Transcriptional Activities of the Orphan Nuclear Receptor ERR{alpha} (Estrogen Receptor-Related Receptor-{alpha})

Jean-Marc Vanacker, Edith Bonnelye1, Sandrine Chopin-Delannoy, Cateline Delmarre, Vincent Cavaillès and Vincent Laudet

Centre Nationale de la Recherche Scientifique UMR 49 (J-M.V., V.L.) Ecole Normale Supérieure de Lyon 69364 Lyon, France
Centre Nationale de la Recherche Scientifique UMR 319 (S.C.-D., C.D.) Institut de Biologie de Lille Institut Pasteur de Lille 59021 Lille, France
INSERM U148 (V.C) 34090 Montpellier, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen receptor-related receptor {alpha} (ERR{alpha}) is an orphan nuclear receptor closely related to the estrogen receptor (ER), whose expression covers various stages of embryonic development and persists in certain adult tissues. We show that ERR{alpha} binds as a homodimer on a specific target sequence, the SFRE (SF-1 response element), already known to respond to the orphan nuclear receptor SF-1. Target sequences that are related to the SFRE and that discriminate between ERR{alpha} and SF-1 were identified. We have also analyzed the transcriptional properties of the ERR{alpha} originating from various species. All ERR{alpha} orthologs act as potent transactivators through the consensus SFRE. ERR{alpha} activity depends on the putative AF2AD domain, as well as on a serum compound that is withdrawn by charcoal treatment, suggesting the existence of a critical regulating factor brought by serum.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The superfamily of nuclear receptors includes transcription factors whose activity depends on the presence of a specific ligand (reviewed in Refs. 1, 2, 3). These ligands are of various chemical natures and include, among others, steroid and thyroid hormones and retinoic and fatty acids. All nuclear receptors share the same overall protein organization. In the N-terminal domain (A/B domain), a hormone-independent transactivation function (AF-1 region) has been identified in certain receptors (1). Immediately downstream, the C domain is strongly conserved among receptors and is responsible for the specific DNA-binding activities. The DNA sequences recognized by nuclear receptors generally consist of a core element (sequence AGGTCA), either single or present as a direct or inverted repeat. In the latter cases, the spacing between both core elements contributes to the specificity of recognition by a given receptor (4). Although also participating to some extent in the DNA-binding function, the D domain is mainly considered a hinge region, bridging the C domain to the most C-terminal ligand-binding domain (E domain). The ligand-binding domain contains the hormone-binding and hormone-dependent transactivation (AF-2 region) functions and is evolutionarily conserved among the receptors (5). On the basis of this domain organization, a growing number of so-called orphan receptors (i.e. for which no ligand has yet been identified) have been characterized (6). It is unclear whether these receptors are regulated by a ligand still to be identified or whether they are constitutively active.

The orphan receptor estrogen receptor-related {alpha} (ERR{alpha} also called ERR1) was one of the first orphan receptors to be identified (7). It is expressed in several tissues during embryonic development (8) and in the adult (7, 9), suggesting a role of prime importance for this receptor throughout life. ERR{alpha} displays high sequence identity with the estrogen receptor (ER), hence its name (7). Indeed, ER and ERR{alpha} share 68% similarity of amino acids in their C domain and 36% in their E domain. Despite this sequence similarity, ERR{alpha} does not bind 17ß-estradiol (E2), the natural ligand of ER. Moreover, ER and ERR{alpha} have been reported to bind to different DNA targets. The estrogen response element (ERE) consists in an inverted repeat of the core elements separated by three variable nucleotides, on which ER binds as a homodimer (10). On the other hand, we and others have shown that ERR{alpha} binds to a single core element extended in its 5'-side by TCA trinucleotide (11, 12, 13). This sequence was originally found to mediate SF-1 transcriptional activity, and consequently termed SF-1 response element (SFRE) (14, 15, 16).

The nature of the transcriptional control exerted by ERR{alpha} is still a matter of controversy. Fusion of the C-terminal part of ERR{alpha} to the DNA-binding domain of the progesterone receptor (PR) generates a potent transactivating protein acting through the PR response element, which indicates that the E domain of ERR{alpha} is transcriptionally functional (17). Moreover, we have shown that a trimer of the SFRE confers a positive ERR{alpha} response to a minimal promoter in a cell-specific manner (11). ERR{alpha} also contributes to the induction of the lactoferrin promoter by ER (18), an effect that is likely to require the physical interaction demonstrated to occur between these two receptors. On another hand, a negative regulation of the SV40 late promoter has been reported to occur through elements that are different from the SFRE (12, 19). Sladek et al. (13) documented a negative effect of ERR{alpha} on the induction of the MCAD gene promoter by retinoic acid. This effect is mediated through a retinoic acid response element, consisting of two core elements separated by five nucleotides. The same report also demonstrated that ERR{alpha} is inactive on a single copy of the SFRE, cloned in front of a minimal promoter.

