Function of N-Terminal Transactivation Domain of the Estrogen Receptor Requires a Potential {alpha}-Helical Structure and Is Negatively Regulated by the A Domain

Raphaël Métivier, Fabrice G. Petit1, Yves Valotaire and Farzad Pakdel

Equipe d’Endocrinologie Moléculaire de la Reproduction UPRES-A CNRS 6026 Université de Rennes I 35042 Rennes cedex, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transcriptional activation by the estrogen receptor (NR3A1, or ER) requires specific ligand-inducible activation functions located in the amino (AF-1) and the carboxyl (AF-2 and AF-2a) regions of the protein. Although several detailed reports of ER structure and function describe mechanisms whereby AF-2 activates transcription, less precise data exist for AF-1. We recently reported that the rainbow trout and human estrogen receptors (rtERs and hERs, respectively), two evolutionary distant proteins, exhibit comparable AF-1 activities while sharing only 20% homology in their N-terminal region. These data suggested that the basic mechanisms whereby AF-1 and the ER N-terminal region activate transactivation might be evolutionary conserved. Therefore, a comparative approach between rtER and hER could provide more detailed information on AF-1 function. Transactivation analysis of truncated receptors and Gal4DBD (DNA binding domain of the Gal4 factor) fusion proteins in Saccharomyces cerevisiae defined a minimal region of 11 amino acids, located at the beginning of the B domain, necessary for AF-1 activity in rtER. Hydrophobic cluster analysis (HCA) indicated the presence of a potential {alpha}-helix within this minimal region that is conserved during evolution. Both rtER and hER sequences corresponding to this potential {alpha}-helical structure were able to induce transcription when fused to the Gal4DBD, indicating that this region can transactivate in an autonomous manner. Furthermore, point mutations in this 11-amino acid region of the receptors markedly reduced their transcriptional activity either within the context of a whole ER or a Gal4DBD fusion protein. Data were confirmed in mammalian cells and, interestingly, ERs with an inverted {alpha}-helix were as active as their corresponding wild-type proteins, indicating a conserved role in AF-1 for these structures. Moreover, using two naturally occurring rtER N-terminal variants possessing or not the A domain (rtERL and rtERS, respectively), together with A domain-truncated hER and chimeric rtER/hER receptors, we demonstrated that the A domain of the ER plays an inhibitory role in ligand-independent activity of the receptor. In vitro and in vivo protein-protein interaction assays using both rtER and hER demonstrated that this repression is likely to be mediated by a ligand-sensitive direct interaction between the A domain and the C-terminal region of the ER.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroid hormones such as estrogen trigger and govern essential functions such as growth, differentiation, and the functioning of many target tissues. They also have dramatic influences on proliferative and metastatic states of breast cancer cells (1). Therefore, it is important to understand the precise mechanisms underlying the action of estradiol (E2). An important advance in this research was the demonstration that estrogenic hormones exert their effects on the regulation of gene transcription through interaction with at least two predominantly nuclear-localized (2) specific receptors [NR3A1 and NR3A2 (3), or estrogen receptor-{alpha} (ER{alpha}) and estrogen receptor ß (ERß), respectively (4)]. ER is a trans-acting factor, binding specific cis-elements (estrogen responsive elements, or ERE) located within the regulatory regions of genes (5). Upon binding of ligand, a conformational change allows stabilization of receptor homodimers and interaction with EREs (6, 7). The importance of estrogen signaling is evident from ER genes knockout mice, which exhibit some striking phenotypes including infertility and defects in bone (for review see Ref. 8 and references therein).

Comparison of steroid hormone receptor sequences permitted the emergence of a superfamily of nuclear receptors (NRs) (9) that includes receptors for steroid and other hormones, as well as orphan receptors for which no ligand is known. There is now some evidence that ligand binding in this family was acquired during evolution (10). Sequence comparisons between ERs from different species have shown that these proteins can be divided into six functionally distinct domains, denoted A to F (11). This division was extended to the entire superfamily. While the C domain containing two zinc fingers is responsible for the specific interaction with steroid-responsive elements (12, 13) and has a constitutive dimerization property (7), the E region located in the C terminus is involved in hormone binding (11) and hormone-dependent dimerization (14) and contains a transactivation function (AF-2) (15, 16). Recently, an activation function called AF2a residing in the boundary region between the hinge (D) and the E domains of the human ER (hER) was reported (17). Although the N-terminal region of ERs is the less conserved domain of the protein during evolution, it contains an additional AF-1 that could function in an hormone-independent manner (11, 18, 19, 20). In the context of a full-length receptor, AF-1 is only active after ligand binding. Full activity of the ER is thought to be due to a synergistic effect between the AFs (20, 21). While no clear role is yet ascribed for the F domain in transcriptional activation by the receptor, it may be important for discrimination between agonists and antagonists (22) and for E domain-mediated dimerization signal (23).

Many studies have focused on AF-2 function in ER, leading to a better understanding of the sequences and mechanisms involved in both ligand recognition and discrimination (24, 25, 26, 27) and in transcriptional activation by the liganded receptor (for review see Refs. 28, 29, 30). The present study focuses on the N terminus of ER, since this region is critical for the antiestrogenic properties of some compounds (18, 31) and may be responsible for interaction with coactivators (32, 33, 34) or corepressors as shown for the thyroid receptor (35). The transactivation function AF-1 begins to be considered as the most important one of the receptor, directing its activity depending upon the cell context (36, 37). Different regions of the ER were previously defined as sufficient for AF-1 activity according to the cell type (19). However, to date, the AF-1 region in ER has not been precisely defined since, in yeast as well as in mammalian cells, regions identified overlap the entire B domain. We have previously shown that rtER, like hER, possesses a functional AF-1 (38), despite poor sequence homology (20%) between the two proteins. Therefore, we postulated that the molecular basis of AF-1 might be conserved in the two species, and we focused our study in a comparative manner, by identification of the regions implicated in rtER AF-1. A minimal sequence of 11 residues possessing intrinsic transactivation potency was identified to be necessary for rtER AF-1. A predictive method for detecting secondary structure indicates that this region could adopt an {alpha}-helix conformation. Such a structure was also found in hER, which exhibited similar characteristics, and the two {alpha}-helices were perfectly interchangeable in terms of transactivation.

Additionally, comparison of the transcriptional activity of a naturally occurring rtER truncated for its A domain (rtERS) with the full-length form (rtERL) and hER{alpha} (39, 40), indicated that the A domain could be involved in repression of ligand-independent activity. In this study, we verified this hypothesis by generation of an hER truncated for its A domain, and interestingly, direct protein-protein interaction assays with ER domains demonstrated that the A domain interacts with the C-terminal region. This interaction appeared to be stronger in the absence of E2, and was weakened by E2 treatment. These data could therefore explain how the A domain represses the hormone-independent activity of the ER. This paper thus reports the characterization of an evolutionary conserved mechanism by which the ER N-terminal region functions, i.e. the requirement for a potential {alpha}-helix, and an indirect repression by the A domain in the absence of ligand.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Comparative Analysis of rtER and hER Transactivation Potency in Yeast
Although a large number of reports describe the role and function of ER domains or subdomains, considerably less attention has been focused on the role of the N-terminal region. Previous reports have implicated large sequences in the N terminus in AF-1 activity in different cell contexts (19, 21). A major influence of this transactivation function is in antiestrogens activity (18, 31). Therefore, we decided to analyze in detail the function of the ER N-terminal region, by employing a comparative approach between the hER and the rainbow trout (rt) ER. We have recently identified and characterized another rtER isoform, called rtERL, from trout ovary produced by the same gene (40), which possesses an additional sequence of 45 amino acids (a.a) (Fig. 1AGo). The earlier isolated rtER form, now called rtERS, showed no homology in its N terminus with the A domain present in mammalian ERs. This observation together with multiple sequences alignments and the recent isolation and characterization of two similar isoforms in chicken (41) led us to the conclusion that this upstream sequence is the rtER A domain (Fig. 1AGo). Note that homology between rtER and hER A domains does not exceed 15%. In yeast, both AF-1 and AF-2 of ER are functional, but with an acute sensitivity to AF-1 (19, 36, 38), providing a powerful model with which to study the ER AF-1. To compare transcriptional activity, the cDNAs corresponding to these proteins were subcloned in the YEpucG yeast expression vector (39, 42). BJ2168 yeast host strain was cotransformed with the pLG{Delta}178/3EREc reporter plasmids (Lac Z gene controlled by the minimal Cyc1 yeast promoter and three consensus EREs, respectively) and the YEpucG constructs. Transformants were selected for auxotrophy on the appropriate media, and then ß-galactosidase activity was measured after a 4-h incubation with or without 10-6 M E2. This comparative analysis of transcriptional activation between the two rtER isoforms and hER in the absence of hormone in yeast showed a differential behavior. Indeed, the shorter isoform (rtERS) lacking the A domain exhibited a consistent E2-independent activity (Fig. 1BGo and Ref. 40), whereas hER and rtERL, which both possess such a region, did not exhibit a similar characteristic (Fig. 1BGo). This ligand-independent activity was ascribed to the AF-1 region since AF-2 is not exposed to the transcriptional machinery in the absence of ligand and thus cannot be active (22, 43). Interestingly, a chimeric rtER containing the hER A-B region instead of its own B domain [named rtER(hAB)] did not exhibit any hormone-independent activity (Fig. 1BGo). However, after E2 treatment, transcriptional activity of this chimera was similar to wild-type receptors. This indicates that the human AF-1 could function in the rtER protein context and that a region in this hAB sequence represses the ligand-independent activity of rtERS. Since the only difference between rtERS and rtERL is the presence of an A domain, we postulated that this domain could be responsible for a repressive effect on AF-1. To test this hypothesis, a truncated hER lacking its A domain (hER{Delta}1–37) was constructed, and, interestingly, it exhibited a basal activity representing 17% of the total activity (Fig. 1CGo). To study directly the AF-1, truncated receptors for N- and C-terminal regions were constructed in both ER species (namely rtBD and hAD), and their transactivation abilities were assayed on the yeast 3EREc-LacZ reporter. As previously shown on other reporter plasmids (38), rtERS possesses, as does hER, a functional AF-1 (Fig. 1CGo), as the two rtBD and hAD constructs constitutively activate transcription, to 75–80% of the maximum induction obtained with the full-length ER. In contrast, the activity of the C-terminal region (rtCF and hCF) represented about 10% the activity of the full-length ER. These data show that the N-terminal region of rtER, like that of the hER, functions in a hormone-independent manner when isolated from its C-terminal region.



