©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Interactions between Retinoic Acid Receptor-related Orphan Nuclear Receptor (ROR) and the Retinoic Acid Receptors in the Regulation of the F-Crystallin Promoter (*)

(Received for publication, March 10, 1995)

Mark Tini (§) Robert A. Fraser (§) Vincent Giguère (¶)

From the Molecular Oncology Group, Royal Victoria Hospital, Departments of Biochemistry, Medicine and Oncology, McGill University, Montréal, Québec, H3A 1A1, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have previously demonstrated that an everted repeat of the hexamer PuGGTCA located within the F-crystallin promoter mediates activation of the murine F-crystallin gene by retinoic acid and thyroid hormone receptors. Here, we show that the recently identified retinoic acid receptor-related orphan nuclear receptor (RORalpha) is expressed in the murine lens and activates the F-crystallin promoter. In contrast to the retinoic acid and thyroid hormone receptors, activation of the F-crystallin promoter by RORalpha requires binding to the single 3` half-site and spacer sequences of F-crystallin hormone response element (F-HRE). We further demonstrate that RORalpha-dependent activation is repressed by the competitive binding of retinoic acid receptor/retinoid X receptor heterodimers to the F-HRE in the absence of all-trans-retinoic acid. These studies suggest that the interplay of retinoid receptors and RORalpha on the F-HRE may constitute an important mechanism regulating F-crystallin gene expression in the murine lens.


INTRODUCTION

The murine -crystallin gene locus contains six closely related but differentially regulated genes encoding lens structural proteins that are expressed concomitantly with lens cell differentiation(1, 2, 3, 4) . Lens-specific expression of the F-crystallin gene is directed by proximal promoter sequences located immediately upstream of the TATA box which are conserved in all -crystallin genes, while upstream enhancer sequences are necessary for proper spatial expression within the lens(5, 6) . We have previously shown that the F-crystallin promoter is activated by retinoic acid (RA) (^1)treatment and have characterized a complex hormone response element (F-HRE) located within the upstream enhancer region(7) . The F-HRE consists of an everted arrangement of two nuclear receptor consensus half-sites motifs (PuGGTCA) separated by 8 nucleotides (referred to as everted repeat-8 or ER-8) and confers RA responsiveness when linked to a heterologous promoter(7) . The F-HRE is bound in vitro by RA (RAR) and retinoid X (RXR) receptor heterodimers, and both classes of retinoid receptors were shown to cooperate in vivo to trans-activate this element. Recently, we have demonstrated that the F-HRE also mediates thyroid hormone (T(3)) responsiveness of the F-crystallin promoter(8) . Although the F-HRE is bound by the T(3) receptor (T(3)R) in the form of heterodimers with either RXR or RAR, only T(3)RbulletRXR heterodimeric complexes appear to be transcriptionally active. These results show that RARalpha exerts a dominant role in the regulation of transcription of the F-crystallin gene and underscore the complexity of the retinoid signal at the level of gene expression (for review, see (9, 10, 11) ).

The retinoid and T(3) receptors are part of a large family of ligand-dependent transcription factors that includes a growing class of related proteins for which regulatory ligands have not been identified(12) . These proteins are referred to as orphan receptors. We have recently identified a novel family of orphan receptors (RORalpha, RAR-related orphan receptor) consisting of different isoforms that share common DNA- and putative ligand-binding domains but possess distinct amino-terminal domains which confer different DNA binding specificities (13) . RORalpha activates transcription constitutively upon binding as a monomer to response elements composed of the PuGGTCA core binding motif preceded by a 6-nucleotide A/T-rich sequence(13, 14, 15) . We have shown that the F-HRE is bound by RORalpha1 and acts as a strong HRE for this orphan receptor(13) . In this report, we have investigated more closely the interaction of RORalpha with the F-HRE and have established that RORalpha can activate the F-crystallin promoter in primary chick lens cell cultures. We demonstrate that RAR/RXR heterodimers compete with RORalpha for occupancy of the F-HRE and that, in the absence of RA, the retinoid receptor complexes block RORalpha activation. Since the retinoid receptors and RORalpha are expressed in the lens, we suggest that both classes of receptors are likely to play important roles in the regulation of the F-crystallin gene.


