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Functional Interactions between Retinoic Acid Receptor-related Orphan Nuclear Receptor (RORα) and the Retinoic Acid Receptors in the Regulation of the γF-Crystallin Promoter

1995; Elsevier BV; Volume: 270; Issue: 34 Linguagem: Inglês

10.1074/jbc.270.34.20156

1083-351X

M Tini, Robert Fraser, Vincent Giguère,

Estrogen and related hormone effects

We have previously demonstrated that an everted repeat of the hexamer PuGGTCA located within the γF-crystallin promoter mediates activation of the murine γF-crystallin gene by retinoic acid and thyroid hormone receptors. Here, we show that the recently identified retinoic acid receptor-related orphan nuclear receptor (RORα) is expressed in the murine lens and activates the γF-crystallin promoter. In contrast to the retinoic acid and thyroid hormone receptors, activation of the γF-crystallin promoter by RORα requires binding to the single 3′ half-site and spacer sequences of γF-crystallin hormone response element (γF-HRE). We further demonstrate that RORα-dependent activation is repressed by the competitive binding of retinoic acid receptor/retinoid X receptor heterodimers to the γF-HRE in the absence of all-trans-retinoic acid. These studies suggest that the interplay of retinoid receptors and RORα on the γF-HRE may constitute an important mechanism regulating γF-crystallin gene expression in the murine lens. We have previously demonstrated that an everted repeat of the hexamer PuGGTCA located within the γF-crystallin promoter mediates activation of the murine γF-crystallin gene by retinoic acid and thyroid hormone receptors. Here, we show that the recently identified retinoic acid receptor-related orphan nuclear receptor (RORα) is expressed in the murine lens and activates the γF-crystallin promoter. In contrast to the retinoic acid and thyroid hormone receptors, activation of the γF-crystallin promoter by RORα requires binding to the single 3′ half-site and spacer sequences of γF-crystallin hormone response element (γF-HRE). We further demonstrate that RORα-dependent activation is repressed by the competitive binding of retinoic acid receptor/retinoid X receptor heterodimers to the γF-HRE in the absence of all-trans-retinoic acid. These studies suggest that the interplay of retinoid receptors and RORα on the γF-HRE may constitute an important mechanism regulating γF-crystallin gene expression in the murine lens. INTRODUCTIONThe murine γ-crystallin gene locus contains six closely related but differentially regulated genes encoding lens structural proteins that are expressed concomitantly with lens cell differentiation(1Murer-Orlando M. Paterson R.C. Lok S. Tsui L.-C. Breitman M.L. Dev. Biol. 1987; 119: 260-267Crossref PubMed Scopus (37) Google Scholar, 2Goring D.R. Breitman M.L. Tsui L.-C. Exp. Eye Res. 1992; 54: 785-795Crossref PubMed Scopus (46) Google Scholar, 3Breitman M.L. Lok S. Wistow G. Piatigorsky J. Tréton J.A. Gold R.J.M. Tsui L.-C. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7762-7766Crossref PubMed Scopus (68) Google Scholar, 4McAvoy J.W. Differentiation. 1980; 17: 137-149Crossref PubMed Scopus (97) Google Scholar). Lens-specific expression of the γF-crystallin gene is directed by proximal promoter sequences located immediately upstream of the TATA box which are conserved in all γ-crystallin genes, while upstream enhancer sequences are necessary for proper spatial expression within the lens(5Liu Q. Tini M. Tsui L.-C. Breitman M.L. Mol. Cell. Biol. 1991; 11: 1531-1537Crossref PubMed Google Scholar, 6Goring D.R. Bryce D.M. Tsui L.