©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of the Amino-terminal Transcriptional Activation Function of the Human Estrogen Receptor in Animal and Yeast Cells (*)

Daniel Metzger (§) , Simak Ali(§)(¶) , Jean-Marc Bornert , Pierre Chambon (**)

From the (1) Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France BP 163-67404 Illkirch-Cedex, C.U. de Strasbourg, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have previously reported that the transcriptional activation function AF-1, located in the A/B region of the human estrogen receptor, exhibits cell-type and promoter context specificity in both animal cells and yeast. To further characterize AF-1, we have constructed a number of deletion mutants spanning the A/B region in the context of either the whole human estrogen receptor or the A/B region linked to the GAL4 DNA binding domain, and tested their transcriptional activity in chicken embryo fibroblasts and in yeast cells, two cell types in which AF-1 efficiently activates transcription on its own. Additionally, we utilized HeLa cells in which AF-1 is poorly active but can synergize with the transcriptional activation function AF-2 located in the hormone binding domain. We show that in animal cells the ``independent'' activity of AF-1 is embodied in a rather hydrophobic proline-rich 99-amino acid activating domain (amino acids 51-149), whereas amino acids 51-93 and 102-149 can independently synergize with AF-2. Interestingly, in yeast, three discrete activating domains (amino acids 1-62, 80-113, and 118-149) are almost as active on their own as the whole A/B region, indicating that multiple activating domains can operate independently in yeast. Our study also demonstrates that, within the context of the whole human estrogen receptor, the same AF-1 activating domains are ``induced'' by either estradiol or 4-hydroxytamoxifen.


INTRODUCTION

In eukaryotes, sequence-specific DNA binding proteins control initiation of transcription and play a major role in tissue-specific and temporal regulation of gene expression (for reviews, see Mitchell and Tjian (1989), Carey (1991), Ham et al. (1992), Hori and Carey (1994), and Tjian and Maniatis (1994)). Functional analyses have shown that transactivators contain separable and interchangeable domains responsible for DNA binding and for transcriptional activation. Little is known concerning the nature of the activating domains (for reviews, see Mitchell and Tjian (1989), Ptashne and Gann (1990), Carey (1991), Hahn (1993), and Tjian and Maniatis (1994)). No strong primary sequence similarity has been found between the activating regions of different transcription factors. However, some activating domains are characterized by their high content in acidic amino acids such as the yeast transactivators GAL4 or GCN4, the human glucocorticoid receptor, or the herpes simplex virus activator VP16 (Gill and Ptashne, 1987; Ma and Patshne, 1987; Hope et al., 1988; Hollenberg and Evans, 1988; Triezenberg et al., 1988; Sadowski et al., 1988; Gill et al., 1990; Cress and Triezenberg, 1991), which may be arranged to form amphipathic -helices or sheets (Giniger and Ptashne, 1987; van Hoy et al., 1993; Leuther et al., 1993). Other activating domains, rich in proline or glutamine residues or in hydroxylated amino acids, have also been described (Mermod et al., 1989; Courey and Tjian, 1988; Theill et al., 1989). However, the importance of these amino acids is not yet clear (see Mitchell and Tjian (1989) and Seipel et al. (1994)). Interestingly, the basic mechanisms controlling initiation of transcription appear to be conserved throughout eukaryotes. Several subunits of RNA polymerase II(B) are highly conserved from yeast to man (Sentenac and Sawadogo, 1990; Young, 1991), and the yeast TATA-binding factor protein can to some extent functionally replace the corresponding HeLa cell factor (Buratowski et al., 1988; Cavallini et al., 1988). Moreover, yeast transcriptional activators can function in animal and plant cells (Kakidani and Ptashne, 1988; Webster et al., 1988a; Fisher et al., 1988; Ma et al., 1988), and many mammalian transcriptional activators have been shown to activate transcription in yeast (see Metzger et al. (1988), Schena and Yamamoto (1988), Heery et al. (1993), and references therein).

The estrogen receptor (ER)() belongs to a superfamily of ligand-inducible transregulators, which includes receptors for steroid and thyroid hormones, vitamin D3, retinoic acid, and peroxisome proliferator-activated receptors (Evans, 1988; Green and Chambon, 1988; Beato, 1989; Gronemeyer, 1991; Laudet et al., 1992; Leid et al., 1992; Kastner et al. 1994; Mangelsdorf et al., 1994). Cloning of ER cDNAs from different species led to the definition of six regions (see Fig. 1 A, A-F) exhibiting different degrees of amino acid sequence conservation (Krust et al., 1986). This division was subsequently extended to all members of the nuclear receptor superfamily. Molecular genetic analyses of ER have identified separable domains responsible for DNA binding, hormone binding, and transactivation (see Fig. 1 A). Region C, which is highly conserved across species, corresponds to the DNA binding domain (DBD). It is responsible for specific binding to estrogen response elements (EREs) of target genes (for Refs., see Gronemeyer (1991)). The carboxyl-terminal region E, which is also well conserved between species, contains the hormone binding domain (HBD) and a hormone-dependent transcription activation function (AF-2, previously called TAF-2) (Webster et al., 1988b; Bocquel et al., 1989; Lees et al., 1989; Tora et al., 1989a; Berry et al., 1990; Tasset et al., 1990; Danielian et al., 1992; Durand et al., 1994).