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Figure 1. Comparative Analysis of Transcriptional Activities of Two Phylogenically Distant ERs in Yeast Saccharomyces cerevisiae

A, Amino acid representation of the divergence between the two rtER isoforms. The arrow represents the point where sequences begin to be identical. B, BJ2168 yeast cells were cotransformed with the pLG178/3EREc (3 ERE-Cyc-Lac Z) reporter plasmid together with the YEpucG constructs expressing the two rtER isoforms, hER, hER{Delta} 1–37, or the chimera rtER(hAB). After auxotrophy selection, clones were cultured and treated during 4 h with 1000 nM E2 or ethanol (EtOH). ß-Galactosidase activity was quantified by liquid assays, and values from at least four separate experiments in triplicate were expressed as percent of E2 stimulation. C, Yeast cells were transformed with expression vectors containing cDNA encoding different parts of the hER and rtER, whereas a control was accomplished by using the empty YEpucG. Results are expressed as percent of E2 stimulation and represent the mean ± SD from at least four separate experiments.

 
This primary analysis highlighted two interesting facts: the ER A domain has a repressive effect on the AF-1 activity of ER, since when it is deleted, a consistent basal activity is detected; the AF-1 of two evolutionary distant ERs are functional in yeast, suggesting a common mechanism of action. This led us to determine precisely which sequences are required for ER AF-1, using a comparative approach to identify evolutionary conserved mechanisms.

Identification of Sequences Involved in rtER AF-1 by Transcriptional Assay in Yeast
To find evolutionary conserved sequences and define the mechanism of AF-1 transactivation function, the shorter isoform of rtER (rtERS) was an ideal starting point, due to its profound ligand-independent activity in yeast. To determine sequences implicated in AF-1, successive deletions in the N-terminal portion of rtERS were constructed either in the full-length receptor context or in the isolated N-terminal region, and their transactivation potencies were analyzed using the yeast 3EREc-Lac Z reporter. Data illustrated in Fig. 2AGo showed that in contrast to the entire rtER B domain, none of the deleted rtBD for the 111, 50, 34, or 17 first amino acids were able to induce the transcription of the reporter gene, indicating that an important sequence for AF-1 resides at the beginning of the B domain. Previous studies have shown that in some cellular contexts, AF-1 activates transcription poorly on its own but could synergize with AF-2 (20, 21). Whereas yeast is well characterized as an AF-1-dependent cell context, it is possible to explain the lack of activity of the truncated receptors used in Fig. 2AGo by a nondetectable AF-1 activity. Thus, to detect these residual AF-1 activities through synergism with AF-2, N-terminally truncated full-length receptors were constructed and expressed in yeast. As shown in Fig. 2BGo, total or successive deletions of N-terminal residues generated receptors that were able to induce the yeast reporter gene in a hormone-dependent fashion. However, the maximum activity obtained with these truncated receptors in the presence of 10-6 M E2 represented only 10–12% of the wild-type receptor activity. Thus, these mutants are transcriptional activators similar to the rtCF protein lacking the A and B domains. To determine more precisely the amino acids necessary for AF-1 activity, receptors with either a.a 5–17 or a.a. 8–17 deleted were constructed and introduced together with the 3EREc-LacZ reporter into yeast. Data illustrated in Fig. 2CGo show that rtERS{Delta}5–17 and rtERS{Delta}8–17 were able to induce reporter gene activity in a hormone-dependent fashion, with a maximal E2 stimulation that only represented 11% of the full-length ER activity. To confirm that the absence of AF-1 activity of these constructs was specific of the amino acids deleted, and not dependent upon the simple fact of deletion, a rtERS lacking the seven first a.a was constructed (rtERS{Delta}1–7). This truncated receptor retained 80–90% of the wild-type ER activity (Fig. 2CGo) and therefore provided a good control for these experiments. These results indicate that a.a. 8–17 of the rtERS isoform are necessary for its AF-1 activity and that they might play an essential role in the full activity of the rtER resulting from the synergistic effect of AF-1 and AF-2 transactivation functions.



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Figure 2. Determination of the rtER Regions Implicated in AF-1 in Yeast

Yeast cells containing the 3EREc-Cyc-Lac Z reporter plasmid were transformed with either no vector or plasmids expressing the different constructs described on the left side of each panel. After growth on medium containing either 10 or 1000 nM E2, or ethanol (EtOH), ß-Galactosidase activity (Miller Units) was measured. Values represent mean ± SD from at least four separate experiments. These experiments demonstrate that a.a. 7–17 of rtERS are necessary for AF-1.

 
Deletion of the rtERS 18 First Amino Acids Is Sufficient to Abolish the Activity of a Gal4DBD Fusion
To confirm the presence of a transactivation function in the rtER N-terminal domain in an alternative system, a series of constructs encoding different parts of the receptor linked to the Gal4DBD were generated. Transactivation potency of these chimeric proteins was tested in the Y190 yeast cells containing the Lac Z reporter gene under control of three responsive elements for the yeast Gal4 transcription factor (UASG) (Fig. 3Go). The rtERS fused to the Gal4DBD did not exhibit any activity in the absence of E2. However, addition of the hormone induced transcriptional activity of this fusion protein, as described for hER (44). The fusion of the rtER N-terminal domain to the Gal4DBD generated a constitutively active protein (rtERS{Delta}153–575/Gal4DBD), confirming the fact that this region contains a transactivation function that could operate independently of the remainder of the receptor (Fig. 1Go). In contrast, the rtER C-terminal region fused to the Gal4DBD (rtCF/Gal4DBD) was not functional with or without hormone, indicating that the AF-2 of rtER is not active by itself in this context. This is in agreement with previous work that has proposed that binding of E2 to full-length hER fused to Gal4DBD induces a conformational change in the chimeric protein enabling AF-1 to mediate transcriptional activation (44). Strikingly, neither the rtERS{Delta}1–111 nor the rtERS{Delta}1–17 truncated receptors fused with the Gal4DBD were able to induce any Lac Z activity, while the rtERS{Delta}1–7 receptor was E2-dependent active (Fig. 3). However, this latter construct exhibited only a weak activation of about 10 Miller Units, although it possessed 80–90% of the full-length receptor transcriptional ability (Fig. 2CGo). This could be explained by the fact that we used a low-level expression vector (pGBT10) in the case of the rtERS{Delta}1–111, rtERS{Delta}1–17, and rtER{Delta}1–7 inserts. Therefore, the lack of transcriptional activity of the rtERS{Delta}1–111/Gal4DBD or rtERS{Delta}1–17/Gal4DBD could be due to a weak expression of these proteins, resulting in an activity below the sensitivity of the system. However, the use of a filter-lift assay for Lac Z activity, known to be more sensitive than a liquid assay, did not enable detection of ß-galactosidase activity, even over a very long incubation. Western experiments shown in Fig. 3BGo indicated that the truncated receptors expressed using the pGBT10 plasmid were produced at equivalent levels in yeast. Because these constructs possess both LBD and DBD, they should be able to dimerize, since the LBD allows ligand-dependent dimerization and the DBD allows a ligand-independent dimerization ability (7, 14). Then, to test the functionality of these mutant receptors, their interaction with the C-terminal region of rtER fused to the Gal4AD (rtERS{Delta}1–223/Gal4AD) was assessed, in the presence of ligand. Results shown in Fig. 3CGo confirmed that these constructs were functional. All these data indicate that the rtERS{Delta}1–111/Gal4DBD or rtERS{Delta}1–17/Gal4DBD have no intrinsic transcriptional activity. Although the rtER AF-2 fused to the Gal4DBD was not active in this system, this domain might influence the potential AF-1 activity of the two truncated fusion proteins. Thus, constructs devoid of AF-2 were generated (rtB{Delta}1–111/Gal4DBD and rtB{Delta}1–17/Gal4DBD) and introduced in this one-hybrid system. As illustrated in Fig. 3Go, neither of the two fusions were transcriptionally active, whereas they were expressed at similar levels in yeast. Therefore, it seems that the rtERS first 17 a.a. are necessary for AF-1 function in this system, since the activity of the rtERS/Gal4DBD in the presence of E2 is thought to be mediated through AF-1.



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Figure 3. Transactivation Potency of Truncated rtER Fused to the Gal4DBD

A, Schematic representation of the different Gal4DBD fusion proteins used (numbers refer to the amino acid positions). Y190 yeast cells containing the Lac Z reporter gene placed under control of three responsive elements for the yeast Gal4 activator (UASG) were transformed with plasmids expressing Gal4DBD alone (control) or the different fusion proteins indicated. The level of expression of these constructs was assayed by Western blot using anti-Gal4DBD antibodies After growth on selective media lacking histidine, transformants were subjected to 1000 nM E2 or ethanol (EtOH) treatment, and ß-galactosidase activity was measured (B). Values represent the mean ± SD from at least four separate experiments. C, To test the functionality of these constructs, their homodimerization property was assayed in two-hybrid experiments using the rtER protein fused to the Gal4 activation domain (Gal4AD). Transformants were selected by growth and treated with 1000 nM E2. ß-Galactosidase activity was measured and values represent the mean ± SD from three experiments.

