Constitutive Activation of Transcription and Binding of Coactivator by Estrogen-Related Receptors 1 and 2

Wen Xie, Heng Hong, Na N. Yang1, Richard J. Lin, Cynthia M. Simon, Michael R. Stallcup and Ronald M. Evans

Howard Hughes Medical Institute (W.X., R.M.E.) Gene Expression Laboratory (W.X., N.N.Y., R.J.L., C.M.S., R.M.E.) The Salk Institute for Biological Studies La Jolla, California 92037
Department of Pathology (H.H., M.R.S.) University of Southern California Los Angeles, California 90033


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this report, we demonstrate that, in contrast to most previously characterized nuclear receptors, hERR1 and hERR2 (human estrogen receptor-related protein 1 and -2) are constitutive activators of the classic estrogen response element (ERE) as well as the palindromic thyroid hormone response element (TREpal) but not the glucocorticoid response element (GRE). This intrinsically activated state of hERR1 and hERR2 resides in the ligand-binding domains of the two genes and is transferable to a heterologous receptor. In addition, we show that members of the p160 family of nuclear receptor coactivators, ACTR (activator of thyroid and retinoic acid receptors), GRIP1 (glucocorticoid receptor interacting protein 1), and SRC-1 (steroid receptor coactivator 1), potentiate the transcriptional activity by hERR1 and hERR2 in mammalian cells, and that both orphan receptors bind the coactivators in a ligand-independent manner. Together, these results suggest that hERR1 and hERR2 activate gene transcription through a mechanism different from most of the previously characterized steroid hormone receptors.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our understanding of the action of steroid and thyroid hormones, vitamin D, and retinoid acid has undergone a rapid development as a consequence of the molecular characterization of their cognate receptors and characterization of the specific recognition of the target DNA element (for review, see Refs. 1, 2, 3). Nearly all members of the nuclear receptor (NR) superfamily share similar structural features, most notably, a DNA-binding domain (DBD) composed of two zinc fingers and an associated carboxyl-terminal stretch of approximately 250 amino acids, which comprise the ligand binding domain (LBD, for review, see Refs. 1, 2, 3). Embedded within the LBD is a hormone-dependent transcription activation domain (AF2, or E domain), and a transcription repression domain that functions only in the absence of hormone. In some NR family members, such as the two estrogen receptors (ER{alpha} and ß) and steroidogenic factor 1 (SF-1), an additional activation function (AF-1, or A/B domain) resides in the NH2-terminal region of the NRs (4, 5, 6, 7). It is possible to exchange the domains between receptors to create chimeras with predictable altered DNA and/or ligand binding properties (8).

An important conceptual advance for NRs was the isolation and characterization of the orphan receptors whose cognate ligands are either unknown or unnecessary. Most orphan receptors exhibit domain structure similar to that of classic NRs (3, 9, 10, 11). At present, orphan receptors are by far the largest subclass of the family, and ligands and synthetic drugs have been identified for some of these (for review, see Refs. 3, 9, 12).

Recent advances have identified several classes of NR cofactors that appear to play key roles in transcriptional activation (for review, see Refs. 13, 14). One well characterized family of coactivators for NRs are the three related p160 proteins: steroid receptor coactivator 1 (SRC-1) (15, 16), glucocorticoid receptor interacting protein 1 (GRIP1)/transcriptional intermediary factor 2 (TIF2) (17, 18, 19), and activator of thyroid and retinoic acid receptors (ACTR) (20) [also known as p/CIP (p300/CBP interacting protein) (21), RAC3 (receptor-associated coactivator 3) (22), AIB1 (amplified in breast cancer 1) (23), and TRAM-1 (thyroid hormone receptor activator molecule-1)(24)]. Coactivators are targeted to the LBD via specific receptor interaction domains or RIDs (for review, see Refs. 13, 14). Both structural and mutational analysis of multiple RIDs revealed a core LXXLL motif (where L is leucine and X can be any amino acid) that mediates the direct association. The LXXLL motif-containing {alpha}-helices located in the central region of the p160 coactivators function as protein-protein interaction modules and contact a hydrophobic groove on the surface of an agonist bound LBD (19, 21, 26, 27, 28). Therefore, the LBD serves as a molecular switch that recruits activator proteins in the presence of ligand and releases them in the absence of ligand. While classical NRs require binding of ligand before they can activate transcription (16, 18, 19), some receptors display constitutive activity and bind coactivator in the absence of any apparently added inducer. For example, orphan receptor CARß apparently functions as a transcriptional activator and associates with coactivator SRC-1 in the absence of ligand, while the binding of its ligand androstane metabolites results in the dissociation of bound coactivators, thus providing a negative regulatory mechanism for the ligand (29).