EXPERIMENTAL PROCEDURES

Reverse Transcriptase-PCR

Total RNA was extracted from 100 mg of frozen murine eye lens tissue using 1 ml of Trizol reagent (Life Technologies, Inc.) according to the manufacturer's protocol. Single-stranded cDNA was synthesized from 2.0 µg of total RNA using Superscript reverse transcriptase (Life Technologies, Inc.) as recommended by the manufacturer, primed with either 100 ng of oligo(dT) (Life Technologies, Inc.), 20 ng of gig1961 oligonucleotide (5`-TCTGAGGTCATCATCGAGTTCCGC-3`), or 20 ng of gig1962 (5`-CCGGCTGCAGAAATGCCTGGCCGT-3`). PCR amplification of 2 µl of each of the three cDNA synthesis reactions were performed simultaneously in 1 PCR buffer (Perkin-Elmer) 200 nM of dATP, dTTP, dCTP, and dGTP (Perkin-Elmer), and 100 ng of gig1961 and gig1962 in a 90-µl final volume. After 4 min of denaturation at 94 °C, 2.5 units of Taq polymerase (Perkin-Elmer) were added to each reaction for a final volume of 100 µl. The reactions were cycled 10 times through 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min and then 30 times through 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min. One fifth of each reaction was then fractionated on a 1.7% agarose gel cast in 1 TBE (Tris-borate-EDTA), then transferred to Hybond N (Amersham Corp.). The membrane was prehybridized for 30 min at 65 °C with Rapid-Hyb (Clonetech) prior to hybridization for 2 h at 65 °C with 1 10^6 cpm of [alpha- P]dCTP (Amersham)-labeled (Quick Prime, Pharmacia Biotech Inc.) 107 bp of XhoI/XcmI human RORalpha fragment. The blot was washed twice for 30 min at room temperature with 2 SSC and 0.1% SDS and then at 65 °C with 0.2 SSC and 0.2% SDS.

Plasmids, Cell Culture, and Transfection

The F-crystallin and TREpalTKLUC reporter plasmids have been previously described(16, 17) . F-HRE reporter constructs were prepared by cloning two copies of double-stranded oligonucleotides (see ``Results'') containing SalI and BamHI cohesive ends into the SalI site of plasmid TKLUC. Primary lens cultures were prepared as described previously(7) . The equivalent of five lenses was plated on 60-mm culture dishes coated with collagen. Cells were transfected with 10 µg of F-crystallin reporter construct and 1 µg of plasmid RSV-betagal, and 0.5-1.0 µg of RSV LTR-based expression vectors directing the synthesis of either the human RORalpha1 or RARalpha1. P19 and CV-1 cells were cultured on alpha-minimum essential medium containing 7% fetal calf serum. These were transfected with 2 µg of thymidine kinase promoter based reporter plasmids, 1 to 2 µg of RSV-betagal, and 100 to 500 ng of appropriate expression vector. Transfected cells were treated for 20 to 48 h with appropriate ligands. beta-Galactosidase and luciferase assays were carried as described elsewhere(18) . CAT assays were performed using equivalent amounts of beta-galactosidase activity using a non-TLC method as described by Amersham.

In Vitro Translation and EMSA

Plasmid pCMXhRARalpha (19) containing the human RARalpha1 cDNA(20) , and plasmid pSKmRXRbeta (21) containing the mouse RXRbeta were linearized with BamHI and AccI, respectively. Plasmid pSKhR5 containing the human RORalpha1 cDNA was linearized with BamHI. Capped mRNAs were synthesized in vitro using linearized plasmids and T7 RNA polymerase for RORalpha1 and RARalpha, while RXRbeta mRNA was synthesized with T3 RNA polymerase. These mRNAs were used to synthesize RORalpha1, RARalpha1, and RXRbeta protein in vitro using rabbit reticulocyte lysates (Promega). EMSAs were performed as described previously(7) . The amount of reticulocyte lysate in each binding reaction was kept constant within a given experiment.