 
Basic Functionality of ER AF-1 Relies on an {alpha}-Helix Possessing Intrinsic Transactivation Potency
The C-terminal tridimensional structure of NRs is now well characterized since the crystal structure of holo-retinoid X receptor, liganded retinoic acid receptor, thyroid hormone receptor, or ER (45, 46, 47) were defined as a canonical structure of 12 {alpha}-helices (48). Similar structural data are not yet available for the N-terminal region and, therefore, no structure can be ascribed for AF-1. However, a performant predictive method such as the hydrophobic cluster analysis (HCA), based upon the hydrophobicity property of the amino acids (49, 50), could be used to define probabilities for the existence of secondary structures. We followed this predictive approach, and the rtER HCA plot represented in Fig. 4Go, A and B, revealed that an {alpha}-helix (existence probability vs. a ß-sheet of 2.2) could be formed between a.a 14 and 22, overlapping the region defined as being required for AF-1 activity (a.a 8–17). Interestingly, the deletion of the first 4 a.a of this {alpha}-helix (FNYL), occurring in the rtERS{Delta}1–17 construct, leads to the disintegration of the structure by the loss of three hydrophobic amino acids. This could indicate that this structure is required for rtER AF-1. In this case, AF-1 being a ligand-independent transactivation function, a major condition for this structure is to activate, on its own, the transcription state of a target gene. Therefore, we linked a.a 9–26 or 11–23 of rtERS to the Gal4DBD (respectively, rtERS9–26/Gal4DBD and rtERS11–23/Gal4DBD, Fig. 4CGo) and introduced these constructs in Y190 yeast cells containing the 3 UASG-Lac Z reporter gene. While the basal activity of the reporter yeast strain did not exceed 0.15 Miller Units, both fusion proteins were able to stimulate the transcription status of the reporter by almost 5 Miller Units. Such an activity was specific to the sequences fused to Gal4DBD since rtB{Delta}1–111/Gal4DBD did not stimulate the reporter (Fig. 4DGo). To demonstrate the link between transactivation potency and the presence of this potential structure, we introduced point mutations in the corresponding sequence fused to the Gal4DBD (a.a 9–26), converting the hydrophobic amino acids of the chain to proline, an amino acid known to disrupt secondary structure without perturbing the chain charge and polarity. Thus, either the phenylalanine or leucine of the {alpha}-helix was substituted. A 70% reduction in transactivation was observed by mutating the first amino acid of the {alpha}-helix (phenylalanine: rtERS9–26F15P/Gal4DBD), whereas no Lac Z activity was detected with the construct mutated for the central amino acids of the structure (leucine: rtERS9–26L18P/Gal4DBD) (Fig. 4DGo). The residual activity obtained with the rtERS9–26F15P/Gal4DBD fusion protein can be explained by the fact that this sequence could still present an {alpha}-helix conformation, but shorter than the original and with a lower probability. As a control, an alanine external to the structure was mutated (Fig. 4CGo). This mutation (rtERS9–26A12P/Gal4DBD construct) induced a slight increase of approximately 50% of the reporter activation compared with the wild-type sequence, which may be due to a better exposition of the structure to the transcription machinery. In fact, this {alpha}-helix is preceded by a succession of glycines and alanines that could give flexibility to the protein chain. Substitution of one of the alanines by a proline may create a bend, perhaps more propitious to an interaction of the structure with the transcriptional apparatus. Western blot experiments carried out with yeast extracts demonstrated an equivalent and correct expression of the fusion proteins (Fig. 4CGo).



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Figure 4. Identification of a Potential {alpha}-Helix in rtERS N-Terminal Region Possessing Intrinsic Transactivation Potency

A, Illustration of the HCA plot analysis for the rtERS N terminus. The nomenclature used is classical: hydrophobic amino acids are circled, proline is represented by *, glycine by {diamondsuit}, serine by {square}, and threonine by {dotsquare}. B, Simplified one-dimensional HCA representation of the HCA plot (1 represents an hydrophobic amino acid while the star is used for prolines and 0 for the others). Numbers reflect the statistical relevance of the secondary structure boxed. C, Gal4DBD fusion proteins (rtERS 9–26 and rtERS 11–23) containing the potential {alpha}-helix, with or without substitution of some residues in proline (A12P, F15P and L18P) were constructed and introduced in Y190 yeast cells. Correct and equivalent expression of these constructs was checked by Western blot using an anti-Gal4DBD antibody. As controls, vectors expressing Gal4DBD alone or the rtB{Delta}1–111/Gal4DBD protein were also included. D, After selection, ß-galactosidase activity was measured. Values represent the mean ± SD from at least four separate experiments.

 
Since our data identified a potential {alpha}-helix located at the beginning of the B domain to be important for rtER AF-1, we investigated whether or not this structure was conserved during evolution. An HCA-based structural study performed on the hER N-terminal region revealed a striking conservation of many potential structures (compare Fig. 5Go, A and B, with Fig. 4Go, A and B). The most evident are the two ß-strands separated by a rich proline sequence and preceded by a coil (a.a. 106–133 and a.a. 63–96 for hER and rtERS, respectively). Interestingly, the hER HCA plot displayed an {alpha}-helix in the beginning of the B domain, located within a.a 39–44 (Fig. 5Go, A and B). Previous reports characterized a region between a.a. 29 and 63 to be involved in hER AF-1 (19, 31). To examine the transactivation potency of this potential {alpha}-helical structure in this region, a.a 35–47 were linked to the Gal4DBD (hER35–47/Gal4DBD), with or without point mutations resulting in substitution of either the leucine or tyrosine residues to proline (hER35–47L39P or hER35–47Y43P, respectively). A control was also generated by substitution of a tyrosine to phenylalanine (hER35–47Y43F), which conserved the basis of structure, charge, polarity, and sterical properties of the chain. These constructs were expressed in Y190 yeast strain, and liquid ß-galactosidase assays were performed. Results illustrated in Fig. 5DGo demonstrate that the hER 35–47 sequence does possess an intrinsic ability to activate the transcription state of the 3UASG-LacZ reporter nearly 6 to 7 Miller units. Moreover, disrupting the structure with point mutations to substitute a proline for either the leucine or the tyrosine abolishes this activity, whereas substitution of the tyrosine to phenylalanine did not affect the constitutive activity of the GAl4DBD fusion protein (Fig. 5DGo). Since the expression of these fusions in yeast is similar (Fig. 5CGo), this indicates that, as in rtER, a small sequence with a specific spatial organization could activate transcription. To confirm the implication of this structure in AF-1 in the whole receptor context, we analyzed the transcriptional ability of an hER deleted for this potential secondary structure (hER{Delta}1–44). As illustrated in Fig. 6Go, the deletion of this region produces a 55% loss of transcriptional activity of the receptor. Note also that this receptor mutant exhibited a more significant reduction (~80%) of its ligand-independent activity, compared with the hER{Delta}1–37 (4.7 Miller units vs. 25.3 for the hER{Delta}1–37). As performed for Gal4DBD fusion proteins, we introduced point mutations in the {alpha}-helix of the hER minus its A domain (hER{Delta}1–37) for which a ligand-independent activity is detectable. More precisely, we mutated the first amino acid of the structure (leucine at position 39) and the central amino acid (tyrosine at position 43) into prolines, and examined the transactivation abilities of the resulting mutant proteins in yeast (hER{Delta}1–37L39P and hER{Delta}1–37Y43P, respectively) (Fig. 6Go). This analysis showed that the mutated receptors have a reduced ability to activate gene transcription, similarly to hER{Delta}1–44, confirming that the structure of this small region is important for AF-1 activity.



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Figure 5. hER B Domain Includes a Transcriptionally Active {alpha}-Helix

A and B, HCA plot for the hER N terminus in classical or simplified one-dimensional representations. Nomenclature is the same as in Fig. 4Go. C, Gal4DBD fusion proteins including the sequence for the potential {alpha}-helix with or without point mutations were expressed in the Y190 yeast cell strain, whereas control was produced by using the Gal4DBD alone. Level of expression of these mutants was also observed. D, Reporter gene activation was quantified, and results are expressed as the mean ± SD from at least four separate experiments.

 


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Figure 6. The hER Potential {alpha}-Helix Is Implicated in AF-1

Yeast strain containing the 3-ERE-Cyc-LacZ reporter was transformed with vectors expressing hER, hER{Delta}1–37, hER{Delta}1–44 that does not possess the {alpha}-helix characterized, or two receptors mutated in the {alpha}-helix without the A domain (depicted in panel A). Correct expression of these constructs in yeast was assayed by Western blot using the H222 antibody. B, After growth on selective media, stable transformants were treated for 4 h with ethanol or 10 or 1000 nM E2, and ß-galactodidase was measured. Values are expressed as the mean ± SD for at least three independent experiments.

 
As shown with the rtERS (hAB) chimera (Fig. 1DGo), the hER AF-1 could function within the rtER context, suggesting that the two AF-1 sequences are interchangeable. To answer the question of a conserved role of these structures in the whole receptor context, two chimeras were constructed by exchanging the two {alpha}-helices either containing or not a crucial point mutation (Fig. 7AGo). Note that one or two flanking amino acids were conserved in these transpositions to preserve the environment of the helical structure. Transactivation ability of these receptor chimeras was tested in yeast containing the 3 ERE-Cyc-Lac Z reporter. As illustrated in Fig. 7BGo, the replacement of the human helix by its rainbow trout counterpart, or vice versa, did not affect the activity of the receptors, either in the absence or presence of E2. Indeed, the hER{Delta}1–37(rt{alpha}) as well as the hER{Delta}1–37 or the rtER{Delta}1–7(h{alpha}) activated the reporter gene state from 150 to 250 Miller Units. Point mutations within the potential helix of these chimeric receptors dramatically reduced their transcriptional activity. This reduction occurred to a greater extent in the case of the rtER{Delta}1–7(h{alpha}Y43P), confirming the absolute requirement of such a structure for activity of rtER in yeast. Together, these data suggest that a small region located in the beginning of the B domain that could adopt an {alpha}-helix conformation is necessary for ER AF-1 in yeast.



