©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Human TR2 Orphan Receptor Response Element in the Transcriptional Initiation Site of the Simian Virus 40 Major Late Promoter (*)

(Received for publication, October 19, 1994)

Han-Jung Lee Chawnshang Chang (§)

From the Department of Human Oncology and the Endocrinology-Reproductive Physiology Program, Comprehensive Cancer Center, University of Wisconsin, Madison, Wisconsin 53792

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A DNA response element (TR2RE-SV40) for the TR2 orphan receptor, a member of the steroid-thyroid hormone receptor superfamily, has been identified in the simian virus 40 (SV40) +55 region (nucleotide numbers 368-389, 5`-GTTAAGGTTCGTAGGTCATGGA-3`). Electrophoretic mobility shift assay, using in vitro translated TR2 orphan receptor with a molecular mass of 67 kilodaltons, showed a specific binding with high affinity (dissociation constant = 9 nM) for this DNA sequence. DNA-swap experiments using chloramphenicol acetyltransferase assay demonstrated that androgen can suppress the transcriptional activities of SV40 early promoter via the interaction between this TR2RE-SV40 and the chimeric receptor AR/TR2/AR with the DNA-binding domain of the TR2 orphan receptor flanked by the N-terminal and androgen-binding domains of the androgen receptor. In addition, this TR2RE-SV40 can function as a repressor to suppress the transcriptional activities of both SV40 early and late promoters. Together, these data suggest the TR2RE-SV40 may represent the first identified natural DNA response element for the TR2 orphan receptor that may function as a repressor for the SV40 gene expression.


INTRODUCTION

Several members of the steroid-thyroid hormone receptor superfamily, including the androgen receptor (AR)(^1), estrogen receptor, glucocorticoid receptor, progesterone receptor, mineralocorticoid receptor, thyroid receptor (T(3)R), 1,25-dihydroxyvitamin D(3) receptor (VDR), and retinoid acid receptor (RAR), play important roles in the control of vertebrate differentiation and development(1) . These nuclear receptors act as trans-regulators of transcription. Upon binding with their cognate ligands, these hormone-receptor complexes interact with cis-acting DNA elements, termed hormone response elements (HREs), to modulate the transcription of target genes. The functional structure and organization of these receptors comprise a variable N-terminal domain involved in the modulation of gene expression, a well conserved DNA-binding domain with two zinc fingers, and a partially conserved C-terminal ligand-binding domain(2) .

Human testicular receptor 2 (TR2) is one of the first orphan receptors identified that share structural homology with members of the steroid-thyroid hormone receptor superfamily(3, 4, 5, 6) . The TR2 orphan receptor cDNA was isolated from human prostate and testis cDNA libraries with a probe homologous to a highly conserved DNA-binding domain common to steroid hormone receptors. The TR2 orphan receptor cDNA encodes a protein of 603 amino acids with a calculated molecular mass of 67 kilodaltons(4, 5) . The expression of the TR2 orphan receptor has been detected in various cell lines and tissues, including the human prostatic carcinoma cell line LNCaP, testis, ventral prostate, and seminal vesical(7) .

The HREs for steroid hormone receptors are structurally related but functionally distinct(8) . Based on the finger model, the first zinc finger in the DNA-binding domain of steroid hormone receptors may determine target HRE specificity. The three amino acids at the C-terminal region of the first zinc finger are categorized as the P (proximal) box, which is important in base interaction. Consequently, HREs can be classified into two main categories of repeat consensus sequences based on the P box, the response element for glucocorticoid receptor and estrogen receptor (ERE)(9) . The glucocorticoid response element group, which includes glucocorticoid receptor, AR, progesterone receptor, and mineralocorticoid receptor, recognizes AGAACA core consensus half-site. On the other hand, the ERE group, which includes estrogen receptor, T(3)R, VDR, RAR, RXR, and many orphan receptors, recognizes AGGTCA half-site. In addition, five amino acids localized in the second zinc finger, referred to as D (distal) box, which is important in dimerization contact forming, are involved in half-HRE spacing functional discrimination. These two structural determinants (P and D boxes) of target gene specificity may suggest a pathway for the coevolution of receptor DNA-binding domains and regulatory gene networks(8, 9) . On the basis of P and D box sequences, the TR2 orphan receptor is assigned to the ERE subfamily and predicted to bind to an AGGTCA repeat.