Even though human estrogen receptor-related protein 1 and 2 (hERR1 and -2) were the first orphan receptors identified, their properties and mechanisms of transcriptional activation remain to be defined. We now describe the characterization of ligand-independent gene activation by each of these receptors and suggest that members of the p160 family of NR coactivators potentiate this activity.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ligand-Independent Activation of Estrogen Response Element (ERE)- and Palindromic Thyroid Hormone Response Element (TREpal)-Controlled Reporter Genes by hERR1 and hERR2
To analyze the transactivation of hERR1 and hERR2, expression vectors pRShERR1 and pRShERR2 were cotransfected into CV-1 cells with a series of hormone-responsive reporter plasmids based on the thymidine kinase (tk) promoter driving luciferase or chloramphenicol acetyltransferase (CAT) gene expression. As shown in Fig. 1AGo, both hERR1 and hERR2 unexpectedly activate transcription through both the ERE and the palindromic thyroid hormone response element (TREpal) (Fig. 1AGo) reporters in a hormone- and serum-independent fashion. Under the same serum-free conditions, other NRs including the ER and TR (Fig. 1AGo) and GR (Fig. 1BGo), activate gene transcription only in the presence of their cognate ligands. To demonstrate that this constitutive activity is promoter independent, another series of reporters based on the glucocorticoid-inducible mammary tumor virus (MTV)-CAT (10) was used in the cotransfection experiments. To generate {Delta}MTV-ERE-CAT and {Delta}MTV-TREpal-CAT (30), the glucocorticoid response element (GRE) in the MTV sequence was replaced by the ERE or TREpal, respectively. As shown in Fig. 1BGo, when this series of reporters were cotransfected in HeLa cells together with the hERR2 expression plasmid, gene transcription is again activated independently of exogenous hormones or serum. In addition, similar activation curves were seen with the ERE-containing Xenopus vitellogenin A2 gene promoter (vitA2 -331/-87CAT) (31) when cotransfected with hERR1 and hERR2 expression vector (data not shown). Induction is specific to ERE and TRE reporter genes as the two hERRs fail to stimulate MTV, a reporter carrying the glucocorticoid response element under the same experimental conditions (Fig. 1BGo). This hormone-independent transactivation activity of hERR2 depends upon the amount of the expression vector transfected into the recipient cells; the activity increased with the amount of hERR1 or hERR2 expression vector transfected. Shown in Fig. 2Go is a saturation curve revealing a dose-dependent transactivation profile.



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Figure 1. Ligand-Independent Activation of ERE- and TREpal-Controlled Reporter Genes by hERR1 and hERR2

A, Two micrograms of expression plasmids encoding ER, TR, hERR1, and hERR2 were cotransfected into CV-1 cells together with 5 µg of either a thyroid-hormone-responsive (tk-TRE-Luc) or an estrogen-responsive (tk-ERE-Luc) reporter plasmid. Luciferase activity was measured and graphed. Where indicated transfected cells were grown with 10-7 M T3 or 10-8 M E2. C, Control expression plasmid. The amount of expression plasmid in each reaction was compensated to a total of 10 µg by addition of pRSerbA-1 control plasmid. The transfections were done under serum-free condition; cells were split into serum-free DMEM 1 day before transfection. B, HeLa cells were cotransfected with 2 µg of the expression plasmid and 5 µg of the reporter plasmid by calcium phosphate precipitation method as indicated. {Delta}MTV, MTV, ERE, and TREpal represent the reporter plasmids {Delta}MTV-CAT, MTV-CAT, {Delta}MTV-ERE-CAT, and {Delta}MTV-TREpal-CAT (10 ), respectively. Cells were treated with ethanol (-), 10-7 M dexamethasone (Dex), or 10-8 M estradiol (E2) or 10-7 M T3 after transfection. CAT activity was normalized by ß-galactosidase activity.