These contradictory results, together with the potential importance of ERR{alpha}, as judged by its broad expression pattern and its capacity of interaction with ER, prompted us to analyze in more detail the transcriptional properties of this receptor. Analysis of the DNA-binding capacities of ERR{alpha} show that it forms a homodimer on the SFRE site in vitro. In addition, the ability of both ERR{alpha} and SF-1 to bind to and to activate transcription through derivatives of the SFRE was evaluated, ascribing a consensus recognition sequence to each of these receptors. The positive effect of ERR{alpha} on promoter activity depends on the number of responsive sites, but not on the reporter gene nor on the minimal promoter used. cDNAs corresponding to ERR{alpha} originating from several species (human, mouse, and zebra fish) all efficiently enhance transcription in transient transfection assays via a consensus SFRE. This effect requires the integrity of the AF-2 domain, which may suggest a ligand-mediated regulation. In agreement with this, cells cultivated in charcoal-stripped medium do not support ERR{alpha}-induced transactivation. In conclusion, this report demonstrates that ERR{alpha} is a potent transcriptional activator.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Target Site Requirements of ERR{alpha}
We questioned in more detail the DNA-binding mode of ERR{alpha} using electrophoretic mobility shift assay (EMSA, Fig. 1AGo). Mouse (m) ERR{alpha} (lane 2) formed a single complex with the SFRE probe (sequence shown in Fig. 1BGo as oligonucleotide A). Using the deletion construct {Delta}A/BmERR{alpha} in the same assay (lane 3) resulted in the appearance of a faster migrating complex. As previously reported (11), a superposition of these patterns was obtained when mixing the independently translated proteins (lane 5). Surprisingly, cotranslation of the two products generated a third, intermediate migrating complex (lane 4, arrow), formed from a heterodimer between mERR{alpha} and {Delta}A/BmERR{alpha}, bound on the SFRE probe. The intensity of this complex could be modulated by varying the respective amounts of mERR{alpha} and {Delta}A/BmERR{alpha} plasmid used in cotranslation (lanes 6–10), reaching its maximum in a 50:50 ratio. We thus reinterpret the complex formed between the SFRE and ERR{alpha} as containing a homodimer of this receptor. Identical results were obtained using the oligonucleotides displayed in Fig. 1BGo as probes, suggesting irrelevence of the sequences flanking the SFRE for homodimer binding. Since ERR{alpha} forms a single complex with the SFRE probe, we conclude that this receptor preferentially binds as a homodimer on the SFRE.



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Figure 1. Dimerization of ERR{alpha}

A, EMSA. mERR{alpha} (wt, lane 2) or {Delta}A/BmERR{alpha} ({Delta}A/B, lane 3) were synthesized in vitro and allowed to bind on 32P-labeled consensus SFRE probe (first sequence displayed on panel B) in EMSA. Proteins were tested together either after cotransfection (cotr., lane 4) or as a mixture of separately translated products (indep. tr., lane 5). Proteins originating from cotranslation in a mERR{alpha} to {Delta}A/BmERR{alpha} plasmid ratio of 0:100, 25:75, 50:50, 75:25, or 100:0 (lanes 6 to 10) were used in EMSA. Lane 1, Unprogrammed reticulocyte used as a control. The arrow indicates a heterodimer between wt- and {Delta}A/BmERR{alpha}. This figure represents one of three independent experiments. B, Sequence of the oligonucleotides supporting ERR{alpha} homodimer binding. Sequence of the probes that have been tested is displayed. Core SFRE appears in capital letters.