Recently, several transcriptional factors from HeLa cell nuclear extracts have been suggested to bind to the transcriptional initiation site of the simian virus 40 major late promoter (SV40-MLP). One of these factors turned out to be the hERR1 orphan receptor(6, 10) . One of the core sequences within the SV40-MLP was identified as AGGTCA, which could be a potential HRE for the TR2 orphan receptor. We, therefore, developed several strategies to test this hypothesis. Herein this paper is the summary of our results showing the TR2RE within SV40-MLP as the first identified natural HRE for the TR2 orphan receptor.


EXPERIMENTAL PROCEDURES

Materials

TNT SP6-coupled reticulocyte lysate system was purchased from Promega. L-[S]methionine, [^14C]chloramphenicol, and ^14C-methylated protein standards were obtained from Amersham Corp. [-P]ATP was from DuPont NEN. Acetyl coenzyme A and poly(dI-dC)bulletpoly(dI-dC) were purchased from Pharmacia Biotech Inc. Synthetic oligonucleotides were either a gift from Dr. J. E. Mertz or made by Bio-Synthesis Inc. (Lewisville, TX). Restriction enzymes and other modifying enzymes were mainly obtained from New England BioLabs and Promega.

Plasmid Construction

All plasmids were constructed by standard recombinant DNA techniques(11) . Plasmid pSPUTK-TR2 contains the full-length coding sequence of the TR2 orphan receptor cDNA with Xenopus beta-globin gene 5`-untranslated region under the control of the SP6 promoter in the pSPUTK vector (Stratagene). Plasmid pSG5-TR2 constitutes the TR2 orphan receptor cDNA under the control of the SV40 early promoter in the pSG5 vector (Stratagene) for transient transfection experiments. Plasmid pBL-SVL-CAT consists of the SV40 late promoter without the TR2RE region (SV40 nucleotides 372-385) derived from pSVL-GR (kindly provided by Dr. A. Mizokami) in the pBLCAT3 vector(12) . On the other hand, plasmid pBL-SVLRE-CAT contains the SV40 late promoter with the TR2RE region and intron of major capsid protein VP1 derived from pSVL (Pharmacia) in the pBLCAT3. Plasmids pSV55wt1 and pSV55mut1 contain one copy of the wild-type TR2RE (SV40 nucleotides 368-389, 5`-GTTAAGGTTCGTAGGTCATGGA-3`) and the mutant TR2RE (SV40 nucleotides 367-389, 5`-CGTTAAGCTTCGTAGCTCATGGA-3`) in the blunted BglII site of the pCAT-promoter plasmid (Promega), respectively. Plasmid pCMV-AR contains the full-length AR cDNA under the control of the human cytomegalovirus promoter(13) . Chimeric AR/TR2/AR plasmid consists of the DNA-binding domain of the TR2 orphan receptor flanked by the N-terminal and androgen-binding domains of AR at both ends(14) . Plasmid pMSG-CAT contains the full-length mouse mammary tumor virus long terminal repeat sequence linked with the CAT gene (Pharmacia).

Coupledin VitroTranscription and Translation

Circular pSPUTK-TR2 plasmid containing the full-length TR2 orphan receptor cDNA was in vitro transcribed and translated simultaneously in rabbit reticulocyte lysate according to the manufacturer's instructions (Promega). Depending on the purpose of the experiment, the reactions were performed either with or without [S]methionine in the transcription-translation mixture. After synthesis, ZnCl(2) was added to a final concentration of 0.5 mM. The in vitro translated products were then analyzed directly by either electrophoresis in SDS-12% polyacrylamide gel or electrophoretic mobility shift assay (EMSA). Plasmid pSG5-TR3 was used to express the TR3 orphan receptor under control of T7 promoter as a negative control in EMSA.