 


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Figure 2. Dose-Dependent Activation of hERR2

A, Increasing amounts (0.001–10.0 µg) of pRShERR2 plasmid were transfected into HeLa cells (lanes 2–8) as indicated under each reaction. Five micrograms of {Delta}MTV-TREpal-CAT reporter plasmid were cotransfected. Two micrograms of pRShTR{alpha} (68) were transfected in lanes 9 and 10. T3 (10-7 M) was added to reaction 10 after calcium phosphate transfection while ethanol was added to reaction 1–9. B, Quantitation of the CAT activity. Percentage of conversion of CAT activity was calculated and graphed. Activity was measured relative to the activity of 10 µg pRShERR2 (maximum conversion was arbitrarily defined as 100%). Relative activity of each conversion was then plotted against the amount of plasmid in the transfection.

 
The Intrinsically Activated State of hERR1 and hERR2 Resides in Their LBDs
To further confirm their constitutively active phenotype and to localize the regions that transmit this property, a series of domain swap mutants were generated. Site-directed mutagenesis was used to introduce common NotI and XhoI restriction sites into the receptor cDNA sequences to generate hGRNX, hERR1NX, and hERR2NX, as illustrated in Fig. 3AGo. The DBDs of hERR1 and hERR2 were then swapped with that of hGR to generate chimeric receptor GE1G and GE2G (Fig. 3AGo). When the resulting chimeric receptor GE2G was transfected into HeLa cells, it functioned as a glucocorticoid-dependent activator of reporter vectors harboring ERE and TRE sequences (Fig. 3BGo). This indicates that the DBD of hERR2 is important for response element recognition but does not embody the dominant constitutive activity of the parental receptor.



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Figure 3. Construction and Activity of GR/ERR Chimera

A, Unique NotI and XhoI sites were introduced into the sequences of hGR, hERR1, and hERR2 flanking the DBDs to create mutants hGRNX, hERR1NX, and hERR2NX. Numbers above the boxes correspond to the amino acid positions. Chimeric receptors were created by exchanging domains through the common XhoI and NotI sites. Hybrids are named by three letters referring to the origins of each domain. All the expression plasmids used in transfections performed in this paper were constructed in the pRS vector. Note the diagrams are not necessarily proportional to the actual domain sizes. B, GE2G activates gene transcription through ERE and TRE DNA sequences. HeLa cells were transfected with 2 µg control plasmid (lanes 1, 2, 5, 6, 9, and 10) or 2 µg of pRSGE2G (lanes 3, 4, 7, 8, 11, and 12) and 5 µg of the reporter plasmids, {Delta}MTV-CAT (lanes 1–4), {Delta}MTV-TREpal-CAT reporter (lanes 5–8), and {Delta}MTV-ERE-CAT reporter (lanes 9–12). Dexamethasone (Dex) was administered after calcium phosphate transfection at 10-7 M as indicated. C, GGE1 and GGE2 activity is hormone independent. HeLa cells were cotransfected with 2 µg of the expression plasmids pRShGRNX (lanes 3 and 4), pRSGGE1 (lanes 5 and 6), and pRSGGE2 (lanes 7 and 8) and 5 µg of the reporter plasmid MTV-CAT by the calcium phosphate coprecipitation method. Cells were then treated with minus (-) or 10-7 M dexamethasone (Dex) as indicated.

 
In the reciprocal experiments, the putative LBDs of the hERR1 and hERR2 were substituted with that from the GR to generate GGE1 and GGE2 (Fig. 3AGo). The resulting hybrids should recognize the GRE of the MTV-LTR. When cotransfected with MTV-CAT reporter genes into HeLa cells, GGE1 and GGE2 activated gene transcription through MTV-LTR in the absence of any exogenous inducers (Fig. 3CGo, lanes 5 and 7) in contrast to the parental hGRNX molecule, which activated MTV only in a hormone-dependent fashion (Fig. 3CGo, lane 3 and 4). Together, these data indicate that the LBDs of the hERR1 and hERR2 are in an intrinsically activated state and that this property is transferable to a heterologous receptor.

