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Volume 270, Number 10, Issue of March 10, 1995 pp. 5427-5433
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a New Enhancer in the Promoter Region of Human TR3 Orphan Receptor Gene
A MEMBER OF STEROID RECEPTOR SUPERFAMILY (*)

(Received for publication, August 24, 1994; and in revised form, December 5, 1994)

Hiroji Uemura ,  Atsushi Mizokami ,  Chawnshang Chang (§)

From the Department of Human Oncology, Comprehensive Cancer Center, University of Wisconsin, Madison, Wisconsin 53792

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Human TR3 orphan receptor is a member of the steroid/thyroid hormone receptor superfamily and is the human homologue of the proteins encoded by the rat NGFI-B and mouse nur77 genes. These genes are induced rapidly by androgens/growth factors and may have functions related to cell proliferation, differentiation, and apoptosis. To investigate the TR3 orphan receptor gene transcriptional regulation, a 2.3-kilobase genomic DNA fragment containing the TR3 orphan receptor gene promoter region was isolated, sequenced, and characterized. Sequence homology search within this promoter region revealed some potential cis-acting elements such as cAMP response element, interleukin-6 response element, estrogen response element, and GC box. Deletion analysis and chloramphenicol acetyltransferase assay also showed a novel cis-acting element of TR3 orphan receptor gene (NCAE-TR3), 200-181 base pairs upstream of the transcriptional start site. Gel retardation assay further demonstrated that some nuclear factors can bind to this NCAE-TR3. Together, our data suggest that NCAE-TR3 could be a new enhancer element associated with the transcription of an early response gene for mitogenesis and apoptosis.

INTRODUCTION

TR3 orphan receptor is a member of the steroid/thyroid hormone receptor superfamily(1, 2, 3, 4, 5) . The cDNA of human TR3 orphan receptor was isolated during the screening of human testis and prostate cDNA libraries for androgen receptor (AR) (^1)cDNAs using an oligonucleotide probe homologous to the highly conserved part of the DNA binding domain of glucocorticoid receptor(6, 7, 8) . This receptor is also a human homologue of the protein encoded by mouse nur77(9, 10) , N10 gene(11) , and rat NGFI-B gene(12) , which are thought to be the early response genes immediately induced by androgens and several growth factors. It has been reported that NGFI-B mRNA can be rapidly induced by nerve growth factor in PC12 cells(12) . Our previous reports also showed that the expression of human TR3 orphan receptor mRNA in human prostate LNCaP cell can be rapidly and biphasically induced by the addition of androgens or growth factors(13) .

Using domain switch strategy, a chimera receptor (TR3/AR/TR3), in which the DNA binding domain of TR3 orphan receptor was replaced by that of AR, exhibited constitutive activity in the absence of exogenously added ligands in COS-1 monkey kidney and human prostate cancer PC-3 cells. The activation was dependent only on the amount of TR3/AR/TR3 vector transfected and appeared to be independent of the concentration of serum supplement(5) . In contrast, TR2 orphan receptor, another orphan receptor isolated from our lab(1, 8) , was not constitutively active under the same system. However, a chimera receptor (PR/PR/TR2), in which the N-terminal domain and DNA binding domain of TR2 orphan receptor were replaced by that of progesterone receptor, could be activated through a signal transduction pathway initiated at the cell membrane by the neurotransmitter, dopamine(14) . With the similar domain switch strategy, we were able to use another chimeric receptor (AR/TR3/AR), in which the DNA binding domain of AR was replaced by that of TR3 orphan receptor to identify a potential TR3 response element in the promoter region of mouse mammary tumor virus long terminal repeat (15) . Other studies also revealed that 21-hydroxylase gene, a target gene for the NGFI-B (TR3), may contain a potential NGFI-B (TR3) response element(16) . These hormone response elements may provide a much needed tool for the study of the potential functions of TR3 orphan receptor.

The TR3 orphan receptor gene, 8.5-9.0 kb in length, is located on human chromosome 12, band q13.1, and is split into six introns and seven exons. With some notable genomic structure that is different from most of the other known steroid receptor genes, we speculate that the human TR3 orphan receptor may be an evolutionary ancestor in the steroid receptor superfamily(13) .