DNA-Protein Binding Assay

DNA-protein binding assay was carried out as previously described(15, 16) . P-Labeled oligonucleotides (0.01-0.2 ng, 2 times 10^4 cpm) were incubated with varying ratios (2-200 ng) of in vitro translated TR2 orphan receptor protein and followed by EMSA as described above. The optimum concentration of protein was determined and fixed (60 ng). For Scatchard analysis, constant amounts of in vitro translated TR2 orphan receptor were incubated with different concentrations of the TR2RE probe (0.002-1.5 ng). DNA-protein complexes were resolved by EMSA. The free probe and retarded band were quantified by PhosphorImager (Molecular Dynamics). The dissociation constant (K(d)) value was determined from the minus reciprocal of the slope of the line generated from the empirical data.

EMSA

EMSA was performed as previously described (17) with some modification. Double-stranded TR2RE primers corresponding to SV40 nucleotides 368-389 (5`-GTTAAGGTTCGTAGGTCATGGA-3`) were end-labeled as a probe. 4 µl of in vitro translated TR2 orphan receptor protein were incubated with the probe (0.02-0.1 ng, 10^4 cpm) in the EMSA buffer (10 mM HEPES, pH 7.9, 100 mM KCl, 1 mM dithiothreitol, 0.05 mM EDTA, 2.5 mM MgCl(2), and 6% glycerol) in the presence of 2 µg of poly(dI-dC)bulletpoly(dI-dC) at room temperature for 30 min. The resulting complexes were then separated on a native 6% polyacrylamide (37:1) gel at 4 °C in 0.5 times TBE (1 times TBE = 50 mM Tris base, 50 mM boric acid, and 1 mM EDTA, pH 8.3) at 10 V/cm. After electrophoresis, gel retardation was either visualized by autoradiography or quantified by PhosphorImager (Molecular Dynamics).

Cell Culture, Transfection, and CAT Assay

HeLa cells were plated at an initial density of 3 times 10^5/60-mm dish in Dulbecco's modified Eagle's medium nutrient mixture (DMEM/F-12) medium supplemented with 5% fetal bovine serum (FBS) or 5% charcoal-treated FBS (CTS) as previously described(13) . Transient transfection of the HeLa cells with plasmid DNA was carried out using a calcium phosphate-DNA precipitation method(18) . To normalize the transfection efficiency, the Rous sarcoma virus-based beta-galactosidase expression plasmid pRSV-beta-galactosidase (kindly provided by Dr. A. K. Verma) was cotransfected. Cell extracts were prepared and assayed for both beta-galactosidase and CAT activities at 48 h after transfection(11) . Each value of CAT conversion represents the mean ± S.D. from at least three independent experiments.


RESULTS

In Vitro Expression of TR2 Orphan Receptor

There are at least three binding sites of nuclear proteins within the transcriptional initiation site of the SV40-MLP(10) . One of these initiator-binding sites consists of core consensus AGGTCA half-site that may represent the potential HRE for the TR2 orphan receptor. In addition, our preliminary data also suggested that the TR2 orphan receptor can be detected in the nuclear extracts of HeLa cells. These data suggested that the TR2 orphan receptor could be one of the candidates that may bind to this region and play some role in SV40 gene expression. To test this hypothesis, we prepared in vitro translated TR2 orphan receptor protein for EMSA. We constructed the full-length TR2 orphan receptor cDNA into pSPUTK, which contains the SP6 promoter with a 5`-untranslated region of the Xenopus beta-globin gene and the consensus Kozak translational initiation site (UTK) as shown in Fig. 1A. The mock-translated control expressed in a coupled in vitro transcription-translation system produced no detectable product (Fig. 1B, lane2). In contrast, in vitro translation of pSPUTK-TR2 yielded a protein of the expected molecular mass of 67 kilodaltons for the TR2 orphan receptor (lane3). The minor product probably arose from internal initiation of translation or limited degradation.


