Identification of a Retinoid/Chicken Ovalbumin Upstream Promoter Transcription Factor Response Element in the Human Retinoid X Receptor gamma 2 Gene Promoter*

(Received for publication, May 30, 1996, and in revised form, October 4, 1996)

Philip M. Barger Dagger and Daniel P. Kelly §

From the Department of Medicine and the Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

To investigate the mechanisms involved in the transcriptional control of retinoid X receptor (RXR) gene expression, the 5'-flanking region of the human RXRgamma 2 isoform was characterized. An imperfect hexamer repeat (gamma  retinoid X response element; gamma RXRE) with a single nucleotide spacer (GGTTGAaAGGTCA) was identified immediately upstream of the RXRgamma 2 gene transcription start site. Cotransfection studies in CV-1 cells with expression vectors for the retinoid receptors RXRalpha and retinoic acid receptor beta  (RARbeta ) demonstrated that the gamma RXRE confers retinoid-mediated transcriptional activation with preferential activation by RXR in the presence of its cognate ligand, 9-cis-retinoic acid (RA). Electrophoretic mobility shift assays demonstrated that RXR homodimer binding to gamma RXRE is markedly enhanced by 9-cis-RA, whereas RAR·RXR heterodimer binding is ligand-independent. DNA binding studies and cell cotransfection experiments also demonstrated that the nuclear receptor, chicken ovalbumin upstream promoter transcription factor (COUP-TF), repressed transcription via the gamma RXRE. Cotransfection experiments revealed that COUP-TF and RXRalpha compete at the gamma RXRE to modulate transcription bidirectionally over a wide range. These results demonstrate that the human RXRgamma 2 gene promoter contains a novel imperfect repeat element capable of mediating RXR-dependent transcriptional autoactivation and COUP-TF-dependent repression.


INTRODUCTION

The retinoid derivatives of vitamin A regulate a wide variety of biological processes, including development, differentiation, and cellular metabolism. The retinoids exert influence at the level of gene transcription by serving as ligands for the retinoid receptor families of transcription factors. The retinoid X receptors (RXRalpha , -beta , and -gamma )1 play a distinctly unique role within the nuclear receptor superfamily in that they may trans-activate not only as 9-cis-retinoic acid (9-cis-RA) activated homodimers but also as obligate heterodimeric partners for retinoic acid receptor (RAR), thyroid hormone receptor (TR), vitamin D receptor, peroxisome proliferator-activated receptor, and several "orphan" receptors (Refs. 1-6; reviewed in Ref. 7). RXR has thus been described as a "master regulator" of a subset of nuclear receptor signaling pathways.

RXRalpha and RXRbeta exhibit ubiquitous expression patterns during murine development and in adult tissues (8, 9). In contrast, RXRgamma expression is restricted both in fetal and adult tissues (8-10). The mouse RXRgamma gene has two known mRNA isoforms (RXRgamma 1 and RXRgamma 2), produced via alternative exon splicing and differential promoter utilization (10). The RXRgamma isoforms exhibit a distinct tissue-restricted expression pattern; RXRgamma 1 is enriched in neural tissue, whereas RXRgamma 2 is cardiac enriched (9, 10). Both transcripts are relatively abundant in skeletal muscle (9, 10). During embryologic development, RXRgamma transcripts are expressed in distinct temporal patterns (8, 9, 11). The RXRgamma gene is therefore unique among the RXR gene family members in that its expression is spatially and temporally restricted, suggesting the possibility that the function of this nuclear receptor is distinct from the other RXRs. Little is known about the mechanisms involved in the control of RXR gene expression or if cross-signaling occurs between members of the RXR gene family.

As an initial step in the investigation of the transcriptional regulatory mechanisms involved in the restricted pattern of expression of RXRgamma isoforms, we have cloned the 5'-flanking region of the human RXRgamma 2 gene. In this report, we describe a novel autoregulatory retinoid X response element (gamma RXRE) located within this promoter and present evidence that this element is capable of conferring transcriptional activation via retinoid pathways and transcriptional repression via the orphan receptor COUP-TF.