-C. Breitman M.L. Liu Q. Dev. Dyn. 1993; 196: 143-152Crossref PubMed Scopus (30) Google Scholar). We have previously shown that the γF-crystallin promoter is activated by retinoic acid (RA) 1The abbreviations used are: RAretinoic acidRARretinoic acid receptorRXRretinoid X receptorRORαretinoic acid receptor-related orphan nuclear receptorγF-HREγF-crystallin hormone response elementT3triiodothyronineT3RT3 receptorPCRpolymerase chain reactionRSVRous sarcoma virusβgalβ-galactosidaseEMSAelectrophoretic mobility shift assayCATchloramphenicol acetyltransferasebpbase pair(s)LUCluciferaseTKthymidine kinaseRAREretinoic acid response elementCRBPcellular retinol binding protein I. treatment and have characterized a complex hormone response element (γF-HRE) located within the upstream enhancer region(7Tini M. Otulakowski G. Breitman M.L. Tsui L.-T. Giguère V. Genes & Dev. 1993; 7: 295-307Crossref PubMed Scopus (128) Google Scholar). The γF-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(7Tini M. Otulakowski G. Breitman M.L. Tsui L.-T. Giguère V. Genes & Dev. 1993; 7: 295-307Crossref PubMed Scopus (128) Google Scholar). The γF-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 γF-HRE also mediates thyroid hormone (T3) responsiveness of the γF-crystallin promoter(8Tini M. Tsui L.-C. Giguère V. Mol. Endocrinol. 1994; 8: 1494-1506PubMed Google Scholar). Although the γF-HRE is bound by the T3 receptor (T3R) in the form of heterodimers with either RXR or RAR, only T3R•RXR heterodimeric complexes appear to be transcriptionally active. These results show that RARα exerts a dominant role in the regulation of transcription of the γF-crystallin gene and underscore the complexity of the retinoid signal at the level of gene expression (for review, see (9Giguère V. Endocr. Rev. 1994; 15: 61-79Crossref PubMed Google Scholar, 10Chambon P. Semin. Cell Biol. 1994; 5: 115-125Crossref PubMed Scopus (499) Google Scholar, 11Mangelsdorf D.J. Umesono K. Evans R.M. Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids. 2nd Ed. Raven Press, New York1994: 319-349Google Scholar)).The retinoid and T3 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(12Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6290) Google Scholar). These proteins are referred to as orphan receptors. We have recently identified a novel family of orphan receptors (RORα, 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 (13Giguère V. Tini M. Flock G. Ong E.S. Evans R.M. Otulakowski G. Genes & Dev. 1994; 8: 538-553Crossref PubMed Scopus (448) Google Scholar). RORα 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(13Giguère V. Tini M. Flock G. Ong E.S. Evans R.M. Otulakowski G. Genes & Dev. 1994; 8: 538-553Crossref PubMed Scopus (448) Google Scholar, 14Giguère V. McBroom L.D.B. Flock G. Mol. Cell. Biol. 1995; 15: 2517-2526Crossref PubMed Google Scholar, 15McBroom L.D.B. Flock G. Giguère V. Mol. Cell. Biol. 1995; 15: 796-808Crossref PubMed Google Scholar). We have shown that the γF-HRE is bound by RORα1 and acts as a strong HRE for this orphan receptor(13Giguère V. Tini M. Flock G. Ong E.S. Evans R.M. Otulakowski G. Genes & Dev. 1994; 8: 538-553Crossref PubMed Scopus (448) Google Scholar). In this report, we have investigated more closely the interaction of RORα with the γF-HRE and have established that RORα can activate the γF-crystallin promoter in primary chick lens cell cultures. We demonstrate that RAR/RXR heterodimers compete with RORα for occupancy of the γF-HRE and that, in the absence of RA, the retinoid receptor complexes block RORα activation. Since the retinoid receptors and RORα are expressed in the lens, we suggest that both classes of receptors are likely to play important roles in the regulation of the γF-crystallin gene.EXPERIMENTAL PROCEDURESReverse Transcriptase-PCRTotal 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 × 106 cpm of [α- 32P]dCTP (Amersham)-labeled (Quick Prime, Pharmacia Biotech Inc.) 107 bp of XhoI/XcmI human RORα 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 TransfectionThe γF-crystallin and TREpalTKLUC reporter plasmids have been previously described(16Lok S. Stevens W. Breitman M.L. Tsui L.