 
Contradictory results have been obtained on the transcriptional effects of ERR{alpha} using different systems. For instance, the lack of transcriptional activity of ERR{alpha} reported by Sladek et al. (13) was documented using a single copy of the SFRE. In contrast, the reporter plasmid that we used in our published experiments (8, 11) contains three copies of the consensus SFRE sequence. To investigate this discrepancy, we analyzed the importance of the number of responsive sites in the reporter plasmid. To this end, SFRE-encompassing oligonucleotides were inserted as mono-, di-, or trimer, upstream of the thymidine kinase (tk) minimal promoter driving the chloramphenicol acetyltransferase (CAT) reporter gene (plasmid pBLCAT4; Ref 20 and Fig. 2AGo). These constructs were cotransfected in ROS 17/2.8 (rat osteosarcoma) cells together with increasing amounts of mERR{alpha}-encoding plasmid (Fig. 2BGo). In agreement with Sladek et al. (13), we did not find any effect of ERR{alpha} on a single copy of the SFRE. Indeed, three copies of this DNA fragment were required to yield a full transactivation by ERR{alpha}. Inserting two copies of the SFRE generated only a moderate ERR{alpha}-response, if any. We previously performed (11) transactivation experiments with a SV40-derived promoter driving the Luciferase gene, whereas the present ones were obtained with a tk promoter driving the CAT gene. We thus conclude that the transcriptional effect of ERR{alpha} is independent of the reporter and of the minimal promoter used. However, we constantly observed a lower effect of ERR{alpha} on pSFREx3CAT than on pSFREx3Luc. Essentially the same results were obtained using HeLa cells (data not shown).



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Figure 2. Influence of the Number of Responsive Sites on ERR{alpha} Transactivation

A, Structure of the constructs used in this study. Consensus SFRE sequences (represented by an arrow) were concatemerized and inserted in front of the minimal tk promoter driving the CAT reporter gene (plasmid pBLCAT4). All constructs were verified by sequencing. B, Cotransfection experiment. ROS 17/2.8 cells were cotransfected with 0.1 µg of the indicated reporter plasmid together with the indicated molar excess (activator over reporter) of mERR{alpha}-encoding plasmid. CAT activities were determined 48 h after transfection. The results are expressed as fold activation over the reporter activity without transactivator and represent the average of three independent experiments. Errors bars are indicated. White bars, Basal reporter activity without transactivator. Gray and black bars, 5- and 10 fold (respectively) molar excess of receptor over reporter.

 
SF-1 and ERR{alpha} were reported to bind to the same DNA sequence (SFRE; Refs. 14, 15, 16). We determined their respective binding specificity by competition EMSAs. To this end, we designed oligonucleotides, each bearing a single difference in the SFRE, relative to the consensus sequence (see Table 1Go). Systematic changes of the initial three and last two nucleotides of the SFRE were generated. In vitro translated SF-1 or ERR{alpha} proteins were incubated with a consensus SFRE radioactive probe in the presence of unlabeled mutant oligonucleotides. Table 1Go summarizes the competition properties of the oligonucleotides, as determined with this method. For both receptors, bases 1, 8, and 9 of the SFRE appeared irrelevant. In contrast, changing bases 2 and 3 of the SFRE not only affected the binding of SF-1 and ERR{alpha}, but also revealed subtle affinity differences between the two receptors. For example, TAAAGGTCA displayed ERR{alpha}- but not SF-1 binding, whereas TGAAGGTCA behaved in the reverse manner. This allowed us to define the indicated consensus binding sequences for SF-1 and ERR{alpha}. We then tried to correlate the binding capacities of the two receptors with their transcriptional activation capacities, taking the two above-mentioned sequences as models. To this end, oligonucleotides were inserted as trimers in front of the minimal SV40 promoter contained in plasmid pGL2-prm. The resulting plasmids (pTAAx3Luc and pTGAx3Luc, respectively) were transiently introduced in HeLa cells together with varying amounts of pSG5-SF-1 or pSG5-ERR{alpha}. As presented in Fig. 3Go, ERR{alpha} efficiently enhanced the reporter activity driven by plasmid pTAAx3Luc, but not by plasmid pTGAx3Luc. SF-1 displayed an opposite behavior. As a positive control, plasmid pSFREx3Luc (which contains a trimer of the consensus SFRE in the same plasmid context) was transactivated by both receptors. These results are in agreement with the EMSA experiments summarized above and suggest that, although generally binding to the SFRE, SF-1 and ERR{alpha} can discriminate between derivatives of this sequence and thus exert convergent but also different transcriptional activities.


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Table 1. Determination of ERR{alpha}- and SF-1-Specific Binding Sites

 


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Figure 3. Effect of ERR{alpha} and SF-1 on SFRE Derivatives

A, Structure of the constructions used in this study. Sequences of the SFRE derivatives are displayed. Bold characters indicate the divergence with respect to the consensus SFRE. B, Cotransfection experiment. HeLa cells were cotransfected with 0.1 µg of the indicated reporter plasmid together with the indicated molar excess (activator over reporter) of mERR{alpha}-encoding or SF-1-encoding plasmid, as stated. Luc activites were determined 48 h after transfection. The results are expressed as fold activation over the reporter activity without transactivator and represent the average of three independent experiments. Errors bars are indicated. White bars, Basal reporter activity without transactivator. Gray and black bars, 5- and 10-fold (respectively) molar excess of receptor over reporter.