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Figure 7. The {alpha}-Helices of Both Receptors Are Perfectly Interchangeable

A, To determine whether the {alpha}-helices identified within the rtER and hER are playing the same role in their AF-1, transposed chimeric receptors containing the wild-type or point mutant sequences were constructed. Human sequence is underlined, and sequences transposed are boxed. Point mutations are indicated with an asterisk. B, Transcriptional activities of these chimeric receptors were tested in a yeast strain containing the 3-ERE-Cyc-LacZ reporter, and ß-galactodidase was measured on stable transformants treated either with ethanol (EtOH) or 10 to 1000 nM E2. Bars represent the mean ± SD of values obtained in three independent experiments.

 
Involvement of the {alpha}-Helix for ER AF-1 in Mammalian Cells
Although yeast is a powerful model with which to study the transactivation abilities of NRs, it remains a simple cellular model. To verify the implication of {alpha}-helices in AF-1 in both ER species in more complex models, transient transfection experiments were conducted. Three cell lines were chosen for this study in accordance with their dependence or not on ER AF-1: HeLa cells in which AF-2 is the dominant or even the only activation function driving ER activity; HepG2 cells, which constitute the opposite cell context (17, 18, 21); and the CHO cell line in which both AFs are functional (our unpublished observations). These three cell lines were transiently transfected by a classical calcium phosphate/DNA coprecipitation method. Transient expression was performed during 36 h in steroid-free media containing 10-6 M E2 or ethanol as vehicle control. The transcriptional activity of the various mutants was assessed by cotransfection of a luciferase reporter driven by the thymidine kinase promoter under control of one ERE (ERE-TK-Luc). Luciferase activities were normalized using the SV/ß-galactosidase reporter (pCH110) as an internal control for transfection efficiency. Results expressed as fold induction of the reporter are shown in Fig. 8Go. The activity of these constructs was identical in HeLa Cells. The luciferase reporter was activated 8- to 10-fold in the presence of ligand, indicating that each of the truncated receptors is functional in mammalian cells. Importantly, activation by the hCF construct was similar to the full-length receptor, confirming that AF-2 is the only transactivation function used in HeLa cells. Moreover, these results show that all of the deletions or point mutations performed in the two ER proteins do not affect their AF-2 activity. In the AF-1-dependent cell context, truncated receptor activities were strikingly different, and differed to a greater extent in HepG2 than CHO cells, illustrating their relative relevance to this transactivation function. For instance, in CHO and HepG2 cells, the two rtER isoforms were similar activators in the presence of ligand, stimulating the reporter up to 10-fold. However, the shorter isoform exhibited a basal activity representing 25% or 45% of the E2-stimulated activity in CHO or HepG2 cells, respectively. Since the same observation could be made in these contexts when comparing hER to the A domain truncated form hER{Delta}1–37, this confirms the hypothesis of a repressive function for the A domain of the ER. In HepG2 cells, deletion of the 111 first amino acids of the rtER resulted in a 80% reduction of reporter induction, whereas disruption of the {alpha}-helix occurring within the rtER{Delta}8–17 construct generated a 50% loss in transcriptional efficiency. This indicates that in mammalian cells, as opposed to yeast, other regions are implicated in rtER AF-1, as it was demonstrated for hER (19). The hER{Delta}1–44, hER{Delta} 1–37L39P, and hER{Delta}1–37Y43P activated the reporter from approximately 4- to 7-fold in CHO or HepG2 cells, respectively, demonstrating that the human helical homolog of the rtER is also important for AF-1 in mammalian cells. On the other hand, these latter constructs were more potent activators than the hCF receptor, which activated 2-fold the reporter in HepG2 cells. This also confirms that other regions in the N terminus of hER are implicated in AF-1. The transposed hER including the rtER helical structure was as active as the wild-type ER in the two AF-1 sensitive cell contexts, whereas the transposed hER, including the mutated version of the structure, stimulated the reporter to the same level as the hER{Delta}1–44 or point mutant constructs. The inverse transposed receptors (rtER with the human structures) gave rise to similar results. These data obtained in transient transfection experiments confirm the implication of the {alpha}-helical structures in their cognate AF-1, but also in the context of evolutionary distant protein. These results also demonstrate that regions other than this structure are involved in ER AF-1 in mammalian cells.



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Figure 8. The ER AF-1 Requires the {alpha}-Helix in Mammalian Cells

To confirm the importance of the rtER and hER {alpha}-helices in a more complex cellular context than yeast, transient transfection experiments were performed using different cell lines depending upon their relative sensitiveness to AF-1: HeLa (A), CHO (B), and HepG2 cells (C). The three cell lines were transfected at 60–70% confluence with a classical calcium phosphate/DNA precipitation protocol using an ERE-TK-Luc reporter and pCH110 as internal control. CHO cells were transfected in 24-well plates with 25 ng of expression vector, whereas 250 ng of expression vector were used in 6-well plates for HeLa and HepG2 cells. After 36 h of transient expression in steroid-free media containing ethanol (EtOH) or 10-8 M E2, luciferase activity was quantified using a luminometer and ß-galactosidase assay was performed. Luciferase activities were normalized for transfection efficiency with the ß-galactosidase activity and expressed as the fold induction vs. the activity obtained with the promoter alone. Results from at least three experiments in triplicates are expressed as the mean ± SEM.

 
Inhibitory Effect of the A Domain on ER Ligand-Independent Activity Could Be Mediated by a Direct Interaction between the A Domain and the C-Terminal Region
As previously shown, the comparison of the rtERS and hER transcriptional activity in the absence of hormone both in yeast and mammalian cells clearly revealed that the A domain of ER could repress the ligand-independent activity of the receptor (Figs. 1Go and 8Go and Ref. 40). This was further substantiated by truncation of the hER A domain (hER{Delta}1–37) and isolation of another rtER isoform, rtERL, possessing this domain (Fig. 1BGo). As the aim of this study is to better comprehend ER N-terminal functions, we attempted to characterize how the A domain could act. As illustrated in Fig. 1CGo, the hAD construct was active independent of E2, sharing the same pattern of transactivation as the rtBD construct devoid of the A domain. Moreover, constructs possessing the entire A+B domains of the rtER or hER fused to the Gal4DBD showed similar activities to the B domain alone (data not shown). Thus, we hypothesized that other domains of the ER could be implicated in the repressive effect of the A domain, by a process implying a direct interaction between the A domain and other parts of the receptor.

Pull-down assays were used first to study the in vitro interaction between glutathione-S-transferase fusion with the ER A domain (GST/rtA and GST/hA, Fig. 9Go) and [35S]-labeled ER constructs translated in rabbit reticulocyte lysate that overlap the entire sequence of the two receptors, namely rtERS, rtERS{Delta}1–220, rtERS{Delta}157–575, hER, hER{Delta}1–178, and hER{Delta}272–595 (Fig. 9Go, A and D). As shown in Fig. 9BGo, the three rtER constructs were expressed in the reticulocyte system and migrated at the expected molecular sizes (e.g. 66, 35.5, and 17.8 kDa for rtERS, rtERS{Delta}1–220, and rtERS{Delta} 157–575, respectively). In the pull-down experiment, the A domain interacted with both rtERS and rtERS{Delta}1–220, whereas no interaction was found with the B domain (rtERS{Delta}157–575). The effect of E2 on this interaction was examined (Fig. 9BGo, lanes 4, 8, 9, and 17), using lysates treated either with ethanol or with 10 or 50 µM E2, concentrations known to induce a conformational change in rtER detectable by protease digest analysis (data not shown). This experiment showed that E2 significantly reduces this interaction process. Indeed, quantification with a phosphoimager showed that in the absence of E2, 10% of rtERS labeled protein was retained vs. 5% after E2 treatment, and 7% vs. 2.5% for rtERS{Delta}1–220. As illustrated in lanes 8 and 9, the effect of E2 was maximal at the two E2 doses tested. Effect of two antiestrogens on this interaction was also tested, by treating rtERS{Delta}1–220 lysate with 100 or 500 µM of 4-hydroxytamoxifen (OHT) or ICI164,384. As shown in lanes 10 and 11, OHT was able to compete the interaction between rtER A domain and rtERS{Delta}1–220, but with a lower efficiency than E2 (6 to 4% vs. 2.5%), irrespective of the dose. In contrast, the pure antiestrogen ICI164,384 was unable to affect this interaction, suggesting that the conformational change induced by this ligand is not appropriate to expose AF-1. Similar results were observed with rtERS treated by antiestrogens (data not shown). Similarly, the three hER constructs were expressed at the correct size (66, 46, and 29.8 kDa), and only the hER and hER{Delta}1–178 labeled proteins were retained on the GST/hA beads (Fig. 9EGo). Moreover, E2 was able to reduce this interaction from 10 to 7% or 9.8 to 6.5% in the case of labeled hER or hER{Delta}1–178, respectively. The OHT was also able to reduce this interaction from 19.8% to 9 or 8.7%, depending on the dose, whereas ICI164,384 had no effect. The validity of the experiments was confirmed by performing a Coomassie blue staining of the gels (Fig. 9Go, C and F), demonstrating equivalent loading on the SDS-PAGE gels.