-C. Nucleic Acids Res. 1989; 17: 3563-3582Crossref PubMed Scopus (41) Google Scholar, 17Umesono K. Giguère V. Glass C.K. Rosenfeld M.G. Evans R.M. Nature. 1988; 336: 262-265Crossref PubMed Scopus (424) Google Scholar). γF-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(7Tini M. Otulakowski G. Breitman M.L. Tsui L.-T. Giguère V. Genes & Dev. 1993; 7: 295-307Crossref PubMed Scopus (128) Google Scholar). The equivalent of five lenses was plated on 60-mm culture dishes coated with collagen. Cells were transfected with 10 μg of γF-crystallin reporter construct and 1 μg of plasmid RSV-βgal, and 0.5-1.0 μg of RSV LTR-based expression vectors directing the synthesis of either the human RORα1 or RARα1. P19 and CV-1 cells were cultured on α-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-βgal, and 100 to 500 ng of appropriate expression vector. Transfected cells were treated for 20 to 48 h with appropriate ligands. β-Galactosidase and luciferase assays were carried as described elsewhere(18Giguère V. Shago M. Zirngibl R. Tate P. Rossant J. Varmuza S. Mol. Cell. Biol. 1990; 10: 2335-2340Crossref PubMed Scopus (145) Google Scholar). CAT assays were performed using equivalent amounts of β-galactosidase activity using a non-TLC method as described by Amersham.In Vitro Translation and EMSAPlasmid pCMXhRARα (19Umesono K. Murakami K.K. Thompson C.C. Evans R.M. Cell. 1991; 65: 1255-1266Abstract Full Text PDF PubMed Scopus (1487) Google Scholar) containing the human RARα1 cDNA(20Giguère V. Ong S.E. Segui P. Evans R.M. Nature. 1987; 330: 624-629Crossref PubMed Scopus (1524) Google Scholar), and plasmid pSKmRXRβ (21Mangelsdorf D.J. Ong E.S. Dyck J.A. Evans R.M. Nature. 1990; 345: 224-229Crossref PubMed Scopus (1251) Google Scholar) containing the mouse RXRβ were linearized with BamHI and AccI, respectively. Plasmid pSKhR5 containing the human RORα1 cDNA was linearized with BamHI. Capped mRNAs were synthesized in vitro using linearized plasmids and T7 RNA polymerase for RORα1 and RARα, while RXRβ mRNA was synthesized with T3 RNA polymerase. These mRNAs were used to synthesize RORα1, RARα1, and RXRβ protein in vitro using rabbit reticulocyte lysates (Promega). EMSAs were performed as described previously(7Tini M. Otulakowski G. Breitman M.L. Tsui L.-T. Giguère V. Genes & Dev. 1993; 7: 295-307Crossref PubMed Scopus (128) Google Scholar). The amount of reticulocyte lysate in each binding reaction was kept constant within a given experiment.RESULTSRORα Is Expressed in the Murine Lens and Activates the γF-Crystallin PromoterTo establish whether RORα 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 RORα-specific primer. The products of cDNA synthesis were amplified using RORα-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′-RORα 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 RORα transcripts in the lens. An additional smaller band was also detected in cDNA primed with a 3′-RORα oligonucleotide which may indicate the presence of different isoforms of RORα in the lens. Bands were not observed when cDNA synthesis was primed with a 5′ RORα 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 RORα probe corresponding to sequences contained within the amplified fragment (Fig. 1).The 5′-flanking sequences of the murine γF-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(6Goring D.R. Bryce D.M. Tsui L.-C. Breitman M.L. Liu Q. Dev. Dyn. 1993; 196: 143-152Crossref PubMed Scopus (30) Google Scholar, 16Lok S. Stevens W. Breitman M.L. Tsui L.