Figure 1: Analysis of in vitro expression of the TR2 orphan receptor. A, schematic structure of a high level in vitro expression plasmid pSPUTK-TR2. The full-length coding sequence of the TR2 orphan receptor cDNA was constructed into pSPUTK vector under the control of the SP6 promoter with a 5`-untranslated region of the Xenopus beta-globin gene and the consensus Kozak translational initiation site (UTK). B, analysis of in vitro translated TR2 orphan receptor by SDS-12% polyacrylamide gel electrophoresis. Lane1 displays ^14C-methylated protein standards. The mock-translated product and the TR2 orphan receptor expressed in a coupled in vitro transcription-translation system are shown in lanes2 and 3, respectively. The expected molecular mass of 67 kilodaltons for the TR2 orphan receptor is indicated on the right.



TR2 Orphan Receptor Can Bind at +55 Region of the SV40-MLP

To investigate the possible interaction of authentic TR2 orphan receptor with the +55 region of the SV40-MLP, we performed an EMSA. The probe used in this study was a double-stranded oligonucleotide corresponding to the SV40 nucleotide numbers 368-389 (5`-GTTAAGGTTCGTAGGTCATGGA-3`)(19) . A specific DNA-protein complex was visualized in the EMSA with the probe and the TR2 orphan receptor protein prepared as described above (Fig. 2, lane2). This DNA-protein complex could be eliminated upon the addition of 100-fold molar excesses of unlabeled oligonucleotides (lane3). In contrast, the DNA-protein complex remained intact in the presence of 100-fold molar excesses of mutated oligonucleotides (mutant TR2RE) with a point mutation on the third nucleotide in both repeat motifs (lane4). Furthermore, there was no specific interaction between these oligonucleotides (TR2RE) and the in vitro expressed TR3 orphan receptor (lane5), another orphan receptor identified in our laboratory, or a mock-translated product (data not shown). These results indicated that the TR2 orphan receptor indeed had the ability to specifically bind to the TR2RE, but not to mutant TR2RE, in the SV40-MLP (Fig. 3).


Figure 2: Binding of the TR2 orphan receptor to +55 region of the SV40-MLP. EMSA was performed with in vitro expressed TR2 orphan receptor and the P-labeled probe corresponding to SV40 nucleotides 368-389 (see ``Experimental Procedures''). Lane1 shows the probe alone. Binding reaction mixtures incubated with in vitro synthesized TR2 orphan receptor and the probe (lane2), in the presence of 100-fold molar excesses of unlabeled wild-type (wt) oligonucleotides (lane3) or mutant (mut) oligonucleotides (lane4), are shown. Lane5 displays binding reaction mixtures incubated with in vitro expressed TR3 orphan receptor and the probe. The retarded complex is indicated by the arrowhead, while nonspecific complexes appear between the retarded complex and the free probe at the bottom.




Figure 3: Schematic structure of the TR2RE among the transcriptional initiation site of the SV40-MLP. The arrow indicates the position of transcriptional initiation site of the SV40-MLP. The sequence of either the wild-type or mutant TR2RE, an imperfect direct repeat of the AGGTCA half-site, is boxed. The TR2RE is located at the +55 region of the major late transcript. Numbers under the sequence show SV40 nucleotide residues(19) .



TR2 Orphan Receptor Can Bind to the TR2RE with High Affinity

To examine the binding affinity between the TR2 orphan receptor and the TR2RE in more detail, we employed Scatchard binding analysis for the DNA-protein complexes in the EMSA (Fig. 4). Scatchard plot analysis revealed a single binding component for this DNA-protein complex with a dissociation constant (K(d)) of 9 nM and B(max) of 0.3 nM to the TR2RE that fit the range of K(d) for steroid receptors and their HREs (16) .


Figure 4: Binding affinity of the TR2 orphan receptor to the TR2RE. Constant amounts of in vitro expressed TR2 orphan receptor were used in a series of EMSAs with varying concentrations of P-labeled TR2RE. The specific DNA-protein complex and the free probe were quantified by PhosphorImager (Molecular Dynamics). We plotted the ratio between TR2-bound (nM) and free DNA with respect to TR2-bound DNA. The dissociation constant (K) value was determined from the minus reciprocal of the slope of the line generated from the experimental data. A Scatchard plot of the results is shown.