RESULTS

RORalpha Is Expressed in the Murine Lens and Activates the F-Crystallin Promoter

To establish whether RORalpha is expressed in the murine lens we extracted RNA from lens tissue (2-week-old mice) and used reverse transcriptase to synthesize single strand cDNA using either an oligo(dT)- or an RORalpha-specific primer. The products of cDNA synthesis were amplified using RORalpha-specific primers designed to generate a 217-bp fragment spanning sequences at the end of the DNA-binding domain and the hinge region. As a control for amplification of genomic DNA, cDNA synthesis was also carried out with the 5`-RORalpha primer. Upon fractionation of the amplified products on an agarose gel, a band of correct size was detected in both the oligo(dT) and 3`-ROR oligonucleotide-primed cDNA reactions consistent with the presence of RORalpha transcripts in the lens. An additional smaller band was also detected in cDNA primed with a 3`-RORalpha oligonucleotide which may indicate the presence of different isoforms of RORalpha in the lens. Bands were not observed when cDNA synthesis was primed with a 5` RORalpha oligonucleotide indicating that genomic DNA was not amplified in this experiment. The identity of the amplified bands was confirmed by mapping with restriction endonucleases (data not shown) and by hybridization with a radioactively labeled 107-bp RORalpha probe corresponding to sequences contained within the amplified fragment (Fig. 1).


Figure 1: Orphan receptor ROR is expressed in the murine lens. 2 µg of total RNA extracted from 2-week-old (postpartum) murine lens tissue were used in three parallel cDNA synthesis reactions primed with either oligo(dT), a ROR-specific 3` oligonucleotide, or a 5`-ROR-specific oligonucleotide. The products of these reactions were amplified using the polymerase chain reaction primed with ROR-specific primers. Amplified products were fractionated on a agarose gel transferred to Hybond membrane and hybridized at high stringency with a radiolabeled 107-bp human RORalpha1 probe. The size of the amplified products was determined by comparison with known size markers.



The 5`-flanking sequences of the murine F-crystallin gene between nucleotides -226 to +47 are sufficient for optimal promoter activity in primary cultures of chick lens cells and for proper developmental regulation of a reporter gene in the lenses of transgenic mice(6, 16) . The F-HRE (located at -210 to -185) mediates activation by both RAR and T(3)R and also contributes to basal promoter activity since deletion of the 3` half-site and spacer sequences (-202 to -185) decreases promoter function(7, 8, 16) . In contrast, mutation of the first half-site eliminates RA response but has minimal effects on basal promoter activity(7) . These data suggest that there are endogenous transcription factors in lens cells that specifically recognize the downstream half-site and spacer of F-HRE to stimulate basal promoter activity. The orphan receptor RORalpha interacts with a single PuGGTCA motif preceded by A/T-rich sequences and binds to a number of characterized response elements including the F-HRE(13) . To determine whether RORalpha could activate the F-HRE within the natural promoter context we performed transfections in chick lens cells using reporter gene constructs containing the bacterial CAT gene under the control of the mouse F-crystallin promoter (-226 to +47). A mutant reporter construct containing a deletion of spacer sequences and the 3` half-site of F-HRE (Delta -202/-185) was also tested. Transfection of RORalpha1 expression vector increased wild-type promoter activity approximately 3.8-fold, while the Delta -202/-185 promoter mutant displayed reduced basal promoter activity and was not significantly activated by transfected RORalpha1 (Fig. 2).


Figure 2: RORalpha1 activates the F-crystallin promoter. Chick primary lens cultures were transfected with 10 µg of CAT gene reporter plasmids containing either wild type F-crystallin 5`-flanking sequence -226 to + 47 (F(-226/+47)) or an equivalent segment containing a deletion of the F-HRE 3` half-site and spacer sequences (Delta -202/-185). 500 ng of an expression vector containing the human RORalpha1 cDNA under the control of the RSV-LTR were cotransfected together with 1 µg of RSV-betagal as a control for transfection efficiency. Cells were harvested 48 h following transfection. The mean value of duplicate determinations is plotted and dots indicate the values of each determination.



RORalpha1 Selectively Activates the F-HRE

We and others have shown that human RORalpha1 and rat RZRbeta(a distinct ROR isoform)bind to a number of natural and synthetic response elements which are composed of two PuGGTCA half-sites arranged in different configurations (Fig. 3A)(13, 22) . We have determined the relative binding affinity of RORalpha1 for these elements by performing competition studies using EMSA (Fig. 3B). Incubation of radioactively labeled F-HRE oligonucleotide with in vitro synthesized RORalpha1 generates a single complex. Inclusion in the binding reactions of different molar excesses of response elements indicated that RORalpha binds with highest affinity to F-HRE and TREpal. CRBPI-RARE competed poorly while betaRARE failed to compete, indicating that RORalpha1 does not bind with high affinity to these elements.