Sequence analysis of the 5`-flanking region of N10 and NGFI-B genes suggested these genes may contain some potential cis-acting elements(11, 17) . Lau and colleagues (9) also reported that some potential cis-acting elements may play a role in growth factor stimulation. By cloning and sequencing of the 2.3-kb 5`-flanking region of the human TR3 orphan receptor gene, we now report the identification of several potential cis-acting elements. Deletion analysis and CAT assay further reveal, for the first time, that a novel cis-acting element of TR3 orphan receptor gene (NCAE-TR3) may be essential for the transcription of the TR3 orphan receptor gene in HeLa cells.

MATERIALS AND METHODS

Isolation of 5`-Promoter Region of Human TR3 Orphan Receptor

A human peripheral lymphocyte genomic EMBL3 library was screened using a P-labeled 400-bp EcoRI-TaqI DNA fragment of human TR3 orphan receptor cDNA as a probe. Five positive clones were isolated and mapped. A 2.8-kb BamHI-BamHI fragment was isolated from one of the five positive clones and subcloned into the BamHI site of pBluescript II SK(-) (Stratagene). This 2.8-kb fragment was further cut with NarI to remove the first intron. The remaining 2.3-kb BamHI-NarI fragment was then subcloned into the EcoRV site of pBluescript (p-2149 TR3) for further characterization.

Construction of Human TR3 Orphan Receptor Gene Promoter CAT Plasmids

A series of deletion mutants of human TR3 orphan receptor gene promoter CAT plasmids (p-1830TR3CAT, p-1500TR3CAT, p-1180TR3CAT, p-940TR3CAT, p-730TR3CAT) were created using Exonuclease III (New England Biolabs). p-427TR3CAT and p-121TR3CAT were constructed by deletion of 1700- and 2000-bp SmaI-SmaI fragments of p-2149TR3CAT. p-314TR3CAT was constructed by deletion of a 1810-bp XbaI-NarI fragment of p-2149TR3CAT. To produce p-2149(-314/-121)TR3CAT, a SacI-ApaI 1810-bp fragment of p-2149TR3CAT was inserted into p-121TR3CAT. Other deletion mutants between -314 and -121 of the promoter region (p-199TR3CAT, p-194TR3CAT, p-184TR3CAT and p-151TR3CAT) were constructed using Erase-a-Base System (Promega). To create p-314(200/-171)TR3CAT, a polymerase chain reaction-amplified fragment (from -314 to -200) was ligated with p-171TR3CAT at BssHII enzyme restriction site. To create p-314/-121CATp and p-121/-314CATp, a NotI-SmaI 190-bp fragment was cut from p-314TR3CAT and inserted into the BglII site in front of SV40 gene promoter of parent pCAT promoter vector (pCATp) (Promega). To produce p-200/-181CATp, an annealed oligomer (5`-TGCGTCAATGGAACCCCGCG-3`) was inserted into the BglII site of pCAT promoter.

Gel Retardation Assay

For the gel retardation assay, annealed oligonucleotides corresponding to the sequence from -200 to -181 (5`-TGCGTCAATGGAACCCCGCG-3`) and AP-1 oligomer (5`-CTAGTGATGAGTCAGCCGGATC-3`) (Stratagene) were P end-labeled with T4 polynucleotide kinase and used as a probe. Binding reactions were carried out in a total of 10 µl of volume containing 11.5 µg of HeLa nuclear extract or 1 footprinting unit of AP-1 (Promega), varying amounts of unlabeled competitor (annealed oligonucleotides of -200 to -181, and AP-1 oligomer), 1 µg of poly(dI-dC), and 10 times binding buffer (160 mM Hepes (pH 7.8), 30% glycerol, 300 mM KCl, 50 mM MgCl(2), 5 mM EDTA, 5 mM dithiothreitol). Mixtures were incubated at room temperature for 10 min, and 0.1 ng of radiolabeled probe (approximately 5 times 10^4 cpm) was added followed by an incubation for another 20 min. The samples were then electrophoresed on a 5% polyacrylamide gel at 200 V at 4 °C using 1 times TAE as a running buffer. The gel was then dried and exposed to x-ray film at room temperature for 16 h.

Other Methods

Cell culture and CAT assay were performed according to the procedure of Mizokami and Chang(18) .