Ligand Effect on the Interaction between the TR2RE and the TR2 Orphan Receptor

All in vitro binding data suggested that TR2RE could be the potential HRE for the TR2 orphan receptor. The next question asked was whether this interaction between the TR2RE and the TR2 orphan receptor was ligand dependent. Unlike classic members of the steroid-thyroid hormone receptors, which have their own ligands to test the ligand dependence, the TR2 orphan receptor is a receptor without a known ligand. To overcome this problem, we developed a ``DNA swap'' strategy to test this hypothesis. A chimeric receptor, AR/TR2/AR, with the DNA-binding domain of the TR2 orphan receptor flanked by the N-terminal and androgen-binding domains of the AR (Fig. 5A) was created here to study the interaction between the TR2RE and the TR2 orphan receptor. This chimeric plasmid was cotransfected into HeLa cells with reporter plasmids, which consist of the CAT gene regulated by the SV40 early promoter containing either the TR2RE or mutant TR2RE, respectively. The induction of the CAT gene by androgen was then tested. As shown in Fig. 5B, androgen clearly suppressed the transcriptional activity of the SV40 early promoter via the interaction between the TR2 orphan receptor and the TR2RE. However, SV40 early promoter construct with a mutant TR2RE was not affected by androgen in similar transient transfection CAT assay in HeLa cells. These data demonstrated that ligand can affect the interaction between the TR2RE and the TR2 orphan receptor and further confirmed the TR2RE is the HRE for the TR2 orphan receptor.


Figure 5: Confirmation of the repressor function of the TR2RE by chimeric TR2 orphan receptor. A, scheme of the expression plasmid AR/TR2/AR chimera. Chimeric AR/TR2/AR plasmid contains the DNA-binding domain of the TR2 orphan receptor in the middle flanked by the N-terminal and androgen-binding domains of AR at both ends in the pSG5 vector(14) . Thus, the DNA-binding domain of the AR is replaced with that of the TR2 orphan receptor (shadedbox). H, HpaI; R, EcoRI; S, SacII; and X, XhoI. B, induction of CAT activity in HeLa cells cotransfected with chimeric AR/TR2/AR plasmid and different reporter plasmids in the presence or absence of 10 nM synthetic androgen (R1881). Both pSV55wt1 and pSV55mut1 reporter plasmids are described in Fig. 7A. All CAT assays were normalized for the level of beta-galactosidase activity. Each value represents the mean ± S.D. of three independent experiments. The relative folds of CAT conversion from the cotransfection with chimera plasmid (lanes1-4) or positive control pCMV-AR (lanes5 and 6) and CAT reporter plasmids pSV55wt1 (lanes1 and 2), pSV55 mut1 (lanes3 and 4), or pMSG-CAT (lanes5 and 6) in the presence of 10 nM R1881 (lanes2, 4, and 6) are shown. All experiments were done in CTS during cell culture.




Figure 7: TR2RE may function as a repressor in vitro. A, schematic structure of the reporter plasmids containing either the wild-type or mutant TR2RE in parent pCAT-promoter vector. One copy of the wild-type and mutant TR2RE with the same direction as the promoter and CAT gene were cloned into the pCAT-promoter vector (a) at the blunted BglII site, termed pSV55wt1 (b) and pSV55mut1 (c), respectively. B, induction of CAT activity in HeLa cells cotransfected with the expression plasmid pSG5-TR2 and different reporter plasmids in the presence of normal FBS or CTS. All CAT assays were normalized for the level of beta-galactosidase activity. Each value represents the mean ± S.D. of three independent experiments. The relative folds of CAT conversion from the cotransfection with pSG5-TR2 and parent pCAT-promoter vector (a, lanes1 and 2), pSV55wt1 (b, lanes3 and 4), or pSV55mut1 (c, lanes5 and 6) in the presence of FBS (lanes2, 4, and 6) or CTS (lanes1, 3, and 5) are indicated.