The Intact LBDs of hERR1 and hERR2 Are Essential for Transcriptional Activation
In the case of the GR and ER, the LBDs function as a contiguous unit such that disruption of this region by deletion or insertions leads to loss of hormone binding. Such mutants are expected to become transcriptionally inactive. In contrast, complete truncation of the hormone- binding domain can lead to constitutively active mutants as previously described for both GR and ER (4). Accordingly, a series of mutations were generated in the LBD of hERR1 to examine their potential effects on transactivation (Fig. 4Go). These include a C-terminal truncation mutant 2681, an internal deletion mutant, {Delta}268–373, and a linker insertion mutant I373 (Fig. 4Go). Mutant 2681 has the entire LBD deleted after amino acid position 268 with an addition of six extra amino acids from the inserted linker. Mutant {Delta}268–373 has an in-frame deletion from amino acid 268 to 373, and mutant I373 contains a linker encoding five amino acids, PHRWG, inserted in-frame after amino acid 373 without disrupting any other parts of the molecule. When each of the mutants was tested, all were transcriptionally silent (Fig. 4Go). Thus, an intact LBD is apparently required for the constitutive activation phenotype. Furthermore, the failure of these mutants in transactivation indicates that, unlike ER, the putative AF-1 domain in the N terminus of hERR1 and 2 may not contribute to their transcription activity.



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Figure 4. Structure and Activity of Mutant hERR1 Gene Products

HERR1 gene and mutants are shown. Construction of the mutants are described in Materials and Methods. Numbers indicate the amino acid positions. CAT activity was assayed using HeLa cell extracts obtained by cotransfection with the indicated mutant hERR1 genes and {Delta}MTV-TREpal-CAT reporter plasmid. The transfection procedures were as described in Fig. 1BGo. C, Control; AC, activation.

 
ACTR, GRIP1, and SRC-1 Are Transcriptional Coactivators of hERR1 and hERR2
NR interacting proteins, including members of the p160 family of coactivators, play key roles in hormone-induced transcription. ACTR, GRIP1, and SRC-1 function as transcriptional coactivators for a wide group of the NRs, both in yeast and in mammalian cells (16, 18, 20, 23, 32, 33). We performed transfection assays to examine whether the p160 family members would also serve as transcriptional coactivators for hERR1 and hERR2. Indeed, in transiently transfected CV-1 cells, the activation of the ERE-controlled reporter gene ({Delta}MTV-ERE-Luc) by hERR1 (Fig. 5AGo) and hERR2 (Fig. 5BGo) was enhanced significantly by cotransfection of ACTR, GRIP1, or SRC-1, while these coactivators did not activate the reporter gene in the absence of hERRs (Fig. 5AGo). Among the examined coactivators, GRIP1 exhibited relatively higher potency as compared with ACTR and SRC-1. In the case of GRIP1, while the wild-type proteins potentiate the activation of transcription by hERR1 (Fig. 5DGo) or by hERR2 (Fig. 5EGo), a mutant GRIP1 containing mutations in NR boxes II and III failed to function as a coactivator (Fig. 5Go, D and E). In the NR box mutant, the second and third LXXLL motifs are changed to LXXAA; these mutations are known to disrupt binding to the hydrophobic pocket created by the carboxyl-terminal AF-2 domain (Ref. 32 and data not shown). In the coactivator transfection assays, treatment with 17ß-estradiol (E2) had no effect on either hERR1 or -2 activation (data not shown).