RESULTS

Cloning and Sequence of the Promoter Region of Human TR3 Orphan Receptor Gene

Five positive genomic clones were isolated from a EMBL3 human peripheral lymphocyte genomic library with a P-labeled 400-bp EcoRI-TaqI DNA fragment of human TR3 orphan receptor cDNA. Restriction mapping and Southern blot analysis further confirmed that one of these five positive clones contained at least a 2.3-kb DNA fragment upstream from the first intron of the human TR3 orphan receptor gene. This 2.3-kb 5`-flanking region was then subcloned into pBluescript and sequenced by dideoxy method. The complete nucleotide sequence of this 2.3-kb 5`-flanking region of human TR3 orphan receptor gene is shown in Fig. 1. Sequence homology search revealed some cis-acting elements in this 2.3-kb human TR3 orphan receptor gene promoter region. A TATA box motif (TATAAAA) (19) was identified at -924 to -930, which is not very close to the transcription initiation site. By CAT assay using p-940TR3CAT and p-427TR3CAT, this TATA box motif was shown to have no functional promoter activity (Fig. 2). Nine GC boxes, known as a putative SP-1 binding site(20) , one AP-2 motif (5`-CC(C/G)C(A/G)GGCA-3`)(21) , and five AP-1 binding site-like sequences (5`-TGAGTCA-3`) (22, 23) are found in the promoter region (Table 1). Other potential cis-acting elements found in the promoter region of human TR3 orphan receptor gene included one cAMP response element (5`-TGACGTCA-3` with one mismatch) (24) at -683 to -676, one estrogen response element (5`-GGTCAN(3)TG(A/T)CC-3` with two mismatches) (25) at -1944 to -1932, two inverted sequences of nuclear factor-1 binding motif (5`-TGGN(7)CCA-3`) (26) at -1193 to -1181 and -790 to -778, as well as four interleukin-6 response elements (5`-AGTGANGNAA-3` and 5`-NTGGNAA-3`)(27) . When we compared the sequence homology among human (TR3 orphan receptor), rat (NGFI-B), and mouse (nur77 or N10) genes at the promoter region, we found that sequences within 220 bp of the promoter region from the transcriptional start site are well conserved. This included four AP-1 binding site-like sequences and a CArG-like sequence (5`-CC(A/T)(6)GG-3`), a core sequence of serum response element(28) . As serum response factor may bind to a CArG-like sequence and then activate these early response genes, our findings of a well conserved CArG-like sequence among human, rat, and mouse may suggest a universal activation of the human TR3 orphan receptor gene.

Figure 1: Nucleotide sequence of the human TR3 orphan receptor gene promoter region. The location of potential cis-acting elements and comparison with rat NGFI-B and mouse nur77 genes are shown. Nucleotides are numbered relative to the transcriptional start site of genes that were published by Chang et al.(13) . All sequences with potential cis-acting elements are underlined and labeled as SP-1, AP-1, AP-2, CRE, ERE, NF-1, CArG-like, DSE-like, and IL-6-RE. Triangle indicates the existence of first intron. Dot indicates the transcriptional start site.


Figure 2: The promoter activity of various deletion mutants of the human TR3 orphan receptor gene. Deletion constructs are numbered and named according to their length upstream of the transcriptional start site. CAT activity, after transiently transfected into HeLa cells, was measured and normalized with beta-galactosidase activity. Relative CAT activity of all deletion plasmids and parent vector was calculated with p-2145TR3CAT as 100%. Results were mean ± S.D. of four separate experiments.


Identification of Novel Cis-acting Element (NCAE-TR3) in the Promoter Region of the Human TR3 Orphan Receptor Gene