TR2 Orphan Receptor Suppresses the Transcriptional Activity of the SV40-MLP via the TR2RE

Thus far, all of our data have supported the idea that SV40-MLP contains the TR2RE, a potential HRE for the TR2 orphan receptor. We then determined if the TR2 orphan receptor directly can play any role in SV40 gene expression via interaction with the TR2RE. CAT assay with the cotransfection of mammalian expression vector containing the full-length TR2 orphan receptor cDNA (pSG5-TR2) and either pBL-SVL-CAT (without the TR2RE) or pBL-SVLRE-CAT (with the TR2RE) reporter into HeLa cells indicated that the TR2 orphan receptor cannot affect the transcriptional activity of the SV40-MLP without the TR2RE (Fig. 6B, lane1). In contrast, the TR2 orphan receptor can suppress the transcriptional activity of the SV40-MLP containing the TR2RE region (lane2). These findings suggested that this TR2RE may function as a repressor for the transcriptional activity of the SV40-MLP.


Figure 6: Suppression of CAT activity of the SV40-MLP via the TR2RE. A, scheme of two CAT reporter plasmids. While pBL-SVL-CAT plasmid (a) consists of the SV40 late promoter without the TR2RE, pBL-SVLRE-CAT plasmid (b) contains the SV40 late promoter with the TR2RE and intron of major capsid protein VP1 followed by the CAT gene. B, induction of CAT activity in HeLa cells cotransfected with the expression plasmid pSG5-TR2 and two different reporter plasmids. All CAT assays were normalized for the level of beta-galactosidase activity. Each value represents the mean ± S.D. of three independent experiments. The relative folds of CAT conversion from the cotransfection with pSG5-TR2 and either pBL-SVL-CAT (lane1) or pBL-SVLRE-CAT (lane2) are shown.



Suppression of SV40-MLP Gene Expression Is Activator Dependent

As our data demonstrated that the TR2 orphan receptor may function as a repressor for the SV40 gene expression, we then attempted to determine if the TR2 orphan receptor always binds to the TR2RE and, therefore, constitutively blocks the SV40 gene expression or if the TR2 orphan receptor needs activator(s) for the activation of its supression function. To test this hypothesis, we applied regular FBS versus CTS in the transient transfection CAT assay. Our assumption was that the TR2 orphan receptor may have some activator(s) in the serum. As shown in Fig. 7, the repressor function of the TR2 orphan receptor was dependent on the presence of a potential activator(s) in the serum (lanes3 and 4). On the other hand, charcoal-treated serum can abolish such suppression by the removal of the potential TR2 activator(s) in the serum. These results further suggested that such activator-dependent suppression of the SV40 gene expression may rely on the interaction between the TR2RE and the TR2 orphan receptor. As expected, the mutant TR2RE had no influence on the suppression of the SV40 gene expression in the presence of either FBS or CTS (lanes5 and 6). Taken together, these data strongly suggested that the TR2RE is a HRE for the TR2 orphan receptor and functions as a repressor in the SV40 gene expression in an activator-dependent manner.


DISCUSSION

It has been previously shown that some orphan receptors may affect the expression of viruses. For example, chicken ovalbumin upstream promoter transcription factor can bind to a negative regulatory region in the human immunodeficiency virus type 1 long terminal repeat(20) . Hepatitis B virus enhancer I contains response elements of enhancer binding factor to polyoma C, hepatocyte nuclear factor 4, and retinoid X receptor (RXR)(21) . The TR3 orphan receptor and nuclear factor I can induce transcription of the mouse mammary tumor virus long terminal repeat(14, 22) . Recently, hERR1 and other orphan receptors have also been suggested to bind to the transcriptional initiation site of the SV40-MLP that may be involved in the early-to-late shift in the SV40 gene expression(10) . Our finding that the TR2 orphan receptor can bind to a negative response element in the transcriptional initiation site of the SV40-MLP will further expand the knowledge of the influence of orphan receptors on the gene expression of viruses.