MATERIALS AND METHODS

Cloning of the Human RXRgamma 2 Gene 5'-Flanking Region

Human RXRgamma cDNA fragments were produced via polymerase chain reaction from a human heart cDNA library template (Clontech) using oligonucleotide primers that were designed based on cross-species homology between mouse (8, 10), chicken (12), and Xenopus (13) RXRgamma cDNA sequences. Several overlapping human RXRgamma partial cDNA clones were obtained, encompassing sequences representing most of the coding region and having an overall nucleotide identity of greater than 90% with the mouse RXRgamma cDNA.2 One partial human cDNA clone (hRX45), representing a fragment colinear with bp 451-669 relative to the published mouse RXRgamma 1 sequence (10), was generated by primers rx4 (5'-ATCAggatccCTTCTGCCATGGGTCCACCCTCA-3') and rx5 (5'-TCTGggatccTCCCCACAGATGGCACAGATGTG-3'). hRX45 was used as a probe to screen a human genomic EMBL3 phage library (Clontech). Three genomic clones were isolated, two of which were characterized (G1SH10 and G2SH1). Restriction endonuclease and Southern blot analysis confirmed that these genomic clones were overlapping and contained sequences recognized by the hRX45 probe. Additional Southern blot analysis using a polymerase chain reaction-generated mouse RXRgamma exon 1b probe (corresponding to the 5'-untranslated region of the RXRgamma 2 cDNA sequence (10)) identified overlapping genomic DNA restriction fragments containing the putative human homolog of exon 1b. Primer extension analysis using total human heart RNA and 32P-end-labeled rx20 (5'-AATCTGCCCATGCGATCCAGAGTC-3') and rx15 (5'-GCCTTTTTTCCAGTGTCATC-3') primers from within the putative human exon 1b mapped the transcription start site to 322 bp upstream of the ATG start codon located in exon 3. To compare the genomic sequence with cDNA sequence, 5'-RACE was performed using a human heart 5'-RACE-Ready cDNA library (Clontech) and primers from within the putative human exons 3 (rx5) and 1b (rx16, 5'- ATCGggatccCATGGGCAGATTATTCC-3', rx20, and rx15). Comparison of the DNA sequence of G1SH10 and G2SH1 confirmed (i) there was 100% nucleotide identity in the 5'-untranslated region region between the 5'-RACE clones and genomic clones G1SH10 and G2SH1, (ii) the human RXRgamma exon 1b was spliced to human exon 3 in a manner identical to that found in mice (10), and (iii) no intervening sequences existed between exon 1b and the transcription start site.

Reporter Plasmids and Eukaryotic Expression Vectors

hRXRgamma 2.luc.-1140 was constructed by cloning a BamHI-PstI fragment of the hRXRgamma 2 5'-flanking region from -1140 bp upstream to +71 bp downstream of the transcription start site into the luciferase reporter plasmid pGL2 basic (Promega). The mutated human RXRgamma 2 promoter reporter plasmid (hRXRgamma 2.luc.-1140.m1) was constructed by replacing the native sequence from -121 to +71 (an NdeI-PstI fragment) with a polymerase chain reaction-generated fragment containing point mutations in the gamma RXRE (G<UNL>C</UNL>TTGAAA<UNL>C</UNL>GTCA; underlined nucleotides represent substitution mutations, compared with Fig. 1). The construction of pTKLuc has been described (14). gamma RXRE.TKluc and gamma RXREm2.TKluc were each constructed by ligating two copies of the corresponding double-stranded oligonucleotide fragments in sense orientation into the BamHI site of pTKLuc (5'-gatccTGGGGTTGAAAGGTCAGATGGAtc-3' for gamma RXRE.TKluc and 5'-gatccTGGG<UNL>C</UNL>TTGAAA<UNL>C</UNL>GTCAGATGGAtc-3' for gamma RXREm2.TKluc (underlined nucleotides represent mutations)). Dideoxy DNA sequencing confirmed the location and orientation of the inserts. The construction of eukaryotic expression vectors for use in cell culture transfection experiments (pCDMRXRalpha , pCDMRARbeta , and pCDMCOUP) have been described (14) and were the generous gifts of Drs. Tod Gulick and David Moore (Harvard University). The murine expression vectors pSG5RXRalpha , -beta , and -gamma were generously provided by Dr. Pierre Chambon, Institut de Genetique et de Biologie Moleculaire et Cellulaire, Strasbourg, France.