Figure 1: Localization of the AF-1 activating domain using the chimeric AB-GAL activator in CEF cells. A, schematic representation of hER and the reporter gene 17M/ERE-G.CAT. The regions containing the AFs, DBD, and HBD of hER are indicated. The numbers refer to amino acid positions for hER and to nucleotides for the reporter gene. The +1 arrow represents the transcriptional start site. B, transcriptional activity of the AB-GAL deletion mutant series. The amino acids of the A/B region are represented in gray. The deletion is indicated by a line, and the deleted amino acids are indicated. Amino acids 1-147 of GAL4 are represented by an open box. The induction of CAT activity obtained with AB-GAL relative to the parental pSG5 vector is taken as 100; the corresponding -fold induction is given in parentheses. Transcriptional activities of deletants are normalized to that of AB-GAL. Each value is an average (±10%) of at least four independent experiments. C, a Western blot analysis of transfected COS-1. Each lane contains 10 µg of cell extract: lane 1, transfection with pSG5 expression vector; lanes 2-12, transfections with the indicated expression vector. The extracts were immunoprobed with 2GV3 and 3GV2 antibodies. Note that in this particular transfection series, 306-GAL and 313-GAL were expressed at a lower level than the other AB-GAL derivatives. However, this decreased expression was not seen in other transfection series. The position of the molecular mass standards are indicated in kDa. D, a representative CAT assay. CEF cells were transfected with the reporter plasmid 17M/ERE-G.CAT together with the expression vector as indicated. Extracts were assayed for CAT activity after normalization for -galactosidase activity (see ``Materials and Methods'').



Another transcriptional activation function (AF-1, previously called TAF-1) was characterized in the ER amino-terminal A/B region and was shown to function in a hormone independent manner when isolated from the HBD (Bocquel et al., 1989; Lees et al., 1989; Tora et al., 1989a; Tasset et al., 1990). This ER amino-terminal region, which is less conserved between species (Krust et al., 1986), corresponds to a region that exhibits little or no conservation among the nuclear receptor superfamily (see Segraves (1991) for references). A number of nuclear receptor genes encode isoforms that differ exclusively in their amino-terminal amino acid sequences (for reviews, see Gronemeyer (1991), Leid et al. (1992), and Chambon (1994)). Interestingly, the two chicken and human progesterone receptor isoforms A and B have been shown to differentially activate transcription of target genes (Tora et al., 1988; Kastner et al., 1990), and we have previously shown that AF-1 of the human ER (hER) exhibits cell-type and promoter context specificity. In HeLa cells, AF-1 activates transcription very poorly on its own, but it can synergize with AF-2 to activate transcription from some promoters. In contrast, in chicken embryo fibroblasts (CEF), AF-1 can efficiently activate transcription on its own (Kumar et al., 1987; Tora et al., 1989a; Berry et al., 1990). In the yeast Saccharomyces cerevisiae, hER transactivates from reporter genes containing the GAL1 promoter into which an ERE has been inserted (Metzger et al., 1988). Interestingly, deletion of the HBD results in a constitutive ER mutant that is almost as active as hER (White et al., 1988), whereas little or no transcriptional activity remains when the A/B region is deleted (Berry et al., 1990; Metzger et al., 1992). However, when tested on different promoters, an A/B region-deleted hER is more active than a region E-deleted hER, indicating that the activities of AF-1 and AF-2 are also promoter context dependent in yeast (Metzger et al., 1992; Pham et al., 1992). We have also shown that the partial agonistic activity of the anti-estrogen 4-hydroxytamoxifen (OHT) can be ascribed to the activity of AF-1, whereas AF-2 activity is inhibited by OHT (Berry et al., 1990; Metzger et al., 1992). Comparative functional analyses of hER AF-1 and AF-2 and of the acidic activation functions of GAL4 and VP16, have shown that their synergistic and transcriptional interference (``squelching'') properties are different and suggested that AF-1 and AF-2 may interact with different intermediary factors (TIF) (mediators or coactivators) also required for mediating the activity of acidic activators, whereas acidic activators may require an additional mediator (Tora et al. (1989a) and Tasset et al. (1990) and references therein).

In the present study, the AF-1 transcriptional activation function present in the hER A/B region has been functionally dissected in animal cells and in yeast. We have identified a hydrophobic, proline-rich, 99-amino acid-long region, which is responsible for AF-1 activity in CEF cells and for synergistic activation with AF-2 in HeLa cells. We also show that shorter segments of the A/B region are sufficient for efficient transcriptional activation in yeast. Finally, we provide further evidence that estradiol (E2) and 4-hydroxytamoxifen have similar effects on AF-1 activity within the context of the whole ER.