Figure 3: Competition analysis of hormone response elements for binding of RORalpha1. A, sequences of response elements used in this study. TREpal is an idealized response element derived from the rat growth hormone gene(38) , betaRARE was identified in the promoter of the human RARbeta2 gene(39) , CRBPI-RARE was identified in the mouse cellular retinol binding protein I gene(40) . B, radiolabeled F-HRE was incubated with reticulocyte lysate (2 µl) programmed with human RORalpha1 mRNA and fractionated on polyacrylamide gel cast in 0.5 TBE. Oligonucleotides corresponding to the different response elements were included in the binding reactions at the indicated molar excess.



We next compared the transcriptional activity of RORalpha1 on F-HRE and TREpal which represent the highest affinity binding sites. Luciferase gene reporter plasmids containing a single copy of either element in front of the viral thymidine kinase promoter (-105 to +51) were tested by transfection in P19 cells. Since both F-HRE and TREpal mediate transcriptional responses to retinoid receptors, duplicate transfected cells were treated with RA as a control. As can be observed from Fig. 4, the TREpal reporter plasmid was efficiently activated by RA treatment, but only the F-HRE reporter could be activated by transfected RORalpha1.


Figure 4: RORalpha1 is transcriptionally inactive on TREpal. Reporter plasmids (2 µg/plate) containing either the F-HRE or TREpal in front of the thymidine kinase promoter linked to the luciferase reporter gene were transfected into P19 cells together with RORalpha1 expression vector (0.5 µg/plate) and plasmid RSV-betagal. Duplicate plates were treated with RA (100 nM). Cells were grown for 24 h following transfection. The mean values of three independent experiments are plotted with the standard error indicated.



The Spacer Region and Downstream Half-site of F-HRE Are Sufficient for High Affinity Binding of RORalpha1

To determine which half-site of F-HRE is occupied by RORalpha1 we tested two oligonucleotides (m1 and m2) containing either the 5` or 3` half-site and spacer sequences for binding of RORalpha1 in competition EMSA (Fig. 5). Oligonucleotide m2 containing the 3` half-site competed as efficiently as the intact element, while oligonucleotide m1 containing the 5` half-site competed less efficiently (Fig. 5B). This indicates that the 3` half-site and spacer sequences are sufficient for high affinity binding of RORalpha. This conclusion is supported by transfection studies indicating that reporter plasmids containing two copies of either the intact F-HRE or the 3` half-site and spacer sequences are transactivated with the same efficiency by RORalpha1 while a reporter containing the 5` half-site and spacer is not activated (Fig. 5C).


Figure 5: The 3` half-site and spacer are sufficient for high affinity binding and transcriptional activation by RORalpha1. Oligonucleotides containing either half-site and spacer sequences (A) were used as competitors in binding reactions (B) at the indicated molar excess. (C) Luciferase reporter plasmids (2 µg/plate) containing two copies of each oligonucleotide in front of the thymidine kinase promoter were transfected in P19 cells together with RORalpha1 expression vectors (500 ng/plate). The mean values of three independent experiments are plotted with the standard error indicated.



Comparison of the spacer sequences preceding each half-site suggests that the cytosines located at the first and second positions (-1 and -2) upstream of the 3` half-site may be required for preferential binding of RORalpha1. As expected from previous analysis of the RORalpha1 binding properties(13) , deletion of dinucleotide CC (m4) decreased binding, whereas replacement with AG (m3) failed to do so (Fig. 6B). The importance of other sequences within the spacer is confirmed by additional mutant oligonucleotides (m5, m6, and m7) (Fig. 6C). In accordance with the consensus RORE determined by binding site selection(13, 14) , replacement of the dinucleotide AA (positions -3 and -4) with thymidines does not affect binding while replacement with CG causes a sharp reduction in binding affinity. Mutation of TT at positions -5 and -6 causes a slight reduction in binding.


Figure 6: Analysis of spacer mutants for binding of RORalpha1. Mutant oligonucleotides (A) were used as competitors in binding reactions (B and C). Each composite was derived from the same experiment.



RAR/RXR Heterodimers Repress RORalpha-dependent Activation by Competing for Occupancy of the F-HRE

We have previously shown that primary cultures of chick lens cells are responsive to RA indicating the presence of endogenous retinoid receptors(7) . Furthermore, mice disrupted for RAR alpha and genes display extensive ocular defects including lens fiber abnormalities (23) . These studies provide evidence for a role of RARs in lens and eye development. In P19 cells, combined transfection of RORalpha1 and RA treatment results in greater levels of transactivation of the F-HRE suggesting that retinoid receptors and RORalpha cooperate in transcriptional regulation (Fig. 4).