Fig. 1. Schematic diagram of the 5'-flanking region of the human RXRgamma 2 gene. The transcription start site (+1) is indicated by the small arrow. The sequence and location of the imperfect repeat (gamma RXRE) is shown with large arrows denoting the potential receptor binding half-sites. The first exon of the human RXRgamma 2 mRNA (EXON 1b) was identified based on homology with the murine exon 1b. The intron 1b donor splice site sequence is shown. Putative binding sites for skeletal muscle- and cardiac expressed transcription factors (E box and CArG) based on published consensus sequences are shown (23, 24).
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Mammalian Cell Transfections

Simian CV-1 cells were employed for all transfection experiments. Cells were maintained and transient cotransfections performed as described (15). In brief, transient transfections were performed by the calcium phosphate coprecipitation method in 12-well tissue culture plates (Falcon) with 4 µg of reporter construct and 1 µg of receptor expression plasmid (as indicated in the figure legends) or an equivalent amount of pCDM without insert (pCDM(-)). Cells were harvested 48 h after transfection. One µg of a Rous sarcoma virus beta -galactosidase expression vector (RSVbeta gal) was included to correct for transfection efficiency with the exception of experiments involving pCDMCOUP. pCDMCOUP was noted to exert a modest repressive effect on the transcriptional activity of the thymidine kinase (TK) promoter. Accordingly, activities were adjusted to the effect of COUP-TF on the TK promoter based on the results of parallel experiments using pTKLuc without insert. In experiments involving the addition of ligand, 9-cis-RA or all-trans-RA was added 48 h prior to harvest, and vehicle was added at the same concentration to control wells. Luciferase activity was measured using the standard luciferin-ATP assay (16), and beta -galactosidase activity was measured using the Galacto-Light chemiluminescence assay (Tropix) in an Analytical Luminescence Monolight 2010 luminometer.

Electrophoretic Mobility Shift Assays (EMSAs)

EMSAs were performed as described (14, 17). The pT7lac-RXRalpha and pT7lac-myc-COUP-TF bacterial expression vectors (14) were generously provided by Dr. Tod Gulick. The nuclear receptors were overproduced in bacterial cells and partially purified as described previously (14). Antibody supershift experiments were performed with monoclonal antibodies to RXR (4RX1D12, directed against the D or E domain of all three murine RXRs; a generous gift of Dr. Pierre Chambon) and a monoclonal antibody directed against an epitope in the c-Myc protein (9E10, Oncogene Science).


RESULTS

Identification of a Retinoid-responsive Element in the Human RXRgamma 2 Gene Promoter

A human EMBL phage genomic library was screened with a partial human RXRgamma cDNA probe (hRX45; see "Materials and Methods") encoding a portion of human RXRgamma gene exon 2 and all of exon 3 (nucleotides 451-669 relative to the published mouse RXRgamma 1 cDNA sequence (10)). The human RXRgamma gene exon 3 was identified by the high degree of cross-species nucleotide identity with the murine exon 3 (>90%) (10). To determine whether the 5'-flanking region of the human RXRgamma 2 gene was contained within either of two genomic clones, Southern blot analysis was performed with a polymerase chain reaction-generated murine RXRgamma 2-specific cDNA probe containing only the 5'-untranslated region sequence encoded by the murine exon 1b (10). A single 4.0-kilobase pair BamHI restriction fragment was identified with this exon 1b probe and DNA sequence analysis defined a 250-bp region with over 70% nucleotide identity with the murine RXRgamma exon 1b sequence. 5'-RACE clones from a human heart library confirmed that, as in mouse, the human exon 1b sequence is spliced to exon 3 (10). Comparison of the genomic DNA sequence with that of multiple 5'-RACE clones revealed 100% nucleotide identity, confirming that no intervening sequences existed between exon 1b and the transcription start site. The 5'-RACE sequence data and primer extension analysis with human heart total RNA using two different antisense oligonucleotides from within the human exon 1b sequence (data not shown) localized the transcription start site to 322 bp upstream of the start codon. Of note, in addition to a high degree of identity, the human and mouse exon 1b nucleotide sequences are nearly colinear, diverging by no more than 4 consecutive nucleotides over the entire length of both sequences (data not shown). These results confirmed that the 4-kilobase pair BamHI genomic fragment contained 1.14 kilobase pairs of RXRgamma 2 gene 5'-flanking sequence, the 250 bp of 5'-untranslated region sequence encoded by the human homolog of murine RXRgamma exon 1b, and approximately 2.5 kilobase pairs of downstream sequence (Fig. 1).