 
Receptor Domain Requirements of ERR{alpha}
Since the discrepancies concerning the transcriptional activities of ERR{alpha} might be due to differences in the species from which the ERR{alpha} cDNA originates, we isolated cDNAs corresponding to ERR{alpha} in human and zebra fish (Danio rerio), hERR{alpha} and zfERR{alpha}, respectively, and analyzed their activity. The transcriptional effects of these constructs were compared with those (previously described in Ref. 11) of the mERR{alpha} cDNA. Increasing amounts of these constructs were cotransfected in ROS 17/2.8 cells together with pSFREx3Luc reporter plasmid. The three ERR{alpha} efficiently enhance the expression of the reporter gene in a dose-dependent manner, indicating that the transactivation properties of ERR{alpha} are evolutionarily conserved, at least from fish to human (Fig. 4Go). Comparable results were obtained when HeLa cells were transfected (data not shown).



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Figure 4. Transcriptional Activities of ERR{alpha} Originating from Various Species

ROS 17/2.8 cells were cotransfected with 0.1 µg of pSFREx3Luc plasmid together with the indicated molar excess (activator over reporter) of ERR{alpha}-encoding plasmid. Luciferase activities were determined 48 h after transfection. The results are expressed as fold activation over the reporter activity without transactivator and represent the average of three independent experiments. Errors bars are indicated.

 
Nuclear receptors are reported to contain two transactivation domains (1). In particular, this is true for the estrogen receptor (ER), which is closely related to ERR{alpha}. The AF-1 function is located in the A/B domain, is hormone independent but promoter and cell type dependent. The AF-2 region is located in the extreme part of the E domain and is hormone dependent in classical receptors. Since these regions are present in ERR{alpha}, we questioned their importance in transactivation driven by this receptor. The mERR{alpha} cDNA was deleted of its A/B domain, and the zfERR{alpha} was truncated in its AF-2 part (resulting in {Delta}A/BmERR{alpha} and {Delta}AF-2zfERR{alpha}, respectively; see Fig. 5AGo). We first determined that both recombinants were not impaired in their DNA-binding ability (Ref. 11 and data not shown). Increasing amounts of these constructs were then independently cotransfected together with the pSFREx3Luc plasmid in ROS 17/2.8 cells. As seen in Fig. 5BGo, the A/B domain (encompassing the putative AF-1 function) was not required for transactivation in our experimental conditions. In contrast, deleting the extreme 3'-part of the E domain, and thus the putative AF-2 region, abolished the transcriptional activity of zfERR{alpha}. Deleting the AF-2 region of nuclear receptors generally results in creating a dominant negative molecule. We thus tested the ability of {Delta}AF-2zfERR{alpha} to repress the activation induced by wild-type ERR{alpha}. pSFREx3Luc plasmid was cotransfected with increasing amounts of zfERR{alpha}, in the presence or absence of plasmid {Delta}AF-2zfERR{alpha}. As shown in Fig. 5CGo, the latter construct totally inhibited the activation driven by wtERR{alpha}, demonstrating its dominant negative feature. As a control, {Delta}AF-2zfERR{alpha} was unable to repress the activation exerted by retinoid X receptor (RXR){gamma} on DR1-containing constructs (right panel in Fig. 5CGo), indicating that the dominant negative effect is specific.



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Figure 5. Effect of ERR{alpha} Deletion Mutants

A, Structure of the mutants used in these experiments. A schematic organization of ERR{alpha} is displayed indicating the different functional domains. Black box indicates the putative AF2 region deleted in {Delta}AF2zfERR{alpha}. B, Transcriptional activities of the deletion mutants of ERR{alpha}. ROS 17/2.8 cells were cotransfected with 0.1 µg of pSFREx3Luc plasmid together with the indicated molar excess (activator over reporter) of plasmid encoding ERR{alpha}-derivatives. Luciferase activities were determined 48 h after transfection. The results are expressed as fold activation over the reporter activity without transactivator and represent the average of three independent experiments. Errors bars are indicated. C, Dominant negative effect of {Delta}AF2zfERR{alpha} construct. ROS 17/2.8 cells were cotransfected with 0.1 µg pSFREx3Luc or pDR1x3CAT plasmid together with the indicated molar excess (receptor over reporter) of zfERR{alpha}-encoding plasmid or RXR{gamma}-encoding plasmid, respectively. Where stated, a 5- or 10-fold molar excess (over reporter) of {Delta}AF2zfERR{alpha} was added. Experiments involving RXR{gamma} were performed in the presence of 10-7 M 9-cis-retinoic acid. Reporter activities were determined 48 h after transfection. The results are expressed as fold activation over the reporter activity without transactivator and represent the average of three independent experiments. Errors bars are indicated.