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Figure 5. ACTR, GRIP1, and SRC-1 Are Transcriptional Coactivators for hERR1 and hERR2

A and B, CV-1 cells were transiently transfected with {Delta}MTV-ERE-LUC reporter gene (0.5 µg), pCMX-hERR1 (panel A) or pCMX-hERR2 (panel B) (0.5 µg), together with 0.5 µg of expression vectors of indicated coactivators, ACTR, GRIP1, and SRC-1a. Total DNA used in each transfection was adjusted to 2 µg by adding the appropriate amount of empty expression vectors. Cells were grown in charcoal/dextran-treated serum with no added hormone after transfection (34 ). Luciferase assays were performed 48 h after transfection. C, Diagram of GRIP1 structure. The vertical filled boxes represent NR boxes (NRB) I, II, and III, each composed of a LXXLL motif. The CBP/p300 interaction domain of GRIP1 is also indicated. D and E, CV-1 cells were transiently transfected with {Delta}MTV-ERE-LUC reporter gene (0.5 µg), pCMX-hERR1 (panel D), or pCMX-hERR2 (panel E) (0.5 µg), together with 0.5 µg of wild-type GRIP1 or a mutant GRIP1 bearing mutations in the LXXLL motifs of NR Boxes II and III.

 
Thus, both hERR1 and hERR2, like classical nuclear hormone receptors, can utilize all three p160 coactivator family members, ACTR, GRIP1, and SRC-1, as transcriptional coactivators.

Ligand-Independent Binding of Coactivators by hERR1 and hERR2
We used a glutathione-S-transferase GST pull-down assay to determine whether hERR1 and -2 can directly interact with p160 proteins in vitro. As shown in Fig. 6AGo, while GST does not bind the full-length hERR1 or hERR2 synthesized in vitro, the bead-bound GST-ACTR-RID621–821 (20) is able to bind the hERRs efficiently. The binding between ACTR and hERRs can also be demonstrated when the LBD of the hERRs, instead of the full-length proteins, was used (data not shown). In the same GST pull-down assays, GST-GRIP1563–1121 also bound full-length hERR2 efficiently (Fig. 6BGo). No hormone was added either in the synthesis of the proteins or in the binding reactions, further supporting the notion that activation of transcription by hERRs is ligand independent.



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Figure 6. Ligand-Independent Binding of Coactivators by hERR1 and hERR2

A and B, The full-length hERR1 or hERR2 was synthesized in vitro with [35S]methionine and then incubated with Sepharose bead-bound GST, GST-ACTR-RID621–821 (panel A), or GST-GRIP1563–1121 (panel B), in the absence of any added hormone. After washing, the bead-bound hERR proteins were eluted and analyzed by SDS-PAGE and autoradiography. For reference a sample equivalent to 10% of the labeled protein in the binding assay is shown along with the total amount of labeled protein bound to the beads. C, The LBD of hERR1 (VP-L-hERR1) or hERR2 (VP-L-hERR2) interacts with ACTR-RID621–821 in mammalian two-hybrid assays (20 ). CV-1 cells were cotransfected with expression plasmids for tk-(MH100)4(UAS)-Luc and ACTR-RID621–821, together with expression vector of VP control, or VP-L-hERR1 and VP-L-hERR2. No exogenous ligand was added in this assay.

 
The interaction between ACTR and hERRs was further demonstrated in a mammalian cell two-hybrid assay. Using tk-(MH100)4(UAS)-Luc (29) as reporter and GAL-ACTR-RID621–821 as bait, VP16 activation domain (VP) itself results only in a low level of basal activation. In contrast, the fusion of VP16 with the LBD of hERR1 or -2 (VP-L-hERR1 and VP-L-hERR2) exhibited substantial activation in the absence of exogenously added ligand (Fig. 6CGo).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Orphan NRs are continuing to increase in number and diversity (3, 9, 12). The large number of currently known orphan NRs, guided by the well characterized paradigm of the classical nuclear hormone receptors, provides an invaluable resource for the elucidation of new aspects of developmental and regulatory biology.