Analysis of the DNA sequence of the RXRgamma 2 5'-flanking region revealed a putative TATA sequence (TATATTA) at bp -16 (relative to the transcription start site, +1), numerous potential E boxes (18), and several putative CArG sites (19) (Fig. 1), consistent with the muscle- and cardiac enriched expression of RXRgamma 2. An imperfect repeat sequence located at -100 to -86 conformed to the binding consensus for class II and class III nuclear receptors (Fig. 1). This sequence contains two potential hexamer binding sites separated by a single nucleotide and thus conforms to the direct repeat-1 (DR-1) group of elements known to confer transcriptional regulation by retinoid receptors (20).

To test the possibility that the putative nuclear receptor response element was retinoid-responsive and to characterize the transcriptional activity of the RXRgamma 2 gene 5'-flanking region from -1140 to +71, transient cell transfection studies were performed with this DNA fragment fused to a luciferase reporter (hRXRgamma 2.luc.-1140). A series of cotransfection studies was performed in simian CV-1 cells with eukaryotic expression vectors for human RXRalpha (pCDMRXRalpha ) and human RARbeta (pCDMRARbeta ) in the presence and absence of the retinoid ligands 9-cis-RA and all-trans-RA. As shown in Fig. 2A, the transcriptional activity of hRXRgamma 2.luc.-1140 was minimally increased in the presence of 9-cis-RA or RXRalpha alone but was induced 7-13-fold upon the addition of both 9-cis-RA and RXRalpha , indicating that this promoter fragment was activated by RXRalpha in a ligand-dependent manner. In contrast, hRXRgamma 2.luc.-1140 transcription was only minimally activated by all-trans-RA or 9-cis-RA in the presence of RXRalpha and RARbeta (Fig. 2A). These results suggest that the RXRgamma 2 promoter is preferentially activated by RXR homodimers rather than RXR·RAR heterodimers.


Fig. 2. The human RXRgamma 2 gene promoter is activated by 9-cis-RA in the presence of RXRalpha . A, the homologous promoter reporter plasmid hRXRgamma 2.luc.-1140 (schematic diagram shown; described under "Materials and Methods") was transfected into CV-1 cells with 9-cis-RA (10-6 M), all-trans-RA (10-7 M), or vehicle and cotransfected with pCDMRXRalpha (1.0 µg) and/or pCDMRARbeta (1.0 µg) as indicated. The bars represent mean (± S.E.) luciferase activity (relative light units; RLU) normalized (=1.0) to the activity of hRXRgamma 2.luc.-1140 cotransfected with pCDM (-) in the absence of ligand. The RLU values were corrected for transfection efficiency using the activity of cotransfected RSVbeta gal as described under "Materials and Methods." B, localization of the retinoid-responsive region of the RXRgamma gene promoter to the gamma RXRE. hRXRgamma 2.luc.-1140 and hRXRgamma 2.luc.-1140 m1 (see "Materials and Methods") were cotransfected into CV-1 cells with 1.0 µg of pCDMRXRalpha in the presence of 9-cis RA (10-6 M). The bars represent mean activation (± S.E.) as a percentage of the 9-cis-RA-mediated activation of hRXRgamma 2.luc.-1140. The data presented in panels A and B represent the mean of at least three independent experiments.
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To localize the region of retinoid responsiveness and to determine whether the imperfect repeat sequence located at -100 bp was indeed an RXR-responsive element, cotransfections were repeated with a 5'-deletion series of hRXRgamma 2.luc constructs. The results of these experiments (data not shown) revealed that the sequences conferring 9-cis-RA-mediated response resided primarily within the fragment flanked by NdeI (-121 bp) and PstI (+71 bp) sites (see Fig. 1), which contained the putative RXR response element. Cotransfection studies were repeated with a mutated hRXRgamma 2.luc.-1140 construct containing cytidine substitutions for the invariant second position guanine within each hexameric half-site of the imperfect repeat sequence (hRXRgamma 2.luc.-1140.m1; Fig. 2B). The 9-cis-RA/RXRalpha -mediated activation of hRXRgamma 2.luc.-1140.m1 was markedly lower (>75%) than that of hRXRgamma 2.luc.-1140, confirming that the imperfect repeat conferred the majority of the 9-cis-RA/RXR-mediated response (Fig. 2B). This retinoid-responsive element is here referred to as the gamma RXRE.