The AGGTCA motif has been proposed to be the primary target element for the members of the ERE subfamily in the steroid-thyroid hormone receptor superfamily(8, 9) . Many family members, including the TR2 orphan receptor identified in the present study, can bind to this core sequence. However, it is not known yet how distinct sets of target genes are regulated by these nuclear receptors. In addition to the primary sequence, recent evidence has suggested that the orientation and spacing of repeat core elements determine the specificity of hormonal regulation of these receptors ((23) ; also, see (24) for review). More recently, a novel cooperative dimer interaction within the DNA-binding domains of family members has been proposed(25, 26) . A region in the first zinc finger of the DNA-binding domain of the T(3)R or RAR can interact with the second zinc finger in the DNA-binding domain of the RXR to promote selective DNA binding to direct repeats spaced by 4 and 5 nucleotides, respectively. The resulting polarity established by this protein-protein interaction places RXR in the 5`-position (5`-RXR-T(3)R-3` or 5`-RXR-RAR-3`) of the direct repeats(25) . In addition, the formation of either 5`-T(3)R-VDR-3` or 5`VDR-T(3)R-3` heterodimeric complex can be controlled by the ligand for the downstream receptor. Thus, polarity is an important regulatory property of heterodimeric nuclear receptor complexes(26) .

The TR3 orphan receptor, another orphan receptor identified initially in our laboratory, was used here as a negative control in the EMSA (Fig. 2). The TR3 orphan receptor, a human homologue of rat NGFI-B and mouse nur77, is also a member of the steroid-thyroid hormone receptor superfamily(27, 28, 29) . It has been demonstrated that rat NGFI-B can bind to a response element (NBRE), 5`-AAAGGTCA-3`, which contains only one AGGTCA half-site(30) . Thus, NGFI-B has been grouped into a special monomer binding subfamily. Herein, we have shown that the TR3 orphan receptor cannot bind to the TR2RE, an imperfect direct repeat sequence. This finding is in good agreement with the data from NGFI-B(30) .

Based on the binding affinity between some members of the ERE subfamily and the TR2RE, we found that the TR2 orphan receptor prefers TR2RE to other natural HREs, such as T(3)RE, RAREbeta, and NBRE. (^2)The binding affinity between the TR2 orphan receptor and the TR2RE is high and specific. Interestingly, sequence analysis of the TR2RE in the GenBank data base indicated that human hepatitis B virus and human papillomavirus type 33 may also contain binding sites for the TR2 orphan receptor. Whether the TR2 orphan receptor has any function in these viral gene expressions could be an interesting question to ask.

Lydon et al. (31) showed that the TR2 orphan receptor is not constitutively active. The chimera receptor (PR/PR/TR2), which replaces the ligand-binding domain of progesterone receptor with that of the TR2 orphan receptor, can be activated by dopamine, a neurotransmitter. Our previous report (28) showed that chimeric receptor TR3/AR/TR3 can be constitutively activated in both COS1 and prostate PC-3 cells but not TR2/AR/TR2. These two independent data all suggested that the TR2 orphan receptor may need its cognate ligand for the activation.

In summary, our data indicated that this TR2RE is indeed the first identified natural HRE for the TR2 orphan receptor. Moreover, our results showing that TR2RE can function as a repressor for the SV40 gene expression may suggest that several potential HREs for the TR2 orphan receptor could exist in other viruses. This prediction may open a new chapter for the study of interaction between the viral expression and the TR2 orphan receptor.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA 55639 and American Cancer Society Grant BE 78a. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Human Oncology, Comprehensive Cancer Center, University of Wisconsin, 600 Highland Ave., K4/632, Madison, WI 53792. Tel: 608-263-0899; Fax: 608-263-8613.

(^1)
The abbreviations used are: AR, androgen receptor; T(3)R, thyroid receptor; VDR, 1,25-dihydroxyvitamin D(3) receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; hERR1, human estrogen-related receptor 1; HRE, hormone response element; ERE, estrogen response element; TR2RE, TR2 response element; SV40, simian virus 40; MLP, major late promoter; EMSA, electrophoretic mobility shift assay; CAT, chloramphenicol acetyltransferase; CTS, charcoal-treated FBS; FBS, fetal bovine serum.

(^2)
T. Lin and C. Chang, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. Janet E. Mertz for valuable discussions and mutant TR2RE primers, Dr. Ajit K. Verma for pRSV-beta-galactosidase plasmid, Dr. Atsushi Mizokami for pSVL-GR plasmid, and Yi-Fen Lee for technical assistance. We also thank Dr. Alan Saltzman and Donald Itkin for helpful comments and editing on the manuscript, respectively.


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