The isolated 2.3-kb promoter region of human TR3 orphan receptor gene was inserted in the front of a CAT gene (p-2149TR3CAT) for the study of the transcriptional regulation of human TR3 orphan receptor gene. When p-2149TR3CAT was transfected into the HeLa cells, a remarkable increase of CAT activity was observed compared with that of CAT vector without promoter (pBsCAT, Fig. 2). To further identify the functionally important cis-acting regions, we constructed several CAT expression plasmids containing a serial progression of 5` to 3`-deletion mutants (p-1830TR3CAT, p-1500TR3CAT, p-1180TR3CAT, p-940TR3CAT, p-730TR3CAT, p-427TR3CAT, p-314TR3CAT, p-121TR3CAT, and p-26TR3CAT). CAT activity was assayed in HeLa cells transfected with the above plasmids using beta-galactosidase activity as the basis to normalize the transfection efficiency. As shown in Fig. 2, a series of plasmids (p-2149TR3CAT, p-940TR3CAT, p-427TR3CAT, and p-314TR3CAT) induced the very strong CAT activities as compared with the pBsCAT. On the contrary, the CAT activity of p-121TR3CAT was dramatically decreased as compared with p-314TR3CAT (14.0 ± 3.4%) (Fig. 2). These results indicate that the region of -314 to -121 may contain some essential cis-acting elements for the transcriptional activity of the human TR3 orphan receptor gene in HeLa cells. To further investigate this important region (-314 to -121), we deleted this region (-314 to -121) from p-2149TR3CAT, named as p-2149(-314/-121)TR3CAT, and assayed the CAT activity following transfection of this plasmid. As shown in Fig. 2, the level of CAT activity of this internal deletion mutant upon transfection was as low as that of p-121TR3CAT and confirmed the importance of this novel cis-acting element for the human TR3 orphan receptor gene transcription.

To further identify cis-acting elements within -314 to -121, several deletion mutants were constructed using exonuclease III (p-199TR3CAT, p-194TR3CAT, p-184TR3CAT, p-151TR3CAT). As shown in Fig. 3, p-314TR3CAT and p-199TR3CAT had considerably higher levels of CAT activities than p-184TR3CAT, p-151TR3CAT, and p-121TR3CAT. Particularly, the CAT activities of p-184TR3CAT and p-151TR3CAT decreased to 23.4 ± 1.3% and 14.8 ± 2.4% of p-314TR3CAT, respectively. The internal deletion mutant, p-314(-200/-171)TR3CAT, in which the region from -200 to -171 was deleted from p-314TR3CAT, reduced CAT activity to 13.7 ± 3.8% of that of p-314TR3CAT and had about the same CAT activity as compared with p-184TR3CAT. These results indicated that the sequence between -200 and -184 is necessary for the transcription of human TR3 orphan receptor gene in the HeLa cells.

Figure 3: The promoter activities of various deletion mutants of the human TR3 orphan receptor gene between -314 and -121. Deletion constructs are numbered and named according to their length upstream of the transcriptional start site. CAT activities, after being transiently transfected into HeLa cells, were measured and normalized with beta-galactosidase activities. Relative CAT activities of all deletion plasmids and parent vector were calculated with p-314TR3 as 100%. Results were mean ± S.D. of four separate experiments.


Functional Characterization of NCAE-TR3

To further characterize the essential cis-acting elements, the -314/-121 fragment was inserted in front of SV40 promoter of pCAT promoter vector (pCATp) and named as p-314/-121CATp and p-121/-314CATp (Fig. 4), based on their opposite orientations. These vectors were transiently transfected into HeLa cells and assayed for CAT activity, as shown in Fig. 4. The p-314/-121CATp and p-121/-314CATp significantly elevated CAT activity (5.7 ± 1.9- and 4.1 ± 0.9-fold) as compared with that of pCATp, the parent vector. Furthermore, from the result of the above CAT assays as shown in Fig. 3, we generated the CAT reporter construct in which synthetic 20-bp DNA oligonucleotide from -200 to -181 was inserted in front of SV40 promoter of pCATp and named as p-200/-181CATp. As shown in Fig. 4, the addition of this 20-bp element caused an increase in CAT activity of approximately 2.5-fold compared with pCATp. These results clearly demonstrated that this 20-bp fragment in human TR3 orphan receptor gene promoter may play an essential role in transcriptional function. We have named this element the novel cis-acting element of TR3 orphan receptor gene (NCAE-TR3). As the enhancer activity of NCAE-TR3 (-200 to -181) on SV40 promoter is about 2.5-fold and the deletion DNA fragment (-314 to -121) can diminish for TR3 orphan receptor gene promoter strength about 7-fold, the sequence outside the NCAE-TR3 (-314 to -200 and -181 to -121) may also play some roles for the induction of TR3 orphan receptor. Our data (Fig. 3) also showed that sequence between -184 and -154 may be responsible for 2-3-fold CAT induction, and sequence -314 to -199 shows no effect on CAT induction.