MATERIALS AND METHODS

Recombinants for Animal Cell Studies

HEG0 has been described previously (Tora et al., 1989b). The AB-GAL expression vector (a gift of D. Tasset) was constructed as follows. The EcoRI- KpnI fragment encoding amino acids 1-184 of hER was excised from HE28 (Green and Chambon, 1987) and inserted 5` of the GAL4 DBD into the EcoRI- HindIII sites of pG4M poly(I) (Webster et al., 1989) using a KpnI- HindIII adaptor; the stop codon between the HindIII site and the ATG of the GAL4 DBD was removed by site-directed mutagenesis leading to the presence of the extra amino acids GTGTSL between the A/B region and the GAL4 DBD. The EcoRI site located 5` to the AB-GAL coding sequences was blunt-ended, and a BglII linker was cloned in, resulting in pSG5bAB-GAL. The deletions were constructed by site-directed mutagenesis of single-stranded pSG1HEG0 or pSG5bAB-GAL using synthetic oligonucleotides (36-mer) complementary to 15 nucleotides located 5` and 3` to the deletion and containing the sequence 5`-CTCGAG-3` corresponding to a XhoI restriction site in the middle. All constructs were verified by sequencing. The reporter plasmids ERE-TATA-CAT and 17M/ERE-G.CAT have been described (Tora et al., 1989a).

Recombinants for Yeast Cell Studies

pY0 was constructed by exchanging the XmaI- XhoI restriction fragment from pYERE1 (Metzger et al., 1988) containing the ERE sequence with a linker 5`-CCGGGTTTGCGGCCGCATC-3` 3`-CAAACGCCGGCGTAGAGCT-5` containing a NotI site.

The oligonucleotide 5`-GGCCGCAAGGTCGGAGGACTGTCCTCCGAAGCC-3` 3`-CGTTCCAGCCTCCTGACAGGAGGCTTCGGAGCT-5` containing a 17M GAL4 binding site was cloned into the NotI- XhoI sites of pY0, resulting in pY017M. The BglII fragments containing AB-GAL and mutant AB-GAL sequences isolated from the pSG5b vectors (see above) were cloned into the BglII site of pY017M. pYERE1/HEG0 has been described (Tora et al., 1989b), and the HEG0 mutant derivatives were cloned into pYERE1 as described for HEG0 (Tora et al., 1989b).

Cell Transfection and CAT Assays

COS-1, CEF, and HeLa cell transfections were performed using the standard calcium phosphate co-precipitation technique (Tora et al., 1989a, Berry et al., 1990). COS-1 cells were transfected with 5 µg of expression vector and 10 µg of carrier Bluescribe M13+ DNA (BSM+, Stratagene). CEF cells were transfected with either 500 ng of 17M/ERE-G.CAT or 2 µg of ERE-TATA-CAT as reporter genes, together with either 500 ng of AB-GAL, HEG0, or mutant derivatives as activator genes. CEF cell transfections included 1 µg of the reference plasmid pCH110 (Pharmacia Biotech Inc.) and Bluescribe M13+ DNA as carrier DNA to make a total of 15 µg of DNA. HeLa cells were transfected with 2 µg of ERE-TATA-CAT reporter gene, together with 1 µg of HEG0 or A/B region deletants as activator genes, 2 µg of pCH110 and Bluescribe M13+ DNA up to 15 µg DNA. Ethanol (as a control), E2 (10 n M) or OHT (100 n M) prepared in ethanol were added 1 h post-transfection, where appropriate. CEF and HeLa cell extracts and CAT assays were performed as described (Berry et al., 1990).

Yeast Transformation and -Galactosidase Assay

Yeast strain TGY14.1 was transformed as described (Metzger et al., 1988), and yeast strain SC30 (MATa, gal4, ura3-52, leu 2-3, 112, his3, ade) was transformed by electroporation (Becker and Guarente, 1991). Transformants were grown in selective medium, ligands were added as appropriate, and -galactosidase activity determined as described previously (Metzger et al., 1988; Tora et al., 1989b).

Immunoblots

COS-1 cells were extracted (100 µl/plate) in high salt buffer containing 400 m M KCl, 20 m M Tris-HCl, pH 7.5, 1 m M EDTA, 1 m M dithiothreitol, 10% glycerol (v/v), 1 m M phenylmethylsulfonyl fluoride, protease inhibitor mixture (2.5 µg/ml of leupeptin, pepstatin, chymostatin, antipain, and aprotinin) by three cycles of freeze (-70 °C)/thaw (+4 °C) and centrifugation at 10,000 g for 15 min at 4 °C. Yeast extracts were prepared in the same buffer, and Western blotting was performed as described previously (Metzger et al., 1988), using the monoclonal antibody F3 directed against the F region of the hER (Ali et al., 1993a) or 2GV3 and 3GV2 directed against the GAL4 DBD (White et al., 1992).