 
Since the AF-2 region is also required for the hormone-binding properties of nuclear receptors, this suggests that ERR{alpha} might be ligand regulated. To address this hypothesis, we tested the serum dependence of the transactivation driven by ERR{alpha}. ER{alpha}, whose transcriptional activity on the ERE is known to rely on the presence of estrogen in the culture medium, was used as a control. ROS 17/2.8 cells were cultured for 2 weeks in phenol red-free medium supplemented with charcoal-treated (hormone-free) serum or, alternatively, in native medium and serum. These conditions are referred to as "stripped" or "normal medium," respectively, on Fig. 6Go. pSFREx3Luc or pEREx2Luc plasmid was then transiently introduced into the cells together with ERR{alpha}- or ER{alpha}-expressing plasmid, respectively (Fig. 6AGo). After transfection, cells were further cultivated for 48 h in the same medium as before transfection. Both ER{alpha} and ERR{alpha} were unable to exert any transactivation on their cognate response element when cells were cultivated in charcoal-treated serum. As expected, transfection performed in normal medium conditions resulted in transactivation by ER{alpha} or ERR{alpha}. The antiestrogen ICI 164,384 inhibited the transactivation ability of ER{alpha} in normal medium, confirming that these culture conditions stimulate this receptor. On the contrary, ICI 164,384 has no effect on ERR{alpha}-driven transactivation. It is noteworthy that ER{alpha} achieved the same level of transcriptional enhancement as when the cells were maintained in stripped serum conditions supplemented with pure E2 (data not shown), suggesting that the amount of estrogen present in the culture medium was fully sufficient. Our data indicate that charcoal treatment withdrew a factor that is essential to the activity of ERR{alpha}. This factor could be a direct ligand (or precursor of a ligand) for ERR{alpha} or, alternatively, could intervene in any other way in ERR{alpha}-induced transactivation (e.g. through phosphorylation). We supposed that if a ligand for ERR{alpha} is present in the serum (as is E2 for ER{alpha}), the transcriptional activity of ERR{alpha} would be restored upon the shift from stripped to normal serum conditions. Transient transfections (pSFREx3Luc minus or plus ERR{alpha}) were thus performed on cells initially cultivated for 2 weeks in stripped serum conditions and shifted to normal serum at various times relative to transfection (Fig. 6BGo). ER{alpha} (cotransfected together with pEREx2Luc) was again used as a control. Changing the medium immediately after transfection (D0) resulted in a moderate enhancement of reporter activity induced by ER{alpha} or ERR{alpha}. The transactivation potential of these receptors increased when normal medium was added 24 h (D-1) or 48 h (D-2) before transfection. Interestingly, the potential enhancement of ERR{alpha} as a transactivator paralleled that of ER{alpha}, suggesting that the same phenomenon (i.e. accumulation of a ligand in the cells) occurred in both cases.



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Figure 6. Serum Requirements for ERR{alpha}-Induced Transactivation

ROS 17/2.8 cells were cotransfected with 0.1 µg pSFREx3Luc or pEREx2Luc plasmid together with a 10-fold molar excess of mERR{alpha}-encoding plasmid or hER{alpha}-encoding plasmid (respectively), as indicated. Luciferase activities were determined and are expressed as fold activation over the reporter activity without transactivator. Results represent the average of three independent experiments. Errors bars are indicated. Stripped medium, Phenol red-free DMEM supplemented with charcoal-treated serum. Normal medium, DMEM supplemented with native serum. White bars, Basal reporter activity without transactivator. Black bars, 10-fold molar excess of receptor over reporter. A, Effect of serum depletion. Cells were cultivated for 2 weeks in untreated serum-containing medium or stripped serum-containing medium, as indicated. Same medium was added after transfection with 10-8 M ICI 164,384 where indicated. B, Kinetics of serum effect. Stripped culture medium was shifted to normal medium at the indicated time relative to transfection (D0, immediately after transfection; D-1, 24 h before transfection; D-2, 48 h before transfection).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ERR{alpha} Binds as a Homodimer in Vitro
Earlier reports have described the SFRE sequence as a preferred binding target for ERR{alpha} (11, 12, 13). Based on experiments in which ERR{alpha} and {Delta}A/BERR{alpha} proteins were mixed after translation, we concluded that this receptor binds as a monomer on the SFRE (11). Here we have used another experimental protocol, namely cotranslation of a wild-type and a mutant form of ERR{alpha}. This generated three complexes with the SFRE probe, the intermediately migrating one representing an association of both protein products. The two other complexes are formed of bound homodimers of either wild-type or deletion-mutant ERR{alpha}. It is noteworthy that these three complexes were also observed upon cotranslation of mouse and human ERR{alpha}, but not upon mixing of their separated translation (data not shown). The same situation has also been observed for Xenopus ER, where a heterodimer between two isoforms can be observed only upon cotranslation of the mRNAs (21). Since we repeatedly observed a single complex between ERR{alpha} and the SFRE probe, our data suggest that this receptor can only bind in vitro as a homodimer on this probe and not as a monomer as previously claimed.