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Figure 9. In Vitro Evidence for an Evolutionary Conserved Interaction between ER A Domain and C-Terminal Region

A and D, Schematic representation of the ER constructs used for in vitro [35S]methionine-labeled translation, and the GST fusion protein with the A domains from rtER (GST/rtA) or hER (GST/hA). B and E, The GST fusion proteins were analyzed by pull-down assays for their binding to the different ER constructs. Pull-down assays were performed in the absence (lanes 3, 7, and 16) or in the presence (lanes 4, 9, and 17) of 50 µM or 10 µM E2 (lane 8), 100 or 500 µM of OHT (lanes 10 and 11), or ICI164,384(ICI, lanes 12 and 13). Input lanes (1 5 14 ) represent 25% of the amount of labeled proteins used in the assay. C and F, Stability of the GST fusion proteins and equal loadings were checked by Coomassie blue staining. Positions of standard markers (S.M.) are indicated.

 
To confirm these interactions in vivo, two-hybrid assays were performed. For this purpose, the A domain from rtERL and hER were fused to the Gal4DBD (rtA/Gal4DBD and hA/Gal4DBD), while the C-terminal (rtERS{Delta}1–223 and hER{Delta}1–178) or N-terminal (rtERS{Delta}149–575 and hER{Delta}181–595) domains were fused to the activation domain of the yeast Gal4 protein (Gal4AD), as well as the rtERS and full-length hER (Fig. 10Go, A and C). Y190 yeast strain was transformed with these fusion constructs alone or in association, and correct expression of the proteins was confirmed by Western blot of whole yeast extracts using anti-Gal4DBD or anti-HA epitope antibodies (data not shown). Positive clones from ß-galactosidase filter-lift assays were cultured and treated or not with 1 µM E2, 50 µM OHT, or 50 µM ICI164,384 for 4 h, and Lac Z reporter gene activation was then quantified in liquid assay. Results illustrated in Fig. 10BGo show that the rtER A domain interacts with the full-length and rtERS{Delta}1–223 constructs, but not with rtERS{Delta}149–575. E2 reduced these protein-protein contacts by approximately 70%. The mixed antiestrogen OHT also reduced this interaction by approximately 45%, whereas ICI164,384 had no effect, as seen in pull-down experiments. Interactions were specific since no transcriptional activation was seen when transforming the Gal4DBD fusions alone or in association with the Gal4AD. Two-hybrid assays using the hER fusions gave similar results. Indeed, the hA/Gal4DBD fusion did not activate transcription alone, or when cotransformed either with the Gal4AD as control or with hER{Delta}181–595 (Fig. 10DGo). On the other hand, significant activity was detected by cotransformation of the hA/Gal4DBD with either hER/Gal4AD or hER{Delta}1–178/Gal4AD constructs. These interactions were not affected by ICI164,384 treatment, but E2 and OHT inhibited this physical interaction by 40% or 25%, respectively. Occurrence of these physical interaction reductions in the presence of E2 and OHT were statistically significant at P < 0.001, as assessed by Student’s t test. From these experiments, we suggest that the ER A domain can, possibly through an intramolecular folding, interact with its own C-terminal region to repress AF-1 in the absence of ligand. The occurrence of this interaction in two phylogenically far-distant species receptors suggests that sequences and/or structures within the A domain and the C-terminal regions of the receptors were conserved during evolution. To confirm this hypothesis, we performed transposed experiments both in vitro and in vivo, by testing the interaction of the rtER A domain with hER subdomains and vice versa. Results of these experiments are shown in Fig. 11Go. The rtER A domain was able, in both the two-hybrid assay (Fig. 11AGo) and pull-down experiments (Fig. 11BGo) to interact with the human receptor and its C-terminal, but not N-terminal, region. The opposite was also verified with the hER A domain. These physical interactions were also reduced by E2 treatment in two-hybrid and pull-down experiments (compare lanes 3 and 4; 7 and 8; 11 and 12; and 15 and 16, Fig. 11BGo). The ratio of this reduction was similar to when we performed the interaction of ER A domain with their C-terminal counterparts. The results of these experiments demonstrate that sequences or structures implicated in the interaction between ER A domain and the C-terminal region are potentially conserved during the evolution.



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Figure 10. In Vivo Interaction of A Domain and C-Terminal Region

A and C, Illustration of the Gal4AD and Gal4DBD fusions used in the two-hybrid assay. B and D, Yeast strain containing 3UASG-LacZ and 2UASG -His reporter genes were transformed with the rtA/Gal4DBD or hA/Gal4DBD in combination with the Gal4AD fusions shown in panel A. Controls were performed by expressing A domain/Gal4DBD fusions alone or with Gal4AD. Transformants were subjected to growth selection (see Materials and Methods) and filter-lift for ß-galactosidase assays, before quantification. For this purpose, cells were grown in liquid media and treated with ethanol (EtOH) or E2, OHT, or ICI164,384 (ICI). Values represent the mean ± SD from five separate experiments. Double asterisks indicate a significant difference between controls (EtOH) and hormonal treatments at P < 0.001 by Student’s t test.

 


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Figure 11. Physical Interaction between ER A Domain and C-Terminal Region Implies Conserved Sequences or Structures

A, Y190 yeast strain was transformed either with the rtA/Gal4DBD or hA/Gal4DBD as probes for the two-hybrid assays. Full-length or deleted receptors fused to Gal4AD were used as baits and cotransformed with the A domain of the other species construct. After selection, transformants were treated with ethanol (EtOH) or 1,000 nM E2 and ß-galactosidase activity was assessed. Results shown are expressed as the mean ± SD from three separate experiments. Double asterisks indicate a significant difference between controls (EtOH) and hormonal treatments at P < 0.001 by Student’s t test. B, Pull-down assays using in vitro [35S]methionine-labeled full-length or C-terminal region of the two receptors were performed with the opposite A domain fused to the GST fusion or the GST alone (lanes 2, 6, 10, and 14). Incubations were in the presence of ethanol (EtOH, lanes 3, 7, 11, and 15) or 50 µM E2 (lanes 4, 8, 12, and 16). Equal loading of GST fusion proteins was checked by Coomassie blue staining. Positions of standard markers (S.M.) are indicated.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
E2 receptor (NR3A1) is a modulatory transcriptional activator that contains three transactivation functions, namely AF-1 in the N-terminal portion of the protein and AF-2a and AF-2 in the C terminus. For several years, AF-2 was the most studied function since ligand directly regulates the exposition of this activation function, providing a useful explanation for the ligand dependency of the protein transcriptional property (22, 43). Since no precise regions or structures were defined for ER AF-1 function, we decided to adopt a comparative approach between evolutionary far-distant proteins for studying ER AF-1, postulating that the molecular basis of this functional domain might be conserved. We used as a model two naturally occurring N-terminal variants of rtER and the hER{alpha} expressed in a yeast system in which AF-1 and AF-2a appear the dominant receptor activation functions (17). Results were then confirmed in the more complex system of mammalian cells.

To define regions of rtER implicated in AF-1, several deletions in the N-terminal domain of either full-length or C-terminal truncated receptors and a series of plasmid constructs containing various portions of the rtER N-terminal region fused to the DBD of the yeast Gal4 transcription factor were generated. From these analyses, a segment of 11 a.a (residues 7–17) responsible for hormone-independent transcriptional activation by the receptor in yeast was delineated. In agreement with reports describing steroid receptor activation functions (16, 51), this segment of rtER has no homology with the acidic transactivation domains of herpes simplex VP16 or yeast Gal4 activators. However, using the HCA plot prediction, an amphipathic {alpha}-helix could be formed between residues 14 to 22 of rtERS (rtERS{alpha}-H14–22), overlapping amino acids required for transactivation. The activity of this region was further substantiated by linking specific peptides encompassing the potential {alpha}-helix to the Gal4DBD. These chimeric proteins exhibited constitutive transcriptional activity at least 25-fold higher than Gal4DBD either alone or linked to 40 a.a of rtER N-terminal lacking the potential structure defined above. Additionally, since introduction of prolines in this sequence abrogated transcriptional activation, this reinforced the idea that the presence of an {alpha}-helical structure is required for transcriptional activation. Thus, alone, this {alpha}-helix can transactivate, as has been demonstrated for VP16-derived or totally artificial acidic structures (52, 53). A comparative analysis of the two receptor HCA plots (Figs. 4Go and 5Go) clearly showed several conserved hydrophobic clusters, not at the sequence level, but in the general organization of the two N-terminal domains. Similar results were obtained with ER{alpha} from other species such as salmon, tilapia, Xenopus, rat, or mouse (data not shown). Importantly, a hER N-terminal HCA plot also revealed an {alpha}-helix located at the beginning of the B domain, between residues 39–44 (hER{alpha}-H39–44). This analysis indicated that the presence of this structure was conserved among the different ER{alpha} cloned. More precisely, the sequence of the potential rtER {alpha}-helix corresponding to the minimal region essential for the rtER AF-1 was conserved during evolution in fish species, and an {alpha}-helical structure lies in a similar position at the beginning of the ER{alpha} B domain in all tetrapod species. This structure seems to be specific for ER{alpha} AF-1 since ERß shows a different organization. It is also worth noting that neither the fish nor other species sequences corresponding to the {alpha}-helix were found in other NRs (data not shown). Interestingly, Metzger et al. (21) characterized a.a 29 to 63 of hER, a region encompassing the hER{alpha}-H39–44 as an activating domain in yeast. By linking the minimal region required to form an {alpha}-helix in this sequence to the Gal4DBD, we demonstrated that this structure could activate the transcription in an autonomous manner. Moreover, the transcriptional activity was dependent upon the helical structure, since substitution of the key hydrophobic amino acids into proline eliminated transactivation potency. The importance of this potential {alpha}-helix was also tested in the full-length hER context, using either truncated (hER{Delta}1–44) or point mutant (hER{Delta}1–37L39P and hER{Delta}1–37Y43P) receptors. Compared with the A domain truncated receptor (hER{Delta}1–37), this led to a 50% decrease of E2-induced activity, and approximately 80% of E2-independent reporter activation. Therefore, this conserved structure seems to be, at least in part, implicated in hER AF-1 in yeast. The suggestion of two structures in ER AF-1 was also demonstrated in mammalian cells by transient transfection experiments. Deletion or point mutations either in the rtER or hER {alpha}-helices abrogated, at least partially, their AF-1 activity in AF-1 sensitive cell contexts, whereas it had no effect in AF-2-sensitive cells such as HeLa. All these results, plus the finding of potential structures in similar positions in two far-distant ERs, led us to propose that this {alpha}-helix is the functioning core of ER AF-1. This hypothesis was further substantiated by the use of rainbow trout and human receptors in which their {alpha}-helices were transposed. Indeed, such chimeric receptors were as active as their corresponding wild-type forms, both in yeast and in mammalian cells. The two point mutant hER receptors also particularly enlightened the link between ER ligand-independent activity and AF-1, since mutation of a sequence implicated in AF-1 dramatically reduces E2-independent activity. Thus, this helical structure seems to be more involved in E2-independent transactivation than E2-dependent activity.