HERR1 and -2 were originally isolated by low-stringency hybridization probed with the DBD of human ER and were the first two orphan receptors identified. Indeed, ERR is also an expanding family of proteins; we have recently cloned the closely related ERR3 gene as a GRIP1 interacting protein (34). While ERRs display significant homology to the ER (hERR1 has 91% amino acid identity with hERR2, 68% with hER in DBD), none bind either natural or synthetic estrogens in vitro (35). In addition, initial ligand screen has ruled out some other traditional hormones as ERR ligands (N. N. Yang and R. M. Evans, data not shown). Interestingly, individual ERRs appear to have unique expression patterns: ERR1 expresses widely in later stages of mouse embryos, although its expression in the heart, skeletal muscles, and nervous system is relatively abundant (36). The expression of ERR2 is restricted to the trophoblast progenitor cells between embryonic days 6.5 and 7.5. Restricted low levels of ERR2 expression were also detected in adult mouse tissues (35). The highest expression of ERR3 occurs around days 11–15 post coitum, a period of very active organogenesis (34). The restricted patterns of their expression suggest ERRs have unique potential significance in developmental and physiological processes. Indeed, ERR1 has been shown to be a transcriptional regulator of the human lactoferrin (37), the human medium-chain acyl coenzyme A dehydrogenase (38), thyroid hormone receptor {alpha} (TR{alpha}) (39), and the osteopontin (40) genes. Homologous recombination studies revealed that ERR2 has an important role in early embryogenesis where ERR2 null mice die at 10.5 days post coitum due to placental abnormalities (41).

Although many members of the steroid receptor superfamily are ligand-dependent transcription factors, the results reported here support the notion that some members may manifest constitutive activity in absence of the addition of a specific ligand. Transfection studies indicate that hERR1 and hERR2 have similar DNA binding properties and are effective activators of estrogen and thyroid hormone target genes. The activation of ERE-controlled gene expression by hERR2 is consistent with the previous observation that ERR2 protein is able to bind ERE in vitro as revealed by gel mobility shift assay (42). Whether either hERR1 or -2 manifests any estrogenic or thyroimimetic type properties in vivo is not yet known. Even though the LBDs of hERR1 and hERR2, like classic hormone receptors, harbor transcriptional activation functions, added ligands are apparently not needed to manifest this property. This appears to be a consequence of the ability of the p160 cofactors to be able to directly bind the LBDs in a ligand-independent fashion.

Based upon these studies, we can consider at least four possible, but not exclusive, models to explain the transcription properties of the hERRs. First, the hERRs may not be exceptions to the rule that all receptors are hormone dependent in activation. Rather, the activating ligands may be produced endogenously by the recipient cells leading to an apparent constitutive property. In this case, the ligand could either be secreted and reabsorbed by the cells in an autocrine fashion or could represent an internal molecule that is not released but is able to interact with the intracellular receptor directly. Such intracellular regulatory systems might have value in modulating metabolic pathways as well as providing direct feedback regulation for intracellular homeostatic systems. Furthermore, even though hERRs exhibit transcriptional activity in cell-based assays in the absence of any exogenously added ligand, this does not exclude the potential existence of an endogenous ligand that modulates this function. For example, the orphan receptor LXR{alpha} manifests some constitutive activity (high basal) on certain target sequences that can be substantially potentiated by the addition of ligands such as 24(S),25-epoxycholesterol and 24(S)-hydroxycholesterol (43). In addition, two other orphan receptors, SF-1 and hepatic nuclear factor 4 (HNF-4), which also display constitutive activity, have recently been proposed to be further activated by oxysterols and fatty acyl-CoA thioesters, respectively (44, 45). However, direct binding of the ligands to the receptor has yet to be confirmed. A second model would suggest that the receptors are ligand-dependent repressors such that in the absence of the appropriate inducing molecule, target genes are constitutively activated. Indeed, we have recently shown that the constitutive activity of the CAR-ß results from a ligand-independent recruitment of transcriptional coactivators, while androstane metabolites bind to and deactivate CAR-ß by promoting coactivator release from the LBD (29). It remains possible that a mechanism of potential ligand-mediated receptor deactivation also applies to hERRs. A third model is that hERRs may be activated by agents other than steroid hormones. Indeed, a number of steroid receptors including ER{alpha}, ERß, and SF-1 have been shown to be activated by nonsteroid agents such as growth factors, protein kinase A, and dopamine (for review, see Ref. 46), and the AF-1 domain of these NRs appears to be the target. It has been shown recently that the mitogen-activated protein kinase-mediated phosphorylation of the AF-1 domain of ERß and SF-1 promotes ligand-independent recruitment of NR coactivators (7, 47). Indeed, most if not all, of the members of the NR family are phosphoproteins (for review, see Ref. 46). However, two pieces of evidence seem to go against the possibility that the phosphorylation mechanism also applies to hERRs: 1) Unlike ERs, the constitutive activation property of hERRs can be destroyed by truncations, deletions, and insertional mutations in the LBD, indicating that the putative AF-1 domain may not contribute the transcription activity of the hERRs; 2) The transactivation activity of hERRs can be observed in serum-free conditions. The fourth possibility is that some receptors do not bind classic ligands and, rather, represent a subgroup of the gene family that have evolved as hormone-independent transcription factors. Interestingly, classical NRs undergo conformational changes after hormone binding, and the resulting conformation allows them to interact with transcriptional coactivators via the LXXLL motif (26, 28, 32, 48). Mutation of this motif blocks ERR activation. The coactivator binding with hERR1 and -2 would appears to reflect a similar conformation to that of an activated receptor. How this is achieved in the absence of ligand is unknown. Therefore, it will be of interest to investigate the structural features of hERRs that enable their constitutive interaction with coactivators. In either case, these orphan receptors provide a unique opportunity to further investigate the diverse mechanisms by which the NRs may contribute to endocrine function.