Evidence for monomer binding also came from deoxyribonuclease (DNase) footprinting experiments on the SV40 major late promoter (12). These data demonstrated that ERR{alpha} contacts a single extented half-site on the promoter. Taken together with the present data obtained with an SFRE probe (also encompassing a single extended half-site), this suggests that only one member of the ERR{alpha} homodimer binds to DNA. The other member of the dimer would develop protein-protein contact but no DNA-protein contact. In this hypothesis, the sequences flanking the SFRE core are predicted to be irrelevant for dimer binding. This is indeed the case since all the probes shown in Fig. 1BGo supported ERR{alpha} dimer binding. In particular, the shorter size of oligonucleotide D excludes the existence of a cryptic second half-site that could be necessary for homodimerization. The SFRE, although monovalent, is thus the only critical determinant in the homodimer binding of ERR{alpha}. Such a situation could be reminiscent of the NGF1-B-RXR heterodimer bound on the monovalent AAAGGTCA sequence, in which RXR does not contact DNA (22). However this observation has not been confirmed (23), and a divalent NGFI-B response element that is a lot more potent has been found in the POMC gene promoter (24). Dimer binding of ERR{alpha} on a monovalent site could thus represent a new, unique feature among nuclear receptors.

The fact that homodimerization can only be observed upon cotranslation suggests that the ERR{alpha}-dimer is a very stable complex as has been pointed out for dimers of the Xenopus ER (21). Alternatively, it could be that physical association between two ERR{alpha} peptides can only be initiated during protein synthesis. Once translation is finished, the conformation adopted by the completed protein could prevent the association between the two species. As the proteins were translated in an in vitro system that is devoid of DNA (other than the plasmid carrying the ERR{alpha} constructs), we suggest that dimerization does not require DNA. However, we must emphasize that the in vivo relevance and significance of this peculiar dimeric mode of binding are presently unknown and will require more investigation.

ERR{alpha} Is an Activator of Transcription
We here describe the positive transcriptional activity of the orphan nuclear receptor ERR{alpha} on the SFRE site. This activity is apparently independent of the A/B domain of the receptor. However, it should be noted that the AF-1 region of nuclear receptors (located in the A/B domain) functions in a promoter- and cell-dependent manner (1). The {Delta}A/B mutant that we used may thus behave in a different manner under other experimental conditions. On the contrary, ERR{alpha} transcriptional activity is dependent on the putative AF-2AD region. This is consistent with published data demonstrating that the putative transactivation domain (domain E) of ERR{alpha} is fully competent since fusion of the E region of this receptor to the DNA-binding domain of the PR generates a chimeric transcriptional activator acting on PR response elements (17).

We have previously reported that ERR{alpha} enhances the expression of the luciferase reporter gene under the control of the SV40 minimal promoter supplemented with three copies of the SFRE (11). Our present data show that the transactivation driven by ERR{alpha} is independent of the minimal promoter and of the reporter gene used. However, it should be noted that the level of transactivation achieved is always lower with the tkCAT system than with SVLuc. Interestingly, three copies of the SFRE sequence must be cloned in front of the minimal promoter to ensure maximum efficiency. In particular, a single copy of the SFRE is unable to confer any response to ERR{alpha}. Our results are consistent with those reported by Sladek et al. (13) who could not demonstrate any ERR{alpha}-driven transactivation, using a single copy of the SFRE sequence. We have shown that the promoter driving the expression of the thyroid hormone receptor-{alpha} is transactivated by ERR{alpha} (9). Interestingly enough, this effect depends on the single SFRE site present on this promoter, pointing to the importance of the promoter context that surrounds the cis- acting sequence. On the contrary, two imperfect SFRE sites are required for maximal activation of the osteopontin promoter by ERR{alpha} (25).