Figure 4: Induction of CAT activities in the SV40 promoter by the NCAE-TR3. Structure orientation of p-314/-121CATp, p-121/-314CATp, p-200/ -181CATp, and pCAT-promoter and their CAT activities. The orientations of the NCAE are illustrated by an arrowhead. CAT activity, after transfection into HeLa cells, was measured and normalized by beta-galactosidase activity. Relative CAT activity was calculated with that of HeLa cells transfected with pCATp as 1.0. Results were mean ± S.D. of four separate experiments.


Gel Retardation Analysis of NCAE-TR3

Gel retardation assay was used to determine if there are any proteins that bind to the NCAE-TR3 in HeLa cells. As shown in Fig. 5(lane2), HeLa cell nuclear extracts could produce protein-DNA complexes that migrated to two different positions. The specific competitor (Fig. 5, lanes3 and 4) with 100- and 250-fold molar excess of unlabeled NCAE-TR3 can efficiently eliminate two complexes. As NCAE-TR3 includes one AP-1 binding site-like sequence (-200 to -194, 5`-TGCGTCA-3`), we used cold AP-1 oligomer (5`-CTAGTGATGAGTCAGCCGGATC-3`) to compete hot NCAE-TR3 oligomer. As shown in Fig. 5(lanes5 and 6), AP-1 oligomer failed to compete NCAE-TR3 for the two specific protein complexes. Our data also showed that while pure AP-1 protein can bind to AP-1 oligomer (Fig. 5, lane8), the pure AP-1 protein could not bind to NCAE-TR3 oligomer (lane7) and may suggest that some unknown transcriptional factors are involved in the formation of NCAE-TR3-protein complexes. Nevertheless, the above data still did not exclude the importance of these AP-1 binding site-like sequences. It is conceivable that the AP-1 binding site can be significantly influenced by the sequence context in the promoter so that AP-1 can be still involved but now has a much higher affinity due to adjacent DNA-protein or protein-protein interactions. CAT assay using mutated-NCAE-TR3 oligomer (-196 to -176 without the AP-1 binding site-like sequence) showed no induced CAT activity (data not shown), and no clear shift band could be identified when we used this oligomer (-196 to -176) as a probe in the gel retardation assay (data not shown). Together, our data suggested that the AP-1 binding site-like sequence (-200 to -194) within NCAE-TR3 (-200 to -181) may play an essential role for the enhancer activity of NCAE-TR3, and NCAE-TR3 enhancer may bind to some unidentified transcription factors in the HeLa cells.

Figure 5: Gel retardation assay of NCAE-TR3 with HeLa cell nuclear extracts. Gel retardation assay of NCAE-TR3 was performed by using 11.5 µg of HeLa cell nuclear extract and varying amounts of competitors. Nuclear extracts were incubated with P-end-labeled NCAE-TR3 (5 times 10^4 cpm) and electrophoresed on a 5% polyacrylamide gel. Lane2 shows probe with 11.5 µg of HeLa nuclear extract. Lanes3 and 4 are the same as lane2 with a 100- and 250-fold molar excess of unlabeled NCAE-TR3, respectively, used as a specific cold competitor. Lanes5 and 6 are the same as lane2 with a 100- and 250-fold molar excess of unlabeled double-stranded oligonucleotide (AP-1 oligomer), respectively, used as nonspecific cold competitors. Lane7 shows NCAE-TR3 probe, and in lane8, AP-1 oligomer probe with 1 footprinting unit of AP-1 protein is shown.


DISCUSSION

Sequence comparison among human, rat, and mouse TR3 orphan receptor gene promoter regions indicated there are some well conserved sequences within 220 bp upstream of the transcriptional start site. There are four AP-1 binding site-like sequences, five SP-1 binding site sequences, and one CArG-box sequence in this region. However, we could not locate perfect TATA or CCAAT boxes in this region. The lack of a perfect TATA box in the human TR3 orphan receptor gene promoter region is very similar to what was found in the promoter region of the progesterone (29) and androgen receptor genes(30) . The other similarity among these nuclear receptor gene promoter regions is the existence of highly G + C-rich region. As shown in Table 1, the TR3 orphan receptor gene promoter region contains four potential GC boxes within 130 bp of the transcriptional start site (-17, -40, -76, and -91). These GC boxes, like other GC boxes in many housekeeping genes, may interact with the SP-1-TFIID complex to start transcription(31) . Interestingly and surprisingly, based on our CAT assay, a CAT reporter plasmid, p-2149(-314/-121)TR3CAT, which contains these four GC boxes, induces very little CAT activity (Fig. 2). This result suggests that other promoter regions (e.g. -314 to -121) may play an important role in human TR3 orphan receptor gene transcription.