RESULTS

Experimental Design

To determine which amino acid sequences of the hER A/B region are responsible for transcriptional activation in animal cells and yeast, we have created deletion mutants spanning the A/B region appended to either the amino-terminal 147 amino acids of the yeast transactivator GAL4 (containing the DNA binding domain, the dimerization domain, and the nuclear localization signal (Silver et al., 1988; Carey et al., 1989)) or regions CDEF of hER (see Figs. 1-4). To minimize variations in expression levels of the mutant proteins (which may be related to the amino acid composition of the amino-terminal extremity of the protein (Bachmair et al., 1986)), the amino-terminal-most two amino acids of hER were maintained in all of the mutants. A restriction site was inserted at the position of the deletion in order to facilitate the screening of the recombinants, thus resulting in the insertion of two amino acids (Leu-Glu) in each mutant. The mutations were verified by DNA sequencing on double-stranded plasmid DNA, and the size and expression levels of the proteins were controlled by Western blotting analysis of transfected COS-1 or yeast cell extracts, using monoclonal antibodies directed against the GAL4 DNA binding domain (2GV3 and 3GV2; see White et al. (1992)) or a monoclonal antibody directed against region F of hER (F3 antibody) (Ali et al., 1993a). All of the mutants used in this study were expressed at levels similar to that of the parental activator (see Figs. 1 C and 2 C, and data not shown).

In animal cells, transcriptional activation by AB-GAL, HEG0 (wild-type hER, see Tora et al. (1989b)) and their mutant derivatives was investigated by transfection of the corresponding expression vectors together with specific reporter plasmids. The amount of expression vector used was such that the reporter plasmid was in excess relative to the transactivator, so that the chloramphenicol acetyltransferase (CAT) activity of the deletants compared with that of the parental activator was not dependent on the amount of expression vector (see Tora et al. (1989a); similar relative levels of expression were obtained by transfection of 20, 100, or 500 ng of expression vectors, data not shown). For CAT activity measurement, cell extracts were standardized for transfection efficiency using the -galactosidase activity obtained from the vector pCH110 (Pharmacia). A representative CAT assay is shown in Fig. 1D.

In yeast, AB-GAL, HEG0, and their respective deletion, mutants were expressed from PY017M (see ``Materials and Methods'') and PYERE1 (Metzger et al., 1988) vectors as appropriate, and transcriptional activation was obtained by determination of the -galactosidase activity generated from chimeric reporter genes containing a single GAL4 response element (17M) or an ERE (see Figs. 3 A and 4 A). The AB-GAL chimera and its derivatives were assayed in the yeast strain SC30 (kind gift of S. Johnston) containing a deleted GAL4 gene in order to avoid problems due to endogenous yeast GAL4 protein, whereas HEG0 and its derivatives were tested in the previously used TGY14.1 strain (Metzger et al., 1988). hER AF-1 Includes Amino Acids 51-149 in CEFs-In CEF cells, GAL4 (1-147) was inactive on a CAT reporter construct placed under the control of a chimeric promoter composed of a globin promoter region containing a single 17M GAL4 binding site (17M/ERE-G.CAT; see Fig. 1A). In contrast, the chimeric AB-GAL construct activated transcription 5-fold (Fig. 1, B and D, compare lanes 3 and 9 with lanes 1 and 8), as did GAL-ER(AB), which was used in previous studies (Tora et al., 1989a) (Fig. 1, B and D, compare lanes 2 and 3). Thus, AF-1 of hER activates transcription when tethered either amino- or carboxyl-terminally to the GAL4 DNA binding domain. To determine which amino acid stretches are involved in transcriptional activation by AF-1, a series of deletion mutants was generated from AB-GAL (Fig. 1 B). Removal of amino acids 3-50 did not affect transcriptional activation (344-GAL: 110%), and deletion to amino acid 61 (343-GAL) maintained most of the transcriptional activation capacity. Further deletions within the A/B region resulted in a progressive decrease in transcriptional activation, and deletion to amino acid 101 (302-GAL) resulted in less than 10% of the transcriptional activation capacity of AB-GAL being retained. Thus amino acids 51-184 are sufficient for efficient transcriptional activation, and amino acids 62-101 appear to be particularly important. Deletion from the carboxyl-terminal end showed that removal of the last 35 amino acids of the A/B region (as well as the six amino acids located between region A/B and the GAL4 DBD) had relatively little effect on transcriptional activation (411-GAL, 80% and 384-GAL, 75%). Note that deletion of the same carboxyl-terminal amino acids from the A/B region of HE15F2 (an hER mutant lacking the HBD (282-552), see Fig. 2 B) had no effect on transcriptional activation using this reporter plasmid (data not shown), indicating that the A/B activating domain may be exposed in a slightly different manner in the chimeric construct (see also below in the case of HEG0). Larger deletions resulted in reduced activity; the transcriptional activity of 306-GAL (64-184) was only 10% that of AB-GAL, and 313-GAL (35-184) was totally inactive. Taken together, these results indicate that in CEF cells, amino acids 1-50 and amino acids 150-184 are dispensable for transcriptional activation by AF-1. Accordingly, deletion mutant 384/344-GAL (3-50/150-184) retains most, but not all, of the wild-type activity, indicating that amino acids 51-149 are crucial for transcriptional activation in CEF cells and that the flanking amino acids contribute to some extent to full activity, perhaps by allowing proper exposure of the activating domain.