To test whether the gamma RXRE could confer retinoid responsiveness to a heterologous promoter and to examine its transcriptional regulatory properties further, including its potential to interact with other class II and class III nuclear receptors, two copies of the gamma RXRE were cloned upstream of the herpes simplex TK promoter fused to a luciferase reporter (gamma RXRE.TKluc). Cotransfection studies showed that gamma RXRE.TKluc was activated 8-10-fold by 9-cis-RA in the presence of RXRalpha (Fig. 3A). Significant RXR-mediated activation of gamma RXRE.TKluc occurred only in the presence of its ligand, 9-cis-RA, as was observed with the homologous promoter (hRXRgamma 2.luc.-1140). When point mutations identical to those present in hRXRgamma 2.luc.-1140.m1 were introduced into both copies of the gamma RXRE in the context of TKluc (gamma RXREm2.TKluc), 9-cis-RA-mediated responsiveness was abolished (Fig. 3A). The cotransfection experiments were repeated with expression vectors for murine RXRalpha , -beta , and -gamma to determine whether the gamma RXRE was capable of conferring 9-cis-RA-mediated transcriptional activation via all known RXRs. All three RXRs mediated 9-cis-RA-dependent activation to a similar level (data not shown).


Fig. 3. The gamma RXRE confers retinoid responsiveness to a heterologous promoter: comparison of transactivation with RXRalpha and RXRalpha /RARbeta . A, the heterologous promoter reporter gamma RXRE.TKluc (shown at the top) and the mutated gamma RXRE reporter gamma RXREm2.TKluc (hatched) were transfected into CV-1 cells in the presence and absence of 1 µg of pCDMRXRalpha (RXRalpha ) and/or 9-cis-RA (10-6 M) (9-c) as indicated. The bars represent mean (± S.E.) RLU normalized to the activity of gamma RXRE.TKluc cotransfected with pCDM (-) in the absence of ligand. B, gamma RXRE.TKluc was cotransfected into CV-1 cells with 1.0 µg of pCDMRXRalpha and/or pCDMRARbeta with 9-cis-RA (10-6 M) or all-trans-RA (10-7 M) as indicated. The bars represent mean RLU normalized to gamma RXRE.TKluc cotransfected with pCDM (-). Data presented in panels A and B represent a minimum of three independent experiments.
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Previous studies have demonstrated that RXR·RAR heterodimers may confer transcriptional activation via DR-1 elements in the presence of either 9-cis-RA or all-trans-RA (21, 22). In contrast to RXR homodimer-mediated activation, the ligand-mediated activation of RXR·RAR heterodimers on a DR-1 element occurs mainly or solely via RAR (23, 24). Accordingly, RXR·RAR heterodimers may function as transcriptional inhibitors of RXR homodimer activation on DR-1 elements. In fact, the transfection studies shown above (Fig. 2A) revealed that, in the context of the homologous promoter, RXRalpha -mediated activation of gamma RXRE was reduced by the presence of RARbeta . To explore the activation of gamma RXRE in the context of a heterologous promoter, cotransfection studies were performed with gamma RXRE.TKluc and pCDMRXRalpha and/or pCDMRARbeta in the presence and absence of either 9-cis-RA (a potential ligand for either RXR or RAR) or all-trans-RA (an RAR ligand) (Fig. 3B). Cotransfection of pCDMRARbeta alone or pCDMRARbeta plus pCDMRXRalpha in the absence of ligand did not significantly alter the transcriptional activity of gamma RXRE.TKluc. Activation of gamma RXRE.TKluc by either all-trans RA or 9-cis-RA in the presence of both RXRalpha and RARbeta was lower than the induction obtained with RXRalpha alone in the presence of 9-cis-RA (mean of 4-5-fold versus 8-fold, respectively). These results and the retinoid-mediated activation studies of the homologous promoter (Fig. 2A) indicate that the gamma RXRE is preferentially activated by RXR and its cognate ligand 9-cis-RA. The retinoid-mediated transcriptional regulatory properties of the gamma RXRE is similar to that of other DR-1 elements such as the cellular retinol-binding protein II gene RXRE (23, 25) in which cotransfection of RARbeta blunts 9-cis-RA mediated transactivation by RXRalpha . Additional experiments demonstrated that several other known RXR partners, including TRalpha 1, TRbeta 1, or peroxisome proliferator-activated receptor alpha , had no effect on gamma RXRE.TKluc transcriptional activity in the presence of appropriate hormone ligands (thyroid hormone) or peroxisome proliferator-activated receptor activators (fatty acids or clofibrate) with or without RXR (data not shown).