A study on the VP16 protein found that an {alpha}-helical structure could be formed from a random coil organization only upon interaction with a component of the basal transcription machinery, hTAFII 31 (54). A related mechanism was very recently observed in the context of a member of the NR superfamily, the glucocorticoid receptor (GR or NR3C1). Indeed, in that study the authors showed that the GR N-terminal domain, which appears to have little intrinsic spatial organization, acquires helicity and tertiary structures upon binding to DNA when linked to the GR DBD (55). This kind of interdomain signaling mechanism could also be considered with the ER {alpha}-helix. In fact, one could speculate that the rtER and hER {alpha}-helical structures could appear only in certain conditions.

Recent findings showed that coactivators such as SRC-1 (32), GRIP1, RAC3, pCAF (33), and p300/CBP (32, 34) could enhance the AF-1 of ER as well as that of other NRs. These coactivators are potential linking factors as they were first identified to interact with the NR C-terminal region through their signal motif NR box LXXLL (56) exhibiting an {alpha}-helical structure. Moreover, these partners could physically interact with the N-terminal region of steroid receptors. The most important region defined in hER for these interactions was Box 1 (33), a region encompassing the {alpha}-helix that we have identified. The direct implication of this structure in the interaction with these coactivators is one of the next questions that we must answer. In respect to the comparison with yeast and mammalian cell contexts, this could lead to an interesting fact: yeast has been shown to possess no homologs to NR box coactivators. Then, if SRC-1 or another coactivator of this family is able to interact with these structures, it could imply that yeast possess functional homologs of these coactivators, which remain to be determined. It should also be remembered that physical interaction between these proteins and the ER N terminus lies on regions other than the NR boxes (33). Studies on VP16 have implicated {alpha}-helical structures in transcriptional activation (57, 58) and shown that targets of a VP16-derived {alpha}-helix could be TFIIB (59). Consequently, interaction of hER{alpha}-39–44 with components of the basal transcriptional machinery such as TFIID, TFIIH, or TFIIB should be tested since these factors are also able to interact with NRs (60, 61, 62).

Studies on hER AF-1 have shown that in yeast, two regions (a.a 1–62 and a.a 118–149) are able to efficiently activate transcription in an autonomous manner, while a third one between a.a 80 and a.a 113 is involved with the other two in synergism between AFs (19). However, rtER constructs containing these regions, namely the rtERS{Delta}1–17 (corresponding to a.a 45–62 in rtERL), rtERS{Delta}1–34 (a.a 45–79), and rtERS{Delta}1–50 (a.a 45–95) did not possess any activation potency in yeast, when expressed alone or fused to the Gal4DBD. This may suggest, that in contrast to the hER {alpha}-H39–44, the rtER {alpha}-H14–22 is absolutely required in yeast for a general conformation and possibly suitable folding of the other regions implicated in AF-1. Alternatively, one could speculate that during evolution, the ER N-terminal transactivation function has evolved in multiple subdomains to provide several surfaces for the recruitment of evolutionary novel coactivator or adaptor proteins. However, our transfection experiments in mammalian cells have also shown that other regions are required for AF-1 and/or total activity of the rainbow trout receptor. Indeed, deletion or point mutations within the potential structure defined by HCA plot only reduced the fold induction by 2- to 3-fold depending upon the AF-1 sensitive cell-context. This fits well with earlier experiments defining three sequences for AF-1 in mammalian cells (19). Other studies on ER AF-1 have shown that a major regulation of this transactivation function in mammalian cells proceeds through phosphorylation of key serine residues, namely the S118, S167, S154, S104,and S106 one (63, 64, 65, 66, 67, 68), linking the AF-1 activity with other signaling pathways (69). Since the rtER N-terminal sequence exhibits some serine residues, and for some, in comparable positions to those in hER, the contribution of these residues to rtER AF-1 could be tested. The p68 helicase protein was recently identified as a specific AF-1 coactivator (70) and was shown by pull-down assays to associate with hER in a region encompassing Ser118, providing a link between phosphorylation and transactivation. Testing the interaction of this protein with rtER should provide information about the importance of some of these residues for AF-1 functioning.

Deletion of the A domain in hER (hER{Delta}1–37) did not modify ligand-dependent transcriptional activity of the receptor, confirming that sequences implicated in AF-1 are located in the ER B domain. Unexpectedly, this deletion markedly increased the ligand-independent activity of the receptor. Comparison with a new rtER isoform possessing an A domain (rtERL), which does not exhibit any ligand-independent activity (Ref. 38 , and see Fig. 1Go) led to the hypothesis that the A domain of ERs exerts an inhibitory role on AF-1. The existence of an inhibitory function, repressing both AF-1 and AF-2, most probably by an intramolecular process, was recently reported for the progesterone receptor (71). Deletion of rtER C-terminal (rtAD construct) led to a substantial increase in the ligand-independent activity of the receptor (75% of the full-length ER activity), confirming that AF-1 is markedly reduced by the presence of the C-terminal domain. This observation might indicate a functional interaction between the ligand-binding and N-terminal domains of ER. Such an interaction was suggested for ER (72), and demonstrated for the androgen and progesterone receptors by pull-down and two-hybrid assays, but this was related to the synergistic effect between the two AFs contained in these regions (73, 74). Here, we demonstrate that the ER A domain specifically interacts both in vitro and in vivo with the C-terminal region of the receptor, masking hormone-independent activity of the full-length receptor. This interaction was conserved during evolution since both rtER and hER displayed this property, and since the A domain of one receptor could interact with the C-terminal region of the other. This highlights the importance of such a mechanism for the ER overall activity and suggests that these interactions rely on conserved residues or structures in the C-terminal domain of ERs. These regions need now to be identified. However, it is of importance to note that this kind of interaction, inhibited by the ligand, which is not required for DNA binding, could absolutely not define a dimerization process. In the absence of ligand, the A domain could therefore function as a negative regulatory domain for ER AF-1 activity by a direct interaction with the C-terminal region. Stoichiometry of the interaction remains also to be determined. Indeed, since ER dimerizes through its DBD in the absence of E2, it is not clear whether the A domain of one monomer interacts with the C-terminal region of the same monomer or the other dimer partner. However, completion of these two kind of interactions in the absence of ligand led to an inactive conformation of the receptor (Fig. 12Go, left, to simplify, hypothesis of an intramolecular process is shown). However, one cannot exclude indirect mechanisms involving eventual cofactors: 1) the A domain may enhance direct interaction between ER and a corepressor such as SMRT (silencing mediator for retinoid and thyroid hormone receptors) or NCoR (nuclear receptor corepressor) (75, 76) (Fig. 12Go, middle); 2) the A domain could act via a steric prevention of AF-1 coactivator recruitment (Fig. 12Go, right). It was also suggested that a synergistic effect between AF-1 and AF-2 of NRs may be a consequence of direct interactions of these activation domains with a common coactivator, as shown for AR with the CREB-binding protein complex and for GR and ER with DRIP150 and DRIP250 (73, 77, 78). The A domain may prevent interaction of the AFs with such coactivators in the absence of ligand. An alternative hypothesis relates to a corepressor pathway. A recent study described the AF-1 regulating role of the yeast Hsp40Ydj1 protein involved in the maturation of heat shock proteins (Hsp) (79). The Hsp, and especially the Hsp90 chaperone pathway, is thought to inhibit steroid receptor transcriptional activities in the absence of ligand (80, 81). Since Ydj1 seems to be involved in an N-terminal/C-terminal interaction resulting in the inhibition of AF-1 in the absence of E2 (79), it would be interesting to investigate whether the A domain is involved in contacts between ER and Ydj1.



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Figure 12. Models for the Repressive Action of the ER’s A Domain

Hypothetical mechanism by which the A domain exerts an inhibitory effect on AF-1 activity. The A domain could physically repress the AF-1, in cooperation with the C-terminal region (left side of the panel). An inactive conformation induced by binding of the A domain to the C-terminal region may recruit a repressor, inhibiting the ligand-independent activity of the receptor (middle). Finally, this conformation may prevent the receptor from interacting with a specific activator (on the right side).