Another potentially important and conserved sequence within human TR3 orphan receptor gene promoter regions is the Dyad symmetry element (DSE)-like sequences (G(A/C)TG(T/C)CCATAT(T/A)TGG(A/C)CA(T/G)CT), which were also found in other immediate early response genes such as c-fos, krox-20, and krox-24(32, 33, 34) . It has been demonstrated that the DSE sequence in these early response genes could be the binding sites for serum response factor and might be needed for serum induction. However, from our data in Fig. 2, this DSE-like sequence at -349 to -333 may not play a significant role in human TR3 orphan receptor gene transcriptional activity, since we obtained similar CAT activity in HeLa cells transfected with either p-427TR3CAT or p-314TR3CAT. Our transient transfection of these CAT reporter genes into HeLa cells also caused constant CAT induction in normal serum; this DSE-like sequence, therefore, may not have an effect on CAT induction like other early response genes.

Other studies (35, 36) suggested that transient transcriptional activation of early response genes by growth factors may involve a region with imperfect dyad symmetry called serum response element (SRE). The SRE is a 22-bp element containing an inner core sequence, CC(A/T)(6)GG, known as the CArG box. Both CArG box and SRE are functional and interchangeable(36) , which are not only required in the transcriptional activation of early response genes but also are required for the transcriptional repression of the c-fos gene(37, 38, 39) .

These important sequences, however, are not the same sequences (-200 to -181) we identified as the NCAE-TR3 in this report. Our NCAE-TR3 may, therefore, represent an essential sequence for the transcriptional activity of TR3 orphan receptor, an early response gene.

Recently, Liu et al.(40) demonstrated that mouse nur77 is necessary for induced apoptosis in T-cell hybridomas and can also be induced during early mitogenesis. The potential sequences needed for mitogenesis and apoptosis could be located at -378 to -332 and -332 to -151 upstream of the transcriptional start site, which share about 85% homology between mouse and human sequence. Surprisingly, we found NCAE-TR3 located in this region, and it was completely conserved among human, rat, and mouse, suggesting that NCAE-TR3 may be also a candidate of cis-acting element related to the apoptosis. Using VP-16 and calcium ionophore as apoptosis-induced reagents, we also found the early induction of TR3 orphan receptor mRNA in the prostate cancer cells, (^2)which suggests that TR3 orphan receptor could be an early response gene involved in the androgen-induced mitogenesis and/or apoptosis in prostate.

Our gel retardation assays prove two DNA-protein complexes at NCAE-TR3, which are different from the AP-1 binding complex. It is unclear whether these two complexes are derived from dimerization of one protein or from two different proteins. As this NCAE-TR3 can also activate transcription in SV40 gene promoter, our identification of NCAE-TR3 may represent an important finding in new enhancers required for transcription of an immediate early response gene. Although we were unable to identify the same NCAE-TR3 in other known early response gene promoters, the existence and significance of enhancers in other early response genes are well documented. For example, Deschamps et al.(41) also identified an enhancer in the c-fos gene promoter region. Within this enhancer (-64 to -404 of the cap site) (41) , SRE and cis-inducible factor response element were further proved to function as regulatory elements that may be needed for the induction in response to the stimulus(42, 43, 44) .

Using NCAE-TR3 sequence and Southwestern techniques, we are in the process of identifying proteins that may bind specifically to this unique enhancer. The identification of new transcriptional factors that bind to this important enhancer may help us to understand more about the gene regulation of TR3 orphan receptor at the transcriptional level and may also help us to know more about the mechanism of how androgens regulate cell growth and death.

FOOTNOTES

*
This work was supported by National Institute of Health Grant CA 55639 and American Cancer 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U17590[GenBank].

§
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; NCAE-TR3, novel cis-acting element of TR3; SP-1, specific transcriptional factor-1; AP-1, activator protein-1; DSE, dyad symmetry element; CAT, chloramphenicol acetyltransferase; SRE, serum response element; bp, base pair(s); kb, kilobase(s).

(^2)
H. Vemura, A. Mizokami, and C. Chang, unpublished data.

ACKNOWLEDGEMENTS

We thank Alan Saltzman for valuable discussions.

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