The situation seems to be different when considering the effect of ERR{alpha} on responsive sites other than the SFRE. For instance, it has also been reported that this receptor represses the activation of the MCAD gene promoter by the retinoic acid receptor (13). A DR5 sequence present on the promoter is necessary and sufficient to mediate this activity. Similarly, ERR{alpha} has a negative effect on the SV40 late promoter, apparently through an ERE present in the genome of this virus (12, 19). On the other hand, activation of the lactoferrin promoter by ERR{alpha} requires both the SFRE and the imperfect ERE present on this promoter (18). Our data show that ERR{alpha} can act as a potent transcription activator in given conditions. Together with results published by others, we conclude, however, that this receptor is a pleiotropic modulator of transcription. This type of phenomenon has already been emphasized for other nuclear receptors and is therefore not surprising. For instance, members of the FTZ-F1/SF-1 subfamily mediate various transcriptional effects, ranging from inhibition to low and high levels of activation (16, 26, 27). A number of potential target genes of ERR{alpha} have been suggested (as reviewed above), and this receptor has been suspected to act as a modulator of bone (8, 25) and fat metabolism (13), where it is highly expressed. However, in the absence of knockout and/or transgenic studies, the precise in vivo functions of ERR{alpha} remain largely unknown.

Is ERR{alpha} a True Orphan Receptor?
ERR{alpha} was (together with ERRß) the first receptor described as an orphan and is, up to now, still viewed as such (7). Whether true orphan receptors indeed exist, or if proteins that are considered as such simply await the identification of their natural ligand, is a question that remains open. ERR{alpha} makes no exception to this rule. The fact that the putative AF-2AD domain of ERR{alpha} is required for transactivation could suggest that this activity is ligand dependent. Indeed, determination of the crystal structure of classical nuclear receptors has shown that the AF-2AD domain blocks the ligand into the hydrophobic pocket, thereby stabilizing the receptor in an "active" conformation (28). However, the AF-2AD domain is also necessary to establish contacts with coactivators (29, 30, 31, 32, 33). The lack of transactivation in the absence of the putative AF-2AD domain in ERR{alpha} might be due to an inability to interact with a coactivator and thus is not a decisive argument in favor of the existence of a ligand. Nevertheless, the experiments presented here suggest that a regulator of ERR{alpha} transcriptional activity is indeed present in the serum and is withdrawn by charcoal treatment. The identity of this factor is still unknown although some hormones can be excluded. Indeed, the lack of activity of ERR{alpha} under stripped serum conditions could not be challenged by addition of thyroid hormone, retinoic acids, E2, raloxifene (a synthetic antiestrogen), vitamin D, dexamethasone, or pregnenolone (data not shown). How the compound present in the serum acts is also an unresolved question. Our experiments suggest only that ERR{alpha} has an activator, and not necessarily a ligand in the sense of E2 for ER for example. The effect can also be indirect and, for example, could potentiate a coactivator (or any factor that contributes to ERR{alpha}-driven transactivation) rather than ERR{alpha} itself. It must be admitted that the effect is ERR{alpha} specific and dispensable for ER{alpha}. The latter is indeed affected by the same charcoal treatment, but E2 addition is sufficient to reverse the inhibition of its transcriptional activity. The lack of activity of ERR{alpha} and ER{alpha} in stripped serum conditions can be overcome by addition of normal medium. Interestingly, the kinetics with which this restoration occurs is the same for ERR{alpha} and ER{alpha}. Since the activator of ER{alpha} in the medium is a direct ligand, our data strongly suggest that ERR{alpha} is also a ligand-regulated receptor and not an orphan one.

SF-1 and ERR
Both SF1 and ERR{alpha} have been shown to bind to the same general element, namely, the SFRE. Nevertheless, our experiments reveal subtle differences in the preferential binding of these receptors on derivatives of the SFRE. This allows the definition of a consensus binding sequence for each of these factors. The reason for these differences is at present unclear. It has been shown that the T/A box of nuclear receptors, a region located immediately downstream of the C domain, directs the specific recognition of the 5'-extension of the core sequence (15). Comparison of the T/A boxes of SF-1 and ERR{alpha} has revealed strong similarities, consistent with their common interaction with the consensus SFRE (15). However, the sequence divergences of these two factors in their T/A box are likely to be responsible for the preferential binding to SFRE derivatives.

At least in two cases (the TGA and TAA variants of SFRE), the difference between SF-1 and ERR{alpha} observed in the EMSA is reflected in their transcriptional behavior. This suggests that naturally occurring SFRE-like sequences might respond to one or the other receptor as a function of its binding affinity to the target site. As an example, seven SFRE-like sequences can be found on the osteopontin promoter, none of which represent a perfect consensus (34). Two of these sequences are bound by ERR{alpha} but none are bound by SF-1 (25). Consistently, we have found that the osteopontin promoter is transactivated by ERR{alpha} but not by SF-1. Conversely, the analysis of SF-1-responsive gene promoters (and particularily those implicated in steroid hydroxylase cascade; Refs. 1, 35 and references therein) could indicate whether these genes are likely to be transactivated by ERR{alpha}.