 
In all these hypotheses, ligand has to induce a conformational change permitting exposure of the AFs (6, 7, 44). Interestingly, our results showed that the interaction between the A domain and the C-terminal region of the ER is reduced in the presence of ligand. More precisely, this reduction was observed in the presence of total or partial agonists (E2 and OHT, respectively) of the ER, but not in the presence of the pure antagonist ICI164,384. This is highly relevant in the view of a key role of the A domain in the regulation of AF-1 since activity of OHT is thought to proceed through AF-1. On the other hand, ICI164,384, which is known to behave as an ER total antagonist, did not affect the A domain/C-terminal region interaction, suggesting an inactive conformation of the ER bound to this antiestrogen. Since ICI induces a C-terminal structural organization distinct from E2, one could speculate that ICI may not be able to derepress the AF-1 function. Thus, this could be another explanation for the potency of ICI as an estrogen antagonist. A previous report suggested that interaction between whole N-terminal and C-terminal regions of ER occurs only after ligand binding, allowing synergism between the AFs leading to ER full activity (72). Since this study was performed using transfection experiments with reporter gene and both AF-containing regions of ER as separate polypeptides, this approach could not detect interactions that are transcriptionally unproductive. Therefore, our data are not inconsistent with the study of Kraus et al. (72), since the interaction of the C-terminal region with A domain does not result in transcriptional activation. However, the way to reconcile a physical interaction enhanced by ER agonists, at least resulting in transcriptional activation, with an interaction of A domain reduced by these same ligands remains to be found. It is conceivable that E2 promotes interaction of the B domain with the C-terminal region, leading to a synergism between AF-1 and AF-2, whereas in the absence of E2, the A domain would mask this interaction.

In conclusion, as AF-1 is an important transcriptional function implicated in the overall activity of ER, it seems to be essential to have a better understanding of the mechanisms involved in the ligand-independent transcriptional activation. Its importance is particularly illustrated by the behavior of some antiestrogenic compounds that appear driven by AF-1. There is now compelling evidence that in spite of sequence divergences throughout evolution, some structural features are conserved in the ER N-terminal region. The model that we propose for the basic working of the ER N-terminal region is based on repression by the A domain in the absence of ligand and the presence of a minimal activation unit that could form an {alpha}-helix located at the beginning of the B domain. Our study highlights the importance of comparison between the activity of phylogenically far-distant NRs, allowing essential mechanisms conserved during evolution to be identified. Predictive methods such as HCA are an essential step that allows a penetrating view of the three-dimensional organization of peptide sequences. However, real evidence for the presence of a secondary structure important for protein folding or protein-protein interactions will come from physical studies such as x-ray crystallography.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Receptor Constructions and Reporter Plasmids for Yeast and Mammalian Cells
All of the primers and oligonucleotides sequences are available upon request. The construction of the YEprtERS vector was previously described (39), while the expression vector YEphER is a gift from Dr. B. S. Katzenellenbogen (25). The YEprtERL was constructed by insertion of the cDNA isolated from a trout ovary cDNA library in the BamHI site of YEpucG (40). Sequences of the two rtER isoforms (rtERS and rtERL) can be retrieved in GenBank/EMBL Data Bank under accession nos. AJ242740 and AJ242741. The truncated receptors rtBD (rtERS{Delta}234–575), rtCF (rtERS{Delta}1–142), rtERS{Delta}1–111, rtERS{Delta}1–50, rtERS{Delta}1–34, rtERS{Delta}1–17, rtERS{Delta}1–7, rtBD{Delta}1–111, rtBD{Delta}1–50, rtBD{Delta}1–34, and rtBD{Delta}1–17 were constructed by PCR as previously described, using specific oligonucleotides and YEprtER as matrix. The hAD (hER{Delta}272–595), hCF (hER{Delta}1–175), hER{Delta}1–37, hER{Delta}1–37L39P, hER{Delta}1–37Y43P, and hER{Delta}1–44 were synthesized in the same way, using YEphER for matrix. The rtERS{Delta}5–17 and rtERS{Delta}8–17 were constructed by PCR from the rtER{Delta}1–17 construct, using oligonucleotides containing the sequences encoding for the first four or seven amino acids, respectively, of rtER. The chimeric receptor rtER(hAB) was constructed by ligating two PCR inserts including the hER AB domains and the CDEF domains of rtER by creating a silent NcoI site between the two regions. The hER{Delta}1–37 (rt{alpha}) and hER{Delta}1–37 (rt{alpha}L18P) were synthesized by PCR using long oligonucleotides possessing the rtER {alpha}-helix sequence with or without mutations, and the hER{Delta}1–37 as the matrix. The rtERS{Delta}1–7 (h{alpha}) and rtERS{Delta}1–7 (h{alpha}Y43P) were constructed in the same way, using the rtERS{Delta}1–7 as matrix. All these constructs were inserted into the unique site BamHI of YEpucG and verified by sequencing. The pLG{Delta}178/3EREc construct was created by inserting double-stranded oligonucleotides containing three adjacent EREs in the XhoI site of the pLG{Delta}178 vector (82).

For transient expression in mammalian cells, the rtERL, rtERS{Delta}1–111, rtERS{Delta}8–17, hER{Delta}1–37, hER{Delta}1–37L39P, hER{Delta}1–37Y43P, and hER{Delta}1–44 inserts were subcloned in the BamHI site of the pSG5 vector, whereas the hER{Delta}1–37 (rt{alpha}), hER{Delta}1–37 (rt{alpha}L18P), rtERS{Delta}1–7 (h{alpha}), and rtERS{Delta}1–7 (h{alpha}Y43P) were subcloned within the EcoRI and BamHI of the pSG5 vector. The pCMV5/rtERS construct used in this study has been previously described (83), as well as the pSG5/hER, pSG5/hER{Delta}1–178, and the ERE-thymidine kinase-luciferase reporter gene, which were gifts from Dr. G. Flouriot and Pr. F. Gannon.

Gal4 Fusions for One- and Two-Hybrid Assays
The rtERS/Gal4DBD and rtERS/Gal4AD fusion proteins were obtained by inserting the whole coding region of rtERS cDNA into the BamHI cloning site of the pAS2–1 or pACT2 vectors (CLONTECH Laboratories, Inc. Palo Alto, CA), while the rtERS{Delta}1–142, rtERS{Delta}1–111, rtERS{Delta}1–17, and rtERS{Delta}1–7 PCR inserts were subcloned in the pGBT10 vector (CLONTECH Laboratories, Inc.), generating the fusions rtCF/Gal4DBD, rtERS{Delta}1–111/Gal4DBD, rtERS{Delta}1–17/Gal4DBD, and rtERS{Delta}1–7/Gal4DBD. The fusions rtERS{Delta}153–575/Gal4DBD (rtB/Gal4DBD), rtB{Delta}1–111/Gal4DBD, and rtB{Delta}1–17/Gal4DBD were constructed by deletion of the 152–575 fragment, respectively, from the rtERS/Gal4DBD, rtERS{Delta}1–111/Gal4DBD, or rtERS{Delta}1–17/Gal4DBD constructions, by a PstI digestion and religation. Gal4DBD fusion proteins with sequences including the rtER or hER potential {alpha}-helix mutated or not were constructed by inserting the corresponding double-stranded phosphorylated oligonucleotide possessing an EcoRI site in 5', and a BamHI site in 3' in the pAS2–1 vector. The rtER and hER A domains were amplified by PCR with specific oligonucleotides containing an EcoRI site for the 5' one and a BamHI site in the 3' one. These inserts were subcloned in the corresponding sites of pAS2–1, generating the rtA/Gal4DBD and hA/Gal4DBD fusion proteins. The rtERS{Delta}1–223/Gal4AD and rtERS{Delta}149–575/Gal4AD fusion proteins were obtained by subcloning the corresponding PCR inserts within the BamHI site of the pACT2 vector (CLONTECH Laboratories, Inc.). The inserts encoding for the hER{Delta}1–178 and hER{Delta}181–595 were synthesized by PCR with a BamHI site at the 5'-extremity and an EcoRI site in 3'. All fusions were verified by sequencing.

Plasmids for GST Fusion and in Vitro Transcription/Translation
The A domain of rtER and hER were amplified by PCR using oligonucleotides containing SmaI site for the 5' one and EcoRI site for the 3' one. These fragments were inserted in the corresponding sites of the pGEX2-T vector (Amersham Pharmacia Biotech, Buckinghamshire, UK), generating the GST/rtA and GST/hA fusions. The Bluescript (BS) or pSG5 vectors were used for the in vitro transcription/translation. The BS/rtERS construct has already been described (83); BS/rtERS{Delta}1–220 and BSrtERS{Delta}157–575 were obtained by excising of the BS/rtERS, the SacII-SacII, or KpnI-KpnI fragments, respectively. The hER cDNA as well as the hER form III cDNA (hER{Delta}1–178) included in the pSG5 vector were a gift from Dr. G. Flouriot, whereas the hER{Delta}272–595 was produced in vitro using a pSG5 vector containing within its EcoRI and BamHI sites the hAD PCR insert. Proteins were synthesized in vitro using the T7 RNA polymerase in the rabbit reticulocyte-coupled transcription/translation kit (TNT, Promega Corp., Madison, WI), as recommended by the manufacturer. Labeled protein expression was monitored by estimating the relative amounts of protein on SDS-PAGE gels. To study the ligand effects, lysates were submitted to ethanol, 10 or 50 µM E2 (Sigma, St. Louis, MO), 100 or 500 µM of OHT or ICI164,384 treatments for 1 h in TEG Buffer (50 mM Tris-HCl, pH 7.4, 1.5 mM EDTA, 50 mM NaCl, 10% glycerol, 5 mM MgCl2 , and 1 mM dithiothreitol).