To investigate the functional relationship between retinoid receptors and RORalpha on this element, we performed transfection studies in CV-1 cells which contain low levels of endogenous retinoid receptors. A sensitive reporter construct containing three copies of F-HRE was activated approximately 20-fold by transfected RORalpha1 (Fig. 7A). Additional RA-dependent stimulation was not observed unless RAR expression vector was cotransfected. In the absence of RA, transfected RAR represses RORalpha mediated activation in a dose-dependent manner. Thus, RAR can either repress RORalpha activation or cooperate in activation depending on the availability of its cognate ligand. Repression in the absence of RA may be explained by competition of retinoid receptor complexes with RORalpha for occupancy of F-HRE. To demonstrate this we performed EMSA with a limiting concentration of radiolabeled F-HRE, a constant amount of RAR and RXR and increasing amounts of RORalpha. An RORalpha mutant with truncated carboxyl terminus (RORalphaDelta235) used in these studies forms a fast migrating complex due to its smaller size. As can be seen in Fig. 7B, increasing the amount of RORalphaDelta235 in the binding reaction causes a dose-dependent decrease in the formation of RARbulletRXR complexes. Consistent with these results, co-transfection of RORalphaDelta235 which is transcriptionally inactive blocks both RA- and RORalpha-dependent activation on F-HRE reporter plasmids (data not shown).


Figure 7: Retinoid receptors and ROR compete for binding to F-HRE. A, a constant amount of RORalpha1 expression vector (1 µg) was transfected with the indicated amounts of RARalpha expression vector. A luciferase reporter plasmid was used containing three copies of the F-HRE. B, binding reactions were carried out with a limiting amount of probe (0.01 ng), a fixed amount of RAR/RXR and increasing amounts of a ROR mutant lacking the carboxyl terminus (RORDelta235). The RAR/RXR band was quantified using an Molecular Dynamics PhosphorImager system. Arbitrary values representing intensity of the RAR/RXR band was plotted on the y axis and amount of RORDelta235 added was plotted on the x axis. Above is a representative experiment in duplicate where the values of each determination did not vary by more than 30%. The experiment was performed three times.




DISCUSSION

During lens development, differentiation of epithelial cells into fiber cells is regulated by factors secreted from the retina(24, 25, 26) . The F-crystallin gene thus provides an excellent model to study the molecules and transactivating factors that control differentiation processes in the developing lens. Recently, we have shown that both RA and T(3) activate F-crystallin gene expression through a novel HRE (F-HRE) located in the upstream enhancer element (-210 to -185) of the F-crystallin promoter (7, 8) and proposed that RA may serve as a regulatory signal in lens development. This suggestion is supported by the analysis of mice bearing disrupted RARalpha and genes which display severe ocular defects including abnormal lens fibers and agenesis of the lens(23) . In the present study, we report the regulation of the F-crystallin gene by RORalpha1, an orphan member of the superfamily of steroid-thyroid-retinoid receptors, via interactions with the F-HRE. We further demonstrate that constitutive activation of the F-HRE by RORalpha1 is repressed by the competitive binding of RAR/RXR heterodimeric complexes to the element in the absence of RA.

The F-HRE consists of an everted repeat of two consensus nuclear receptor half-sites (PuGGTCA) separated by 8 nucleotides. This element is bound by all three heterodimeric combinations of RAR, T(3)R, and RXR and these binding activities require that both half-sites be intact(7, 8) . In contrast, the 3` half-site and six adjacent nucleotides (TTAACCAGGTCA) of the F-HRE are sufficient for proper recognition by RORalpha1. Since transcriptional activation by transfected RORalpha1 is constitutive, these findings are consistent with the mutational analyses of the F-crystallin promoter. Specifically, previous experiments indicated that alterations of the 5` half-site of the F-HRE abolished binding of lens nuclear factors but had only a marginal effect on basal promoter activity while deletion of the spacer sequences and 3` half-site significantly reduced promoter function(7, 16) . Our mutational analysis also demonstrates that the two A residues at positions -3 and -4 relative to the 3` half-site are crucial for binding activity. The A/T-rich sequences in RORalpha binding sites may allow local distortion of DNA upon binding which may optimize protein:DNA interactions. Consistent with this notion, RORalpha1 has been shown recently to induce a large DNA bend (130°) upon binding to a consensus ROR binding site(15) .

