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

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 -helix within this minimal region that is conserved during evolution. Both rtER and hER sequences corresponding to this potential -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 -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 (rtER_L and rtER_S, 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- (ER) 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 -helix conformation. Such a structure was also found in hER, which exhibited similar characteristics, and the two -helices were perfectly interchangeable in terms of transactivation.

Additionally, comparison of the transcriptional activity of a naturally occurring rtER truncated for its A domain (rtER_S) with the full-length form (rtER_L) and hER (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 -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 rtER_L, from trout ovary produced by the same gene (40), which possesses an additional sequence of 45 amino acids (a.a) (Fig. 1A). The earlier isolated rtER form, now called rtER_S, 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. 1A). 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 pLG178/3EREc 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 (rtER_S) lacking the A domain exhibited a consistent E₂-independent activity (Fig. 1B and Ref. 40), whereas hER and rtER_L, which both possess such a region, did not exhibit a similar characteristic (Fig. 1B). 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. 1B). 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 rtER_S. Since the only difference between rtER_S and rtER_L 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 (hER1–37) was constructed, and, interestingly, it exhibited a basal activity representing 17% of the total activity (Fig. 1C). 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), rtER_S possesses, as does hER, a functional AF-1 (Fig. 1C), 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.

View larger version (29K): [in this window] [in a new window]   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 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.

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 (rtER_S) 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 rtER_S 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. 2A 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. 2A 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. 2B, 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. 2C show that rtER_S5–17 and rtER_S8–17 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 rtER_S lacking the seven first a.a was constructed (rtER_S1–7). This truncated receptor retained 80–90% of the wild-type ER activity (Fig. 2C) and therefore provided a good control for these experiments. These results indicate that a.a. 8–17 of the rtER_S 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.

View larger version (31K): [in this window] [in a new window]   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.

View larger version (34K): [in this window] [in a new window]   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 -Helix Possessing Intrinsic Transactivation Potency

View larger version (34K): [in this window] [in a new window]   Figure 4. Identification of a Potential -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 , serine by , and threonine by . 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 -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 rtB1–111/Gal4DBD protein were also included. D, After selection, ß-galactosidase activity was measured. Values represent the mean ± SD from at least four separate experiments.

View larger version (34K): [in this window] [in a new window]   Figure 5. hER B Domain Includes a Transcriptionally Active -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. 4. C, Gal4DBD fusion proteins including the sequence for the potential -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.

View larger version (42K): [in this window] [in a new window]   Figure 6. The hER Potential -Helix Is Implicated in AF-1 Yeast strain containing the 3-ERE-Cyc-LacZ reporter was transformed with vectors expressing hER, hER1–37, hER1–44 that does not possess the -helix characterized, or two receptors mutated in the -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.

View larger version (43K): [in this window] [in a new window]   Figure 7. The -Helices of Both Receptors Are Perfectly Interchangeable A, To determine whether the -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 -Helix for ER AF-1 in Mammalian Cells

View larger version (38K): [in this window] [in a new window]   Figure 8. The ER AF-1 Requires the -Helix in Mammalian Cells To confirm the importance of the rtER and hER -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.

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. 9) and [³⁵S]-labeled ER constructs translated in rabbit reticulocyte lysate that overlap the entire sequence of the two receptors, namely rtER_S, rtER_S1–220, rtER_S157–575, hER, hER1–178, and hER272–595 (Fig. 9, A and D). As shown in Fig. 9B, 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 rtER_S, rtER_S1–220, and rtER_S 157–575, respectively). In the pull-down experiment, the A domain interacted with both rtER_S and rtER_S1–220, whereas no interaction was found with the B domain (rtER_S157–575). The effect of E₂ on this interaction was examined (Fig. 9B, 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 rtER_S labeled protein was retained vs. 5% after E₂ treatment, and 7% vs. 2.5% for rtER_S1–220. 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 rtER_S1–220 lysate with 100 or 500 µM of 4-hydroxytamoxifen (OHT) or ICI_164,384. As shown in lanes 10 and 11, OHT was able to compete the interaction between rtER A domain and rtER_S1–220, but with a lower efficiency than E₂ (6 to 4% vs. 2.5%), irrespective of the dose. In contrast, the pure antiestrogen ICI_164,384 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 rtER_S 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 hER1–178 labeled proteins were retained on the GST/hA beads (Fig. 9E). 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 hER1–178, respectively. The OHT was also able to reduce this interaction from 19.8% to 9 or 8.7%, depending on the dose, whereas ICI_164,384 had no effect. The validity of the experiments was confirmed by performing a Coomassie blue staining of the gels (Fig. 9, C and F), demonstrating equivalent loading on the SDS-PAGE gels.

View larger version (36K): [in this window] [in a new window]   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.

View larger version (17K): [in this window] [in a new window]   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.

View larger version (33K): [in this window] [in a new window]   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

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 -helix could be formed between residues 14 to 22 of rtER_S (rtER_S-H14–22), overlapping amino acids required for transactivation. The activity of this region was further substantiated by linking specific peptides encompassing the potential -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 -helical structure is required for transcriptional activation. Thus, alone, this -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. 4 and 5) 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 from other species such as salmon, tilapia, Xenopus, rat, or mouse (data not shown). Importantly, a hER N-terminal HCA plot also revealed an -helix located at the beginning of the B domain, between residues 39–44 (hER-H39–44). This analysis indicated that the presence of this structure was conserved among the different ER cloned. More precisely, the sequence of the potential rtER -helix corresponding to the minimal region essential for the rtER AF-1 was conserved during evolution in fish species, and an -helical structure lies in a similar position at the beginning of the ER B domain in all tetrapod species. This structure seems to be specific for ER AF-1 since ERß shows a different organization. It is also worth noting that neither the fish nor other species sequences corresponding to the -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-H39–44 as an activating domain in yeast. By linking the minimal region required to form an -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 -helix was also tested in the full-length hER context, using either truncated (hER1–44) or point mutant (hER1–37_L39P and hER1–37_Y43P) receptors. Compared with the A domain truncated receptor (hER1–37), 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 -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 -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 -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 -helical structure could be formed from a random coil organization only upon interaction with a component of the basal transcription machinery, hTAF_II 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 -helix. In fact, one could speculate that the rtER and hER -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 -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 -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 -helical structures in transcriptional activation (57, 58) and shown that targets of a VP16-derived -helix could be TFIIB (59). Consequently, interaction of hER-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 rtER_S1–17 (corresponding to a.a 45–62 in rtER_L), rtER_S1–34 (a.a 45–79), and rtER_S1–50 (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 -H39–44, the rtER -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 (hER1–37) 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 (rtER_L), which does not exhibit any ligand-independent activity (Ref. 38 , and see Fig. 1) 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. 12, 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. 12, middle); 2) the A domain could act via a steric prevention of AF-1 coactivator recruitment (Fig. 12, 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.

View larger version (12K): [in this window] [in a new window]   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).