift Assays
Cell extract containing wtER or ER mutant proteins were isolated from yeast (BJ5457). Labeled probe (10,000 cpm) was incubated with yeast extract in a binding buffer consisting of 10 mM HEPES, pH 7.9, 100 mM KCl, 1 mM dithiothreitol, 0.5 mM EDTA, 2.5 mM MgCl2, 6% glycerol, and 2% Ficoll. The reactions were allowed to proceed at room temperature for 10 min. Addition of antibody or competing DNA to the protein extract occurred before incubation with the labeled probe. The samples were then subjected to electrophoresis (150 V at room temperature) in a nondenaturing 5% polyacrylamide gel in 0.5x Tris-borate-EDTA. Autoradiograms were prepared from dried gels.

Cell Culture and Transient Transfection Assays
HepG2, CV-1, and Hela cells were maintained in modified Eagle’s medium (Life Technologies, Grand Island, NY) supplemented with 10% FCS (Life Technologies). Cells were plated in 24-well plates (coated with gelatin for transfections of HepG2 cells) 24 h before transfection. DNA was introduced into the cells using lipofectin (Life Technologies) as described previously (20).


    ACKNOWLEDGMENTS
 
The authors would like to thank Maria Huacani, Markus O. Imhof, and Xiao-Fan Wang for kindly providing plasmids 3X-ERE-tata-Luc, pBK-cmv-GAL4 (DBD), and 5X-GAL4-tata-Luc, respectively.


    FOOTNOTES
 
Address requests for reprints to: Donald P. McDonnell, Department of Pharmacology, Box 3813, Duke University Medical Center, Durham, North Carolina 27710.

This work was supported by NIH Research Grant DK-48807 (to D.P.M.).

Received for publication February 5, 1997. Accepted for publication March 14, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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