The fact that hERRs can activate synthetic ERE-controlled genes raises several interesting questions. Is the ERE a natural response element of hERRs? And if so, what is the role of ERRs, relative to ER, in gene regulation. Alternatively, do hERRs have other perfect or imperfect response elements? For the role of hERRs in ERE-controlled gene expression, one possible regulatory mechanism could involve the formation of ER-ERR heterodimers, which may have a different level of activity from ER-ER homodimers. Indeed, cotransfection of hERR1 increased estrogen-dependent activation mediated by the ERE in the lactoferrin promoter, suggesting that heterodimerization between ERRs and ER could play a role in the attenuation or potentiation of estrogen-dependent transcription (37). On the other hand, ERR1 has been shown to bind a monomeric response element (38, 39, 49, 50), and the technique of selected and amplified binding (SAAB) (51) predicts that ERR1 can bind a response element containing a single consensus half-site, 5'-TNAAGGTCA-3' (ERRE). Indeed, most of the previously characterized ERR1 response elements in the promoters of its responsive genes are this single core element (38, 39, 49, 50). We observed that all three ERR family members can activate gene transcription when tk-ERRE-Luc was used as a reporter (W. Xie and R. M. Evans, data not shown). Interestingly, in some promoters, such as those of the oxytocin, PRL, and lactoferrin genes, a putative ERRE overlaps a known ERE (for review, see Ref. 49). Therefore, it is conceivable that ERRs may play a role as transcriptional modulators whose contribution to promoter activity could be determined both by the context of the ERE or ERRE within a complex hormone response element and by potential interactions between ERRs and other nuclear hormone receptors.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
To generate pRShERR1 construct, hERR1 coding sequence (35) was constructed into a BamHI-digested and Klenow filled-in vector fragment derived from a double BamHI linker-insertion mutant pRShER{Delta}160–1825 (V. Giguere, unpublished data). The construct with the correct orientation was named pRShERR1–1. To generate pRShERR1, a SacII-ATtIII fragment of pRShERR1–1 was inserted into a BamHI and ATtIII-digested pRShGR214 (10) vector. To generate pRShERR2, the coding sequence of the hERR2 (35) was prepared as a KpnI-BamHI fragment and ligated into the same pair of enzymes digested vector of pRSER1825 (30). In the assays of coactivators, the coding sequences of hERR1 or hERR2 were released from pRShERR1 or pRShERR2 as an Asp718-BamHI fragment, or an EcoRI-BamHI fragment, respectively, and cloned into the same enzyme-digested pCMX-PL2 expression vector (20) to generate pCMX-hERR1 and pCMX-hERR2 constructs. To generate the NX mutants, NotI and XhoI sites were introduced into the cDNA sequence of hERR1 and hERR2 by site-directed mutagenesis as described previously (8).