Several parameters thus contribute to the specificity of response to SF-1 or ERR{alpha}. In addition to the differences in the pattern of expression of these two receptors, which have already been noted (8, 36), the relative binding affinity for an SFRE derivative can thus contribute to the specificity of response to SF-1 or ERR{alpha}. Given the cell specificity of action displayed by both receptors and the pleiotropic effects that can be observed according to various parameters, we conclude that, although initially described as binding to the same consensus sequence, SF-1 and ERR{alpha} can mediate a vast array of transcriptional responses.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Constructions
The zebra fish homolog of ERR{alpha} was isolated by cDNA library screening and will be described elsewhere (E. Bonnelye, V. Laudet, and B. Thisse, manuscript in preparation). Deletion of the putative AF2AD region was performed using the PCR technique. cDNAs were subsequently subcloned in the EcoRI site of plasmid pSG5. Plasmid {Delta}A/BmERR{alpha} has been described previously (11). Partial human ERR{alpha} cDNA was isolated from a library of K562 cells using the mERR{alpha} cDNA as a probe. An in-frame ATG was added by PCR. PCR was performed using Gold Taq polymerase (Perkin Elmer Corp., Norwalk, CT). PCR fragments were sequenced to confirm the lack of undesired mutations introduced during the amplification process.

SFRE derivatives containing 30-bp long oligonucleotides flanked by BglII and BamHI restriction sites were ligated in the presence of both restriction enzymes to ensure a correct orientation. Concatemers were purified on a native polyacrylamide gel. Products were inserted in the BglII site of plasmid pGL2 PRM (Promega Corp., Madison, WI) or in the BamHI site of plasmid pBLCAT4 (20). All constructions were verified by sequencing. In all cases, SFREs are separated by 21 nucleotides that are identical from one construct to another.

EMSAs
ERR{alpha} or SF-1 proteins were translated in vitro using TNT kit (Promega Corp.). Consensus SFRE probe (A sequence in Fig. 1BGo) was end labeled with {gamma}-32P ATP. Reticulocyte lysates were incubated with 40 103 cpm of probe as described previously (37). Unlabeled oligonucleotides were added as competitor where stated. Reactions were run on a native polyacrylamide gel.

In the dimerization experiment, mERR{alpha} and {Delta}A/BmERR{alpha} plasmid were cotranslated in a 1:1 (cotr. lane in Fig. 1Go) or on a 0:100, 25:75, 50:50, 75:25, or 100:0 ratio as indicated. Equal quantities of individually translated mERR{alpha} and {Delta}A/BmERR{alpha} were used in the "indep. tr." lane in Fig. 1Go.

Cells and Transfections
Cell lines (HeLa and rat osteosarcoma ROS17/2.8) were maintained in DMEM with 10% FCS. For serum test, cells were maintained for 2 weeks in phenol red-free medium supplemented with charcoal-treated serum. Transient transfection was performed using ExGen (Euromedex, Soufflersheim, France), in which 0.1 µg of the reporter plasmid was introduced together with the indicated molar excess of receptor-producing plasmid. When necessary, pSG5 plasmid was added as a carrier up to 1 µg per transfection. Cells were washed after 4 h and fresh medium was added. Cells were then lysed 48 h after transfection and processed for reporter quantitation using standard methods.


    ACKNOWLEDGMENTS
 
We thank Vincent Giguère for the generous gift of mERR{alpha}, Philippe Berta for SF-1, and Frank Delaunay for ER{alpha}. We are indebted to Olivier Gandrillon, Marc Robinson, and Hector Escriva-Garcia for critical reading of the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Jean-Marc Vanacker, Centre National de la Recherche Scientifique, UMR 49, Ecole Normale Supérieure de Lyon, 46 allée d’Italie, F-69364 Lyon Cedex 07, France. E-mail: Jean-Marc.Vanacker{at}ens-lyon.fr

This work was supported by Centre Nationale de la Recherche Scientifique, Ministère de l’Enseignement Supérieur et de la Recherche and Association pour la Recherche Contre le Cancer.

1 Present address: Department of Anatomy and Cell Biology, University of Toronto, M5S 1A8 Canada. Back

Received for publication November 3, 1998. Revision received February 1, 1999. Accepted for publication February 22, 1999.


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