Figure 2: Localization of the AF-1 activating domain using human ER (HEG0) in CEF and HeLa cells. A, schematic representation of the reporter gene. The numbers refer to nucleotides, and the arrow refers to the transcription start site. B, transcriptional activity of HEG0 deletion mutants. The A/B region and deletions are represented as in Fig. 1. The induction of CAT activity obtained with HEG0 relative to the parental pSG1 vector in the presence of E2 is taken as 100 (the corresponding -fold induction is given in parentheses). The average values (±10%) from at least four independent experiments are given. C, a representative Western blot analysis of transfected COS-1 cells. Each lane contains 10 µg of cell extract: lane 1, transfection with 1 µg of pSG1 vector; lanes 2-13, transfection with 1 µg of the indicated expression vector. The extracts were immunoprobed with the F3 monoclonal antibody. The arrowhead points to a nonspecific interaction. The position of the molecular mass standards are indicated in kDa.



To investigate whether AF-1 activity could be influenced by the presence of the ER hormone binding domain, a similar deletional analysis was performed with mutants derived from HEG0. The mutants were tested in CEF cells on the reporter plasmid ERE-TATA-CAT, on which AF-1 is highly active and AF-2 is inactive (see Fig. 2) (Tora et al., 1989a; Berry et al., 1990). Mutants containing deletions of amino acids 3-50 (HE 344) or 150-178 (HE 384) were as active as HEG0 and, furthermore, were active only in the presence of E2 (data not shown). The activity of mutants containing larger deletions was decreased depending on the size of the deletion, and deletion of amino acids 3-101 (HE 302) abolished transcriptional activity, indicating that amino acids 51-101 are crucial for transcriptional activation by HEG0 from this promoter. When deleting into the A/B region from the carboxyl-terminal end, removal of amino acids 94-178 (HE 314) almost totally abolished transcriptional activation, indicating that amino acids 94-149 are also required for transcriptional activation. Accordingly, deletion mutant HE 344/384, which retains amino acids 51-149 only, was as active as HEG0, while deletion of additional 11 amino acids from the amino terminus or nine residues from the carboxyl terminus of this region greatly decreased transcriptional activity (HE 384/343 and HE 344/368; 40% activity of HEG0). Removal of amino acid stretches within this region also resulted in a significant decrease in transcriptional activity (see HE 354, HE 359, HE 353, and HE 363, Fig. 2B). Thus, we conclude that hER amino acids 51-149 include all of the sequences involved in AF-1 transcriptional activity in CEF cells.

Characterization of the hER A/B Sequences Required for Synergism with AF-2 in HeLa Cells

Previous studies have shown that both AF-1 and AF-2 are inactive on their own on a minimal promoter containing only an ERE and a TATA box (ERE-TATA-CAT) in HeLa cells and that synergism between AF-1 and AF-2 is required for HEG0 to activate transcription from this promoter (Tora et al., 1989a; see also Fig. 2 ). The activity of HEG0-derived constructs deleted in the A/B region was tested on this promoter to investigate whether the sequences required for AF-1 activity in CEF cells are the same as those necessary for synergism with AF-2 in HeLa cells. We found that amino acids 51-149 were required for synergism with AF-2 (Fig. 2 B). However, in contrast to what was seen in CEF cells for the activity of AF-1 on its own, the decrease in synergistic activity was less dramatic, such that deletion of amino acids 3-101 (HE 302) gave 40% activity in HeLa cells, while transcriptional activation was abolished in CEF cells (Fig. 2 B). Similarly, although deletion of amino acids 141-178 (HE 368) had no effect on AF-1 activity in CEF or in HeLa cells, deleting amino acids 94-178 (HE 314) resulted in little activation by AF-1 on its own in CEFs, but 45% of the synergistic activity was retained in HeLa cells. Further amino- and carboxyl-terminal deletions caused a drastic fall in transcriptional activation in HeLa cells (deleting from the amino terminus to amino acid 117 (HE 303) or from the carboxyl terminus to amino acid 85 (HE 310) resulted in very little transactivation). Note, however, that deletion of amino acids 91-118 (HE 353) resulted in a 50% loss of transcriptional activity in CEF cells but did not affect the synergistic AF-1 activity in HeLa cells. We have previously shown that there is ligand-inducible phosphorylation of serine 118 in HeLa, COS-1, and CEF cells. We have also shown that in HeLa and COS-1, but not in CEF cells, mutation of Ser-118 to an alanine residue reduces transactivation, whereas mutation to a glutamic acid residue increases transactivation (Ali et al. (1993b) and data not shown). Deletion of amino acids 91-118 leads to the replacement of Ser-118 by a glutamic acid (see ``Experimental Design'' and ``Materials and Methods''). This suggests that, in HeLa cells but not in CEF cells, the presence of a glutamic acid residue at this position may compensate for a loss of transcriptional activity due to deletion of amino acids 91-118. In any event, the above results indicate that deletants that have only a weak AF-1 in CEF cells can synergize with AF-2 in HeLa cells and that amino acids 51-93 and 102-149 are involved in the synergistic activity.