The gamma RXRE Is Bound by RXRalpha Homodimers in a Ligand-dependent Manner and by RXR·RAR Heterodimers in a Ligand-independent Manner

To characterize the interaction of RXR with the gamma RXRE, EMSAs were performed with a 32P-radiolabeled gamma RXRE oligonucleotide probe and bacterially overexpressed, partially purified RXRalpha . As shown in Fig. 4A, RXRalpha homodimers bound the gamma RXRE as a single complex with an affinity that was significantly increased by the addition of 9-cis-RA (Fig. 4A, lane 3) compared with vehicle (Fig. 4A, lane 2). The specificity of the RXR homodimer-gamma RXRE interaction was demonstrated by competition studies showing complete inhibition of formation of the complex by the addition of a 100-fold molar excess of unlabeled gamma RXRE but no reduction in complex formation with an equivalent molar amount of unlabeled, unrelated double-stranded oligonucleotide (Fig. 4A; lanes 3-5). In addition, the specific complex was "supershifted" with anti-RXR antisera (Fig. 4A; lanes 6 and 7). Finally, when a mutated gamma RXRE probe, containing the same point mutations previously shown to abolish functional activity, was incubated with RXRalpha and 9-cis RA, no complex formed (Fig. 4A, lanes 8 and 9). These data confirm that RXRalpha homodimers can interact directly and specifically with the gamma RXRE and that ligand increases binding affinity.


Fig. 4. RXRalpha homodimers and RXRalpha ·RARbeta heterodimers bind gamma RXRE. A, autoradiograph of EMSA performed with a 32P-labeled gamma RXRE oligonucleotide probe and RXRalpha overproduced in bacteria. 9-cis-RA (10-6 M) was added to the incubation mix of lanes 3-7 and 9. Me2SO vehicle was added at an equal volume in lane 2. A 100-fold molar excess of unlabeled, size-matched unrelated competitor oligonucleotide DNA (NS) or unlabeled gamma RXRE DNA (Sp) were added to the incubations as indicated at the top. Preimmune ascites (PI) and anti-RXR antisera (RXR) were added to the incubations in lanes 6 and 7, respectively. A mutated gamma RXRE probe (mt), described under "Results," was used in lanes 8 and 9. B, EMSA performed with gamma RXRE and bacterially overproduced RXRalpha and RARbeta . Competition was performed as in Fig. 3A with a 100-fold excess of competitor (NS 100× and Sp 100×). Nonspecific complexes are designated NS.
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To characterize RXR·RAR heterodimer binding to the gamma RXRE, EMSA was performed with bacterially overexpressed RARbeta and RXRalpha (Fig. 4B). A minimal complex was observed when either RAR or RXR alone was added to gamma RXRE probe in the absence of ligand. In contrast, incubation of the probe with both receptors resulted in a marked increase in complex formation. Competition experiments confirmed that this interaction was specific (Fig. 4B; lanes 4-6). Accordingly, RXR·RAR heterodimers bind the gamma RXRE in a cooperative manner. In contrast to the interaction of gamma RXRE with RXR homodimers, the RXR·RAR-gamma RXRE interaction was not influenced by the addition of 9-cis-RA or all-trans-RA (data not shown).

RXRalpha and the Orphan Receptor COUP-TF Compete on the gamma RXRE

A significant body of evidence indicates that RXR and the known orphan nuclear receptor COUP-TF often compete at a single DR-1-type element (26-31). To examine the potential binding of COUP-TF to the gamma RXRE, EMSAs were performed using COUP-TF tagged with an NH2-terminal Myc peptide overproduced in bacteria (COUP-TFMyc). COUP-TFMyc formed a specific complex with the gamma RXRE, as demonstrated by competition studies (Fig. 5). "Supershift" experiments with an anti-Myc antibody provided additional evidence for the specificity of the COUP-TF-gamma RXRE interaction. Thus, COUP-TF binds the gamma RXRE with high affinity.