The orphan receptor RORalpha1 activates the F-crystallin promoter in transfected lens epithelial cells without addition of exogenous ligands suggesting either that it functions in a ligand-independent manner or that a putative ligand is present in the culture medium or metabolized by lens cells. The identification of an ROR ligand would be important not just in the regulation of -crystallin genes and lens development but probably in many developmental processes since ROR is widely expressed(27, 28) . Recently, it has been reported that human RZRalpha and RZRbeta (isoforms of ROR) can be activated by melatonin and a synthetic ligand, CGP 52608(29, 30) . Numerous attempts to demonstrate activation of RORalpha1 by melatonin on F-HRE or consensus RORalpha1 binding sites reporter plasmids have failed. (^2)It is clear from our results that additional experiments are needed in order to ascribe a possible role for melatonin in the activation of RORalpha. Nonetheless, the regulation of the F-crystallin promoter by ligand activated transcription factors (7, 8) suggests the possibility that their ligands may constitute the signals originating from retina that control lens development.

We have shown that in the absence of RA, increasing levels of RARalpha blocks RORalpha1 activation of F-HRE reporter genes in a dose-dependent manner. These results are similar to those obtained in our study of the activation of the F-crystallin promoter by T(3)R/RXR heterodimers(8) . Transfected RARalpha potently represses T(3) response from F-HRE reporter plasmids apparently by competition for binding site occupancy. Taken together, these data suggest that RAR/RXR heterodimers exert a dominant role in the regulation of the F-crystallin gene by nuclear receptors. These results contrast to those observed for the interactions of orphan receptors ARP-1/COUP-TFII, EAR3/COUP-TFI, and RXR/RAR on the Oct-3/4 promoter where the orphan receptors completely abolish promoter activation by RXR/RAR heterodimers(31, 32, 33) . These findings clearly demonstrate that the specificity of the retinoid signal is dependent on the configuration of the response element as well as the repertoire of competing orphan receptors.

A number of orphan receptors have been identified which like RORalpha can regulate transcription by binding as monomers to target sequences consisting of a single half-site and a few adjoining bases(14, 28, 34, 35, 36) . Since response elements for RA, T(3), and vitamin D(3) receptors consist of two or more half-sites, it is likely that monomeric-binding orphan receptors interact with many of these response elements possibly modifying hormonal responses(37) . The combined action of RORalpha and related nuclear receptors in the regulation of the F-crystallin gene is likely to be of primary importance in the developmental expression in the lens. The identification of putative RORalpha binding sites in other -crystallin genes (^3)suggests a comprehensive role of this orphan receptor in the regulation of -crystallin gene family.


FOOTNOTES

*
This work was supported by Medical Research Council of Canada Grant MT10561 and National Cancer Institute of Canada Grant 3430 (to V. G.), and by a National Science and Engineering Research Council of Canada Post-doctoral Fellowship (to R. A. F.). 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.

§
Present address: IGBMC, U184 de l'INSERM de Biologie Moléculaire et Génie Génétique, BP163, 67404, Illkirch, Cedex, France.

To whom correspondence should be addressed: Molecular Oncology Group, Royal Victoria Hospital, 687 Pine Avenue West, Montréal, Québec, H3A 1A1, Canada. Tel.: 514-843-1479; Fax: 514-843-1478; vgiguere{at}dir.molonc.mcgill.ca.

(^1)
The abbreviations used are: RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; RORalpha, retinoic acid receptor-related orphan nuclear receptor; F-HRE, F-crystallin hormone response element; T(3), triiodothyronine; T(3)R, T(3) receptor; PCR, polymerase chain reaction; RSV, Rous sarcoma virus; betagal, beta-galactosidase; EMSA, electrophoretic mobility shift assay; CAT, chloramphenicol acetyltransferase; bp, base pair(s); LUC, luciferase; TK, thymidine kinase; RARE, retinoic acid response element; CRBP, cellular retinol binding protein I.

(^2)
M. Tini, I. Dussault, and V. Giguère, unpublished data.

(^3)
M. Tini, unpublished observations.


ACKNOWLEDGEMENTS

We thank David Mangelsdorf for the gift of the mouse RXRbeta cDNA and Robert Sladek for critical reading of the manuscript.


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