For the construction of hERR1 mutants, the C-terminal truncated hERR1–2681 was generated by digesting pRShERR1 with SmaI and Eco47III enzymes, which recognize two unique sites in the ligand-binding region of hERR1. Ligation of the larger fragment resulted in a plasmid coding for hERR1 that stops at amino acid position 268. Six extra amino acids were added upon the sequence before it runs into a stop codon. The in-frame internal deletion mutant hERR1-{Delta}268–373 was created by inserting an oligo linker coding for ClaI recognition site (CATCGATG) into the SmaI and Eco47III enzymes’ double-digested pRShERR1 fragment. The linker was ligated with the larger fragment. The linker restores the reading frame; therefore, {Delta}268–373 encodes an hERR1 protein with the sequence between amino acid 268 and 374 in the LBD replaced by a sequence of four amino acids, Pro-Ile-Glu-Gly. HERR1-I373 was generated by inserting an oligo linker with ClaI recognition site (CCCATCGATGGG) into the Eco47III site in the LBD. The resulting hERR1 mutant protein, therefore, has four extra amino acid residues, Pro-His-Arg-Trp, inserted between amino acid 372 and 373.

MMTV-LUC, MTV-ERE-LUC, and MTV-TREpal-LUC (30), tk-ERE-Luc and tk-TREpal-Luc (52), MTV-CAT, {Delta}MTV-CAT, {Delta}MTV-ERE-CAT, and {Delta}MTV-TREpal-CAT (10), and expression vectors pSG5-GRIP1 (32), pCMX-ACTR, pCMX-SRC-1a (20), tk-(MH100)4(UAS)-Luc (29), and pRShTR{alpha} (8) were described previously.

Bacterial expression vector for GST-GRIP1 fusion protein, pGEX.2TK.GRIP1563–1121, was made by inserting a PCR-amplified GRIP1 fragment into BamHI/EcoRI sites of pGEX.2TK (Pharmacia). pGEX-ACTR-RID 621–821 was described before (20).

Cell Culture and Transient Transfection
CV-1 cells and HeLa cells were maintained in phenol red-free DMEM supplemented with 10% FBS (Gemini Bio-Products, Inc.). The serum condition after transfection is presented in the figure legends. Calcium phosphate precipitation-based ( Figs. 1–4GoGoGoGo) or liposome-based (Figs. 5Go and 6Go) transient transfections were performed as described previously (8, 17, 20). Total DNA used in each transfection was adjusted to the same amount by adding the appropriate amount of empty expression vectors. Luciferase assays (30, 34) or CAT assay (10) on cell extracts were performed 48 h after transfection. Data shown represent the mean and SD for three or four transfected cultures.

In vitro transcription and translation of proteins and GST pull-down assays were performed as described previously (20, 53). The cDNAs of hERR1 and 2 were subcloned into pCMX vector (20) to generate vectors for in vitro transcription and translation.

Mammalian two-hybrid assays were performed as described previously (20). pCMX-VP16-L-hERR1 and pCMX-VP16-L-hERR2 were constructed by cloning LBDs from hERRs into the pCMX-VP16 vector.


    ACKNOWLEDGMENTS
 
We thank Hongwu Chen and Hung-Ying Kao for SRC-1 expression vector; Henry Juguilon for help in tissue culture; and Elaine Stevens and Lita Ong for administrative assistance.


    FOOTNOTES
 
Address requests for reprints to: Ronald M. Evans, Howard Hughes Medical Institute, Gene Expression Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037.

This work was supported by grants from NIH. M.R.S is supported by NIH Grant DK-43093. W.X. is supported by the California Breast Cancer Research Program (5FB-0117). R.M.E. is an Investigator of the Howard Hughes Medical Institute at the Salk Institute for Biological Studies and March of Dimes Chair in Molecular and Developmental Biology.

1 Present address: Endocrine Research, Lilly Research Laboratories, Eli Lilly & Co., Indianapolis, Indiana 46285. Back

Received for publication July 9, 1999. Revision received August 12, 1999. Accepted for publication August 19, 1999.


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 MATERIALS AND METHODS
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