Fig. 5. COUP-TF binds gamma RXRE. EMSA performed with gamma RXRE and bacterially overproduced Myc-tagged COUP-TF. 50- and 100-fold molar excesses of unlabeled gamma RXRE DNA (Sp 50× and Sp 100×) and 100-fold molar excess of unlabeled, size-matched unrelated competitor oligonucleotide DNA (NS 100×) were added to the incubations as indicated. Anti-Myc (MYC) antibody (Ab) or preimmune sera (PI) were added as designated.
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Cotransfection mixing experiments were performed with gamma RXRE.TKluc, pCDMRXRalpha , and pCDMCOUP to determine whether these transcription factors could compete at the gamma RXRE to modulate transcription (Fig. 6). For these experiments, increasing amounts of pCDMCOUP were transfected into CV-1 cells with a fixed amount of pCDMRXRalpha in the presence of 9-cis-RA. Because parallel experiments with pTKLuc alone demonstrated a modest repressive effect of COUP-TF on TK transcription, all data presented for gamma RXRE.TKluc have been corrected for the effect on the TK promoter. COUP-TF blunted the 9-cis-RA-mediated RXR activation via the gamma RXRE in a dose-dependent fashion (Fig. 6). With the highest amounts of pCDMCOUP transfected, transcription of gamma RXRE.TKluc was repressed below basal levels, indicating that, in addition to competing with RXR, at higher levels COUP-TF actively represses transcription via the gamma RXRE, a property shown for most known COUP-TF response elements (14, 26-37). The transcriptional activity of gamma RXRE.TKluc varied over 50-fold in these cotransfection experiments. These results, together with the binding studies, indicate that COUP-TF modulates retinoid-mediated activation of gamma RXRE and suggest a mechanism whereby transcriptional activity can be modulated over a wide range.


Fig. 6. Bidirectional modulation of transcription by the competitive interaction of RXRalpha and COUP-TF on gamma RXRE. The heterologous promoter reporter gamma RXRE.TKluc was cotransfected into CV-1 cells with 1.0 µg of pCDMRXRalpha plus 9-cis RA (10-6 M) and a range of pCDMCOUP (from 250 ng to 2.0 µg). The bars represent mean (± S.E.) RLU normalized to gamma RXRE.TKluc cotransfected with pCDM (-). Data represent a minimum of three independent experiments.
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DISCUSSION

This report demonstrates that the human RXRgamma 2 gene promoter contains a RXR response element, a mechanism for the regulation of RXRgamma 2 gene expression by retinoid-mediated pathways. The presence of autoregulatory elements within the promoters of genes encoding other nuclear receptor isoforms, including RARbeta 2, RARalpha 2, RARgamma 2, and TRbeta 1, suggests that the expression of a subset of nuclear receptor genes are controlled by this mechanism (38-43).

The transcriptional regulatory properties of gamma RXRE are similar to those of previously reported DR-1-type retinoid response elements (21, 23, 25, 29-31, 44-46). A comparison of the gamma RXRE sequence with the relatively few known natural DR-1 RXREs is shown in Fig. 7. Although the 5'-half-site sequence of the gamma RXRE is novel compared with other known elements, it conforms to the known consensus (PuG(G/T)TNA) for binding class II or class III nuclear receptors (reviewed in Ref. 7). Furthermore, the gamma RXRE sequence, including the extended heptamer of the 5'-half-site (GGG<UNL>T</UNL>T<UNL>G</UNL>A) resembles the RXRalpha binding site (GGGGTCAaAGGTCA) and the high affinity RXRgamma consensus (RGRNCAaAGGTCA) determined by nonbiased random oligonucleotide selection (47-49). Interestingly, comparison of the sequences shown in Fig. 7 reveals that the 5'-half-site sequence often diverges from the idealized class II/III sequence (AGGTCA). To our knowledge, the role, if any, of such sequence differences in dictating transactivation properties of RXR isoforms has not been established.