Analysis of AF-1 Activity in Yeast

We have previously shown that in yeast an hER truncated for the HBD (HE15 or HE15F2, 282-595 and 282-552, respectively, see Fig. 2B) activates transcription constitutively from chimeric GAL-1 gene-derived promoters containing an ERE (White et al., 1988; Berry et al., 1990; Metzger et al., 1992). In order to demonstrate that the A/B region can activate transcription in yeast when linked to a heterologous DBD, we tested the above described AB-GAL chimera on a reporter gene in which -galactosidase expression is driven by a GAL-1 gene-derived promoter containing a single GAL4 binding site (17M; see 17M-GAL1-LacZ in Fig. 3 A). AB-GAL efficiently activated transcription from this promoter, whereas GAL1-147 was inactive (data not shown, and see Fig. 3 B). The stimulation by AB-GAL was clearly mediated through the 17M GAL4 binding site, since its deletion in the reporter gene rendered the promoter nonresponsive (data not shown). Thus, AF-1 is transcriptionally active in yeast independent of any other hER domain. To determine whether the same amino acid 51-149 region, which is responsible for AF-1 activity in CEF and HeLa cells, is also involved in transactivation in yeast, A/B region deletions were investigated using AB-GAL-derived constructs (Fig. 3 B). Interestingly, mutants bearing extensive deletions from the amino terminus showed little loss in AF-1 activity, while they were totally inactive in CEF and HeLa cells (303-GAL). Only deletions beyond amino acid 117 caused a drastic decrease in transactivation, showing that amino acids 118-184 are sufficient for full activation by the A/B region in yeast. Strikingly, when deleting from the carboxyl terminus, this region could be deleted with no loss in activity relative to AB-GAL, and amino acids 1-84 appeared to be sufficient for full transactivation. Thus sequences located between amino acids 1-84 and 118-184 could independently activate transcription in yeast. This conclusion was further supported by the results obtained with mutants 384/303-GAL (3-117/150-184), 368/303-GAL (3-117/141-184), and 306/346-GAL (3-28/64-184), which retained 100, 30, and 45% of the AB-GAL activity, respectively. We conclude that the hER A/B region contains at least two discrete, apparently redundant activation functions (amino acids 29-63 and 118-149), which can activate transcription in yeast.


Figure 3: Localization of the AF-1-activating domain using the chimeric AB-GAL activator in yeast. A, schematic representation of the reporter gene. 17M corresponds to the GAL4 binding site. The numbers refer to nucleotides (+1 corresponds to the first nucleotide of the relevant coding sequence), the triangle and the arrow refer to the TATA box and RNA start site, respectively. B, activators and their corresponding -galactosidase activity ( -gal) obtained from 17 M-GAL1-LacZ (activators are represented as in Fig. 1). The induction of -galactosidase activity obtained with AB-GAL relative to the parental vector pY017M is taken as 100% (the corresponding -fold induction is given in parentheses). The transcriptional activity of the deletants is normalized to that of AB-GAL. Each value is an average (±20%) of at least three independent experiments.



The transcriptional activity of the A/B region was also investigated in yeast using deletion mutants of the complete receptor (HEG0) and the ERE1-GAL1-LacZ reporter gene (Fig. 4, A and B). Deletion of amino acids 3-79 (HE 304) or 85-178 (HE 310) resulted in mutants having a transcriptional activity comparable with that of HEG0 (Fig. 4 B). Larger deletions progressively decreased the transcriptional activity of the mutants, and deletion of amino acids 3-139 (HE 307) or 12-178 (HE 356) resulted in near complete loss of transactivation. Internal deletions of up to 66 amino acids (HE 354; see Fig. 4 B) did not decrease the transcriptional activity of the mutant, and some internal deletion mutants were even more active than HEG0. Further functional dissection of the A/B region showed that it contains several discrete stretches of amino acids that result in levels of activation greater than 60% relative to HEG0 (Fig. 4 B, see for example mutants HE 311/388 (containing amino acids 18-62), HE 315/304 (containing amino acids 80-113), and HE 303/384 (containing amino acids 118-149)). Interestingly, these mutants and even some mutants with smaller deletions ( e.g. HE 354, HE 303, and HE 310) did not activate transcription in CEF or HeLa cells (compare with Fig. 2) (data not shown).


Figure 4: Localization of the AF-1 activating domain using human ER (HEG0) in yeast. A, schematic representation of the reporter gene. The symbols are the same as in Fig. 3 A. ERE corresponds to the ER response element. B, activators and their corresponding -galactosidase activity ( -gal). The -galactosidase activity obtained with HEG0 in the presence of E2 relative-pYERE1 is taken as 100 (the corresponding -fold induction is given in parentheses). The transcriptional activity of the deletants is normalized to HEG0. Each value is an average (±20%) of at least three independent experiments.