In Vitro Protein-Protein Interaction Assay: GST Pull-Down Assay
E. coli DH5{alpha} (250 ml log-phase culture) containing the pGEX2-T/rtA or pGEX2-T/hA constructs were grown in LB medium and induced by adding 0.1 mM isopropyl ß-D-thiogalactoside for 4 h. After recovery by centrifugation, cells were resuspended in NETN buffer (100 mM NaCl, 20 mM Tris, pH 8, 1 mM EDTA, 0.5% Nonidet-40, 1 mM phenylmethylsulfonylfluoride (PMSF), and the protease inhibitors leupeptin, pepstatin, and aprotinin at a final concentration of 10 µg/ml), and lysed by sonication 4 times 15 sec. Lysates were clarified by centrifugation at 12,000 rpm for 2 min at 4 C and immediately placed in contact with 50 µl of a 50% suspension of glutathione-agarose beads (Sigma, St Quentin Fallavier, France) in NETN. Incubation was performed overnight at 4 C under rotation. The fusion proteins bound to the beads were recovered by centrifugation at 500 x g for 5 min at 4 C, and washed five times in NETN before being resuspended in 500 µl of binding buffer (50 mM NaCl, 50 mM Tris, pH 8, 0.02% BSA, 0.02% Tween 20, 1 mM PMSF and 10 µg/ml of the proteases inhibitors). For quantification, Bradford dosage was performed, together with analysis on SDS-PAGE, allowing an evaluation of the stability of the fusion protein and the amounts of protein recovered. Approximately 4 µg of the GST fusion proteins bound to the beads were incubated at 4 C for 3 h with equal amounts of [35S]methionine-labeled proteins (2–4 µl of the hormone or ethanol-treated lysates) in the presence or not of 10 or 50 µM E2, 100 or 500 µM of OHT or ICI164,384, in a total volume of 300 µl of binding buffer. Beads were collected by centrifugation at 55 x g for 5 min at 4 C, and washed 10 times in 300 µl of washing buffer (50 mM Tris, pH 8, 150 mM NaCl, 1 mM PMSF, 0.05% Tween 20, and 10 µg/ml of the protease inhibitors). Washed beads were suspended in 10 µl of 1 x SDS-PAGE sample buffer, boiled for 5 min, and pelleted in a microcentrifuge. Five microliters of the supernatant were subjected to SDS-PAGE on a 12% acrylamide gel. To control equal loading, gel was stained with Coomassie blue before autoradiography. Quantification was performed using a Packard phosphoimager (Packard Instruments, Meriden, CT).

Host Strains
The bacteria strain used for subcloning and protein expression was E. coli DH5{alpha} (supE44 {Delta} lacU169 (Ø80 lacZ{Delta}M15) hsdR17 Rec A1 end A1 gyrA96 thi-1 relA1). The yeast strains used in this study were BJ2168 (a leu2 trp1 ura3–52 prb 1–1122 pep4–3 prc1–407 gal2) (Yeast Genetic Stock Center, Berkeley, CA) for the transactivation experiments, and Y190 (MATa, ura 3–52, his 3–200, ade 2–201, lys 2–801, trp 1–901, leu 2–3, 112, gal 4{Delta}, gal 80{Delta}, cyhr 2, LYS2::GAL1UAS- HIS3TATA-HIS3, URA3::GAL1UAS-GAL1TATA-LacZ) (CLONTECH Laboratories, Inc.) for the examination of the Gal4 fusion activity and two-hybrid assays. Yeast cells were transformed using a modified lithium acetate method (41), and BJ2168 transformants were selected by growth on complete minimal medium [0.13% dropout powder lacking uracil and tryptophan, 0.67% yeast nitrogen base, 0.5% (NH4)2SO4 and 1% dextrose]. Y190 transformants were selected on the same media lacking histidine and including 25 mM of 3-AminoTriazole (Sigma). This treatment was performed to inhibit the low endogenous activity of the his3 gene placed under the control of two response elements for the yeast Gal4 activator (UASG). This step allows selection of the true positive clones. Hormone-dependent activity of the Gal4DBD fusion proteins or the dimerization state of the Gal4 fusions were tested in the presence or absence of 10-6 M E2 or 100 µM of OHT or 10 µM of ICI164,384 in the plates.

ß-Galactosidase Assays for Transcriptional Activity in Yeast
Y190 transformants were first selected for growth on selective medium lacking histidine, and before quantification by a liquid assay, Lac Z activity was tested in a filter-lift assay. The Lac Z reporter gene being under the control of 3 UASG, the detected activity is strictly dependent upon DNA recognition by the ER/Gal4DBD chimera proteins. When no growing colony was evident on the plates, the transformation mixture was replated on contrast medium, and a liquid assay was performed on colonies after 2 to 3 days of incubation at 30 C. Liquid assays were performed as described previously (39) in the presence of either ethanol or E2 at 10-6 or 10-8 M or 100 µM of OHT or 10 µM of ICI164,384. ß-Galactosidase activity was measured using O-nitrophenyl ß-D-Galactopyranoside substrate (Sigma) and quantified at 420 nm with a spectrophotometer.

Cell Culture and Transient Transfection Experiments
CHO and HeLa cells were routinely maintained in DMEM-F12 (Sigma) supplemented with 10% FCS (Life Technologies, Inc., Gaithersburg, MD), whereas HepG2 cells were grown in DMEM/10% FCS. All the cell lines were cultured at 37 C and 5% CO2, and all media contained 100 U/ml penicillin, 100 µg/ml streptomycin, and 25 µg/ml Amphotericin (Sigma). One day before transfection, cells were dispatched in six-well plates in the case of HeLa and HepG2 cell lines and 24-well plates for CHO. The three cell lines were transfected at 60–70% confluence with a classical calcium phosphate/DNA precipitation protocol after replacing the media 1 h before transfection by a DMEM-F12/8% of charcoal/dextran-treated FCS. Transfection in six-well plates was performed using 3 µg of total DNA per well and containing 250 ng of expression vector, 500 ng of ERE-TK-Luc reporter, and 1.5 µg of the internal control pCH110. For CHO cells, 1 µg of total DNA per well was used, containing 25 ng of expression vector, 50 ng of ERE-TK-Luc reporter, and 150 ng of pCH110. In all cases, the total amount of DNA was maintained constant by adding Bluescript plasmid. Cells were kept in contact with the precipitate one night at 37 C with 2% of CO2, then washed once by PBS, and replaced in fresh DMEM-F12/8% desteroided FCS media supplemented with 10-8 M E2 or ethanol. After 36 h of transient expression, cells were harvested and 10% of the cellular extract was used to measure the luciferase activity. Half of the remaining extract was used for the ß-galactosidase assay. Luciferase activities were normalized for transfection efficiency with the ß-galactosidase activity and expressed as the fold induction vs. the activity obtained with the promoter alone. All transfections were done in triplicate, at least three times.

Western Blot Analysis
Western blots were carried out on yeast whole-cell extracts prepared according to a previous report (39). Briefly, yeast cells were grown in 5 ml of selective medium to a density of 5 million cells per ml. Yeast cells were harvested and resuspended in 1 M Sorbitol, and cell walls were removed using 15 U of lyticase (Sigma). Protein extracts were then obtained by lysis of the spheroplasts as described previously (39). Whole extracts (35 µg) prepared from yeasts expressing the Gal4DBD fusions were fractionated on polyacrylamide-SDS gel and transferred on Hybond-C (Amersham Pharmacia Biotech, Buckinghamshire, UK). Blots were then incubated overnight with either a polyclonal rabbit anti-Gal4 DBD or monoclonal anti-Gal4 DBD antibodies (TEBU, Le-Perray en Yvelines, France) diluted at 1:500 or 1:2000, respectively, washed, and then incubated for 1 h with a 1:10,000 dilution of respective secondary antibodies conjugated to the horseradish peroxidase phosphatase. After several washes in PBS, blots were revealed by means of the enhanced chemiluminescence (ECL) method (Amersham Pharmacia Biotech). Blots were visualized by autoradiography for 15 sec to 5 min. Whole yeast extracts (30 µg) containing the hER wild-type or point mutant receptors were incubated overnight with H222 (a gift from Dr. Katzenellenbogen) diluted at 1:1,000 after migration and transfer on Hybond-C. Blots were washed, incubated for 1 h with rabbit antirat IgG diluted at 1:1,000 and then a further 1 h with 1:5,000 dilution of anti-rabbit IgG conjugated to the horseradish peroxidase phosphatase. Blots were visualized by autoradiography after using the ECL revelation method.

Structural Analysis
Structural segmentation of ER N-terminal regions was determined by HCA plot. One- and two-dimensional representations were done in accordance with the nomenclature previously defined (49, 50).


    ACKNOWLEDGMENTS
 
We thank Dr. J. Duval and Dr. H. Wroblewski for HCA plot analysis and valuable discussion; Dr. G. Flouriot for helpful comments and the gift of cDNAs; and Dr. B. Katzenellenbogen for the gift of vectors and antibodies. We acknowledge Dr. O. Kah and Dr. G. Salbert for reviewing this manuscript. We thank also F. Gay for her helpful advice on GST pull-down experiments.


    FOOTNOTES
 
Address requests for reprints to: Farzad Pakdel, Equipe d’Endocrinologie Moléculaire de la Reproduction UPRES-A CNRS 6026, Université de Rennes I, Rennes cedex, France. E-mail: Farzad.Pakdel{at}univ-rennes1.fr

This work was supported by the Centre Nationale de la Recherche Scientifique and by the Ministere de l’Education et de la Recherche to R.M. and F.G.P. Technical support was funded by the Fondation Langlois.

1 Present address: Department of Molecular and Cellular Biology, Baylor College of Medicine, Texas Medical Center, One Baylor Plaza, Houston, Texas 77030. Back

Received for publication December 17, 1999. Revision received July 24, 2000. Accepted for publication July 26, 2000.


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