Fig. 7. Comparison of natural DR-1 RXREs with the gamma RXRE from the human RXRgamma 2 gene promoter. RXREs of the DR-1 arrangement from rat cellular retinol-binding protein II gene (rCRBP II; Ref. 25), mouse cellular retinol-binding protein II gene (mCRBPII; Ref. 30), hepatitis B virus enhancer (HBV; Ref. 45), major histocompatibility class I genes (MHC; Ref. 44), alpha -fetoprotein gene (alpha FP; Ref. 29), lactoferrin gene (31) and the human RXRgamma 2 gene (RXRgamma 2) are shown.
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We show here that the interaction of gamma RXRE with RXR homodimers but not RXR·RAR heterodimers is induced by the ligand 9-cis-RA, a unique property. Although others have shown ligand-dependent binding of RXR homodimers to DR-1 elements with receptor produced in reticulocyte lysate expression systems, to our knowledge this is the first example of ligand-induced binding with receptor protein produced in bacteria. In fact, several other groups have shown that RXR homodimer (produced in bacterial expression systems) binding to perfect DR-1 elements is ligand-independent (25, 26, 48). We have also shown that binding of RXRalpha (produced in our bacterial expression system) to an idealized DR-1 (AGGTCAaAGGTCA) is not dependent on or induced by 9-cis-RA.2 Taken together, these results suggest that the unique sequence of the gamma RXRE dictates the relative affinity by which RXR homodimers bind this element and thus require ligand for this interaction.

Our results also demonstrate that the orphan receptor COUP-TF competes with RXR homodimers on the gamma RXRE to repress transcription. This finding is consistent with the known role of COUP-TF as a negative modulator of RXR-mediated transcriptional regulatory pathways through competition for DNA binding. This competitive interaction has been demonstrated for a variety of natural and synthetic retinoid-responsive elements, including DR-1 (26, 27, 29-31) and DR-5 elements (27, 33) as well as complex retinoid-responsive elements (14, 27, 28, 33, 36, 50). Our data also indicate that, in addition to interference with RXR homodimer binding to the gamma RXRE, COUP-TF represses transcription via this element as it does on the majority of other COUP-TF response elements.

A major unanswered question in nuclear receptor biology involves the specific biological roles of multiple RXR and RAR isoforms generated by differential promoter utilization and/or alternative splicing. The RXRgamma 2 isoform is an excellent focus for the study of the function of nuclear receptor isoforms because of its tissue- and developmental stage-restricted expression pattern (8-10). The recent characterization of mice homozygous for targeted ablation of retinoid receptors demonstrates the importance of retinoids in murine cardiac development (51-55). In addition, recent studies by us and others suggest that retinoids play a role in the control of postnatal cardiac energy metabolism (56) and antagonize the cardiac hypertrophy program (57). The known cardiac enriched expression of RXRgamma 2 and our identification of the gamma RXRE raises the intriguing possibility that the cardiac specific effects of retinoids occur via retinoid signaling pathways that converge on this gene. Given that RXRalpha and RXRbeta are expressed prior to RXRgamma in the developing heart and somites, it follows that the RXRgamma 2 gene promoter could be a downstream target during embryologic development. Cotransfection studies performed in our laboratory indicate that the gamma RXRE is activated similarly by RXRalpha , RXRbeta , or RXRgamma (data not shown), suggesting that the human RXRgamma 2 promoter is a potential target for any of the known RXRs.


FOOTNOTES

*   This work was supported in part by the Lucille P. Markey Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Supported by a National Institutes of Health Training Grant, T32 HL07275-17.
§   An Established Investigator of the American Heart Association. To whom correspondence should be addressed: Cardiovascular Div., Box 8086, Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-8919; Fax: 314-362-0186; E-mail: kelly{at}im.wustl.edu.
1    The abbreviations used are: RXR, retinoid X receptor; RXRalpha , -beta , and -gamma , retinoid X receptor alpha , beta , and gamma  isoforms, respectively; RXRE, retinoid X response element; gamma RXRE, human retinoid X receptor gamma 2 gene retinoid X response element; RAR, retinoic acid receptor; RARalpha , -beta , and -gamma , retinoic acid receptor alpha , beta , and gamma  isoforms, respectively; RA, retinoic acid; COUP-TF, chicken ovalbumin upstream promoter transcription factor; TR, thyroid hormone receptor; TRalpha 1 and TRbeta 1, thyroid hormone receptor alpha 1 and beta 1 isoforms, respectively; TK, thymidine kinase; DR, direct repeat; EMSA, electrophoretic mobility shift assay; bp, base pair(s); RACE, rapid amplification of cDNA ends; RLU, relative light units.
2    P. M. Barger and D. P. Kelly, unpublished results.

Acknowledgments

We especially thank Tod Gulick and David Moore for helpful discussions and Kelly Hall for expert secretarial assistance.


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