4-Hydroxytamoxifen and Estradiol Play a Similar Role in AF-1 Activity

We have previously ascribed the agonistic activity of the OHT observed in CEF cells and yeast to AF-1 (Berry et al. 1990; Metzger et al., 1992). The above hER mutants were used to investigate whether the same amino acid sequences are involved in transcriptional activation induced by E2 and OHT. The transcriptional activity of HEG0 and of some key mutants was determined in CEF cells in the presence of 10 n M E2 or 100 n M OHT using the ERE-TATA-CAT reporter gene (Fig. 2 A). In agreement with previous results (Berry et al., 1990), in the presence of OHT, HEG0 was about 30% as active as in the presence of E2 (). Deletions of amino acids from HEG0 that did not affect the E2-induced transcriptional activity, also did not affect transcriptional activity obtained with OHT (HE 344, HE 384, and HE 344/384 were as active as HEG0 in the presence of OHT, ). On the other hand, deletions that reduced activation in the presence of E2 (HE 302, HE 314, and HE 354) had a similar effect on the presence of OHT.

The transcriptional activity obtained in the presence of E2 or OHT was also determined in yeast using the most informative mutants on the estrogen-responsive chimeric GAL1 promoter. In the presence of 1 µ M OHT, HEG0 was 80% as active as in the presence of 10 n M E2, in agreement with previous results (Metzger et al., 1992). Mutants containing large deletions in the A/B region of hER, such as HE 303, HE 311, HE 315/304, HE 311/388, and HE 303/384, which were active in the presence of E2, were also highly active in the presence of OHT. Reciprocally, mutants poorly active in the presence of E2, ( e.g. HE 307 and HE 356) were also poorly active in the presence of OHT.

These results demonstrate that, in both animal and yeast cells, the hER A/B segments, which are responsible for activation of transcription in the presence of estradiol, are also those responsible for activation in the presence of 4-hydroxytamoxifen.


DISCUSSION

The estrogen receptor contains two activation functions, which activate transcription in a constitutive manner (AF-1) or in a hormone-dependent manner (AF-2) when isolated from the rest of the ER sequences. Previous studies have shown that both transcriptional activation functions act in a promoter context and cell-type specific manner (see Introduction) and that AF-1 is located within amino acids 1-180 (region A/B) of the human ER. In particular, the A/B region is transcriptionnally active on its own in CEFs (Kumar et al., 1987; Webster et al., 1988b; Tora et al., 1989a; Berry et al., 1990), as well as in yeast (White et al., 1988; Berry et al., 1990; Metzger et al., 1992).

Using systematic deletional mutagenesis, we have now localized the amino acid sequences within the A/B region responsible for AF-1 activity in CEF cells. A/B region deletion mutants were created using both the chimeric activator AB-GAL and HEG0, in order to test AF-1 activity in the absence and in the presence of the hormone binding domain (containing AF-2), respectively. We show that the amino-terminal 149 amino acids contain all of the sequences required for transcriptional activation by the A/B region. Thus, AF-1 is encoded within the first exon of the ER gene (amino acids 1-150; see Ponglikitmongkol et al. (1988)). Amino acids 151-180, which are not involved in AF-1 activity, are encoded in the same exon as the first zinc finger of ER DBD. Furthermore, deletion of amino acids 1-50 has little effect on transactivation using the present responsive promoters. A mutant that contains only amino acids 51-149 of the A/B region was as active as HEG0, whereas deletions within amino acids 51-149 caused a drastic reduction in transactivation, showing that the whole of this region is required for the activity of AF-1 on its own (Fig. 5).


Figure 5: Localization of the activating domains in hER AF-1. The hER A/B region is represented by a line, and the numbers refer to the amino acid positions. The amino acids responsible for AF-1 transcriptional activity in animal and yeast cells are boxed; those responsible for synergism with AF-2 in HeLa cells are dotted.



We have previously demonstrated that AF-1, on its own, is unable to activate transcription in HeLa cells from any promoter (Kumar et al., 1987; Tora et al., 1989a; Berry et al., 1990) and that AF-2, which activates transcription very weakly from minimal promoters, requires the synergistic effect of AF-1 to activate transcription efficiently from such promoters in HeLa cells (Tora et al., 1989a). We show here that deletion mutants of AF-1, which are only weakly active on their own, can nevertheless synergize with AF-2 to transactive in HeLa cells. Our data suggest that amino acids 51-93 and 102-149 are sufficient for synergism between AF-1 and AF-2 (Fig. 5).

We and others have shown that steroid receptors can activate transcription when expressed in yeast cells (see Introduction). We have also shown that a hER mutant, which contains a deletion of the HBD, can activate transcription constitutively and as efficiently as the wild-type receptor (White et al., 1988; Berry et al., 1990; Metzger et al., 1992). In fact, as it is the case for a minimal promoter in CEF cells, AF-2 contributes very little to transcription activation in yeast from a chimeric GAL1-LacZ reporter construct. Accordingly, we found here that the A/B region appended to the GAL4 DBD efficiently activates transcription in yeast. To further characterize the AF-1 function in this simple eukaryotic system, we have tested deletion mutants of the A/B region linked to the GAL4 DBD or to CDEF regions of hER. We have identified three small regions that can activate transcription efficiently on their own: amino acids 1-62, 80-113, and 118-149 are 80, 60, and 70% as active as the whole A/B region, respectively, indicating that multiple activating domains can operate independently in yeast. Two of these regions are comprised within the region (51-149) required for transcriptional activity in CEF cells (Fig. 5). However, the 62 amino-terminal amino acids, which contribute very little, if at all, to the activity of AF-1 in CEF cells are highly active in yeast, which raises the possibility that this region may be involved in some cell-specific and/or promoter-specific transcriptional activation in animal cells.

None of the sequences that are required for AF-1 activity in animal or in yeast cells are significantly similar to those that have been characterized as activating domains in other transactivators, if one excepts similarities in amino acid composition. Examination of the amino acid composition of the core of the activating domain in CEF cells (amino acids 51-149) reveals significantly high proline (16% versus 5.8% for the complete protein) and tyrosine (8% versus 3.8%) content, and low levels of isoleucine (0% versus 3.1%), aspartic acid (0% versus 4%), lysine (0% versus 5%) and cysteine (0% versus 2%). Proline-rich regions have been identified in the transcriptional activation domains of several transcription factors ( e.g. CTF/NF1, progesterone receptor, Oct2, AP2) (Mermod et al., 1989; Gerster et al., 1990; Williams and Tjian, 1991; Meyer et al., 1992). A comparison of the amino acid composition of the 99-amino acid core of AF-1 with such proline-rich activation regions did not reveal any primary sequence similarity. The small amino acid stretches of the A/B region ( i.e. 1-62, 80-113 and 118-149), which are highly active in yeast, are also characterized by a high proline content (10, 18, and 19%, respectively). One phosphorylation site (Ser-118) involved in the transcriptional activity of the A/B region has been identified in hER expressed in COS-1 cells and HeLa cells (Ali et al., 1993b; Le Goff et al., 1994). However, Ser-118 is not phosphorylated in yeast, and mutation of this amino acid to an alanine does not significantly affect the transcriptional activity of A/B region in yeast.()

We have previously shown that the agonistic activity of OHT can be ascribed to AF-1 (Berry et al., 1990; Metzger et al., 1992). Using A/B deletants in CEF cells and in yeast, we further show here that the mechanisms underlying the induction of AF-1 activity are most probably the same in the presence of E2 or OHT, since the same sequences of the A/B region are required in the two instances.

We and others have proposed that different classes of activators may interact with the basic transcriptional machinery through TIFs (Tasset et al., 1990; Martin et al., 1990; Goodrich et al., 1993; Hoey et al., 1993; Gill et al., 1994). In this respect, the observation that the minimal hER AF-1 domain, which is efficient in animal cells, contains two activating domains that can independently activate transcription in yeast suggests that some higher eukaryotic activating domains were formed by the combination of primordial activating modules. The presence of the corresponding primordial TIFs in yeast may account for the observation that many higher eukaryotic transactivators are functional in yeast (see Introduction for references). These primordial TIFs may not be expressed in all cell types of higher eukaryotes, which instead would contain new TIFs recognizing activators generated through multiple combinations of a limiting number of primordial activating modules. In the course of evolution this may have led to a combinatorial generation of the numerous cell-specific transcriptional activators, which are required for the complex spatio-temporal control of gene expression in higher eukaryotes.

  
Table: Comparison of transcriptional activity of HEG0 and A/B deletants in the presence of E2 or OHT in CEF and yeast cells

Transcriptional activities of the activators in CEF and yeast were determined as described in Figs. 2 and 4, respectively, in the presence of either E2 (10 n M) or OHT (1 µ M) as indicated. The -fold induction (ratio between transcriptional activation obtained in the presence and absence of ligand) is given in brackets.



FOOTNOTES

*
This work was supported by funds from CNRS, INSERM, the Centre Hospitalier Universitaire Régional, the Association pour la Recherche sur le Cancer, the Fondation pour la Recherche Médicale, the Human Frontier Science Program, and the Collège de France. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipients of a fellowship from the Université Louis Pasteur.

Present address: CRC Laboratories, Dept. of Medical Oncology, Charing Cross Hospital, Fulham Palace Rd., London W68RF, United Kingdom.

**
To whom correspondence should be addressed. Tel.: 33 88 65 32 13; Fax: 33 88 65 32 03.

The abbreviations used are: ER, estrogen receptor; DBD, DNA binding domain; ERE, estrogen response element; HBD, hormone binding domain; AF, transcriptional activation function; hER, human estrogen receptor; CEF, chicken embryo fibroblast; OHT, 4-hydroxytamoxifen; TIF, transcriptional intermediary factor; E2, 17-estradiol; CAT, chloramphenicol acetyltransferase.

D. Metzger, S. Ali, J.-M. Bornert, and P. Chambon, unpublished results.


ACKNOWLEDGEMENTS

We thank D. Tasset for the gift of AB-GAL and J. White and Y. Lutz for the gift of 2GV3 and 3GV2 antibodies. We also thank Dr. A. Wakeling and ICI (UK) for provinding 4-hydroxytamoxifen and S. Johnston for the gift of the yeast strain SC30. We also thank the cell culture group for providing cells, F. Ruffenach and A. Staub for oligonucleotide synthesis, the secretarial staff for typing the manuscript, C. Werlé, S. Metz, B. Boulay, and J. M. Lafontaine for preparing the figures, and J. Clifford for critical reading of the manuscript.


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