-C. Nucleic Acids Res. 1989; 17: 3563-3582Crossref PubMed Scopus (41) Google Scholar). The γF-HRE (located at −210 to −185) mediates activation by both RAR and T3R and also contributes to basal promoter activity since deletion of the 3′ half-site and spacer sequences (−202 to −185) decreases promoter function(7Tini M. Otulakowski G. Breitman M.L. Tsui L.-T. Giguère V. Genes & Dev. 1993; 7: 295-307Crossref PubMed Scopus (128) Google Scholar, 8Tini M. Tsui L.-C. Giguère V. Mol. Endocrinol. 1994; 8: 1494-1506PubMed Google Scholar, 16Lok S. Stevens W. Breitman M.L. Tsui L.-C. Nucleic Acids Res. 1989; 17: 3563-3582Crossref PubMed Scopus (41) Google Scholar). In contrast, mutation of the first half-site eliminates RA response but has minimal effects on basal promoter activity(7Tini M. Otulakowski G. Breitman M.L. Tsui L.-T. Giguère V. Genes & Dev. 1993; 7: 295-307Crossref PubMed Scopus (128) Google Scholar). These data suggest that there are endogenous transcription factors in lens cells that specifically recognize the downstream half-site and spacer of γF-HRE to stimulate basal promoter activity. The orphan receptor RORα interacts with a single PuGGTCA motif preceded by A/T-rich sequences and binds to a number of characterized response elements including the γF-HRE(13Giguère V. Tini M. Flock G. Ong E.S. Evans R.M. Otulakowski G. Genes & Dev. 1994; 8: 538-553Crossref PubMed Scopus (448) Google Scholar). To determine whether RORα could activate the γF-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 γF-crystallin promoter (−226 to +47). A mutant reporter construct containing a deletion of spacer sequences and the 3′ half-site of γF-HRE (Δ−202/-185) was also tested. Transfection of RORα1 expression vector increased wild-type promoter activity approximately 3.8-fold, while the Δ−202/-185 promoter mutant displayed reduced basal promoter activity and was not significantly activated by transfected RORα1 (Fig. 2).Figure 2:RORα1 activates the γF-crystallin promoter. Chick primary lens cultures were transfected with 10 μg of CAT gene reporter plasmids containing either wild type γF-crystallin 5′-flanking sequence −226 to + 47 (γF(-226/+47)) or an equivalent segment containing a deletion of the γF-HRE 3′ half-site and spacer sequences (Δ -202/-185). 500 ng of an expression vector containing the human RORα1 cDNA under the control of the RSV-LTR were cotransfected together with 1 μg of RSV-βgal 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)RORα1 Selectively Activates the γF-HREWe and others have shown that human RORα1 and rat RZRβ(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)(13Giguère V. Tini M. Flock G. Ong E.S. Evans R.M. Otulakowski G. Genes & Dev. 1994; 8: 538-553Crossref PubMed Scopus (448) Google Scholar, 22Carlberg C. van Huijsduijnen R. Staple J.K. DeLamarter J.F. Becker-André M. Mol. Endocrinol. 1994; 8: 757-770Crossref PubMed Scopus (191) Google Scholar). We have determined the relative binding affinity of RORα1 for these elements by performing competition studies using EMSA (Fig. 3B). Incubation of radioactively labeled γF-HRE oligonucleotide with in vitro synthesized RORα1 generates a single complex. Inclusion in the binding reactions of different molar excesses of response elements indicated that RORα binds with highest affinity to γF-HRE and TREpal. CRBPI-RARE competed poorly while βRARE failed to compete, indicating that RORα1 does not bind with high affinity to these elements.Figure 3:Competition analysis of hormone response elements for binding of RORα1. A, sequences of response elements used in this study. TREpal is an idealized response element derived from the rat growth hormone gene(38Glass C.K. Holloway J.M. Devary O.V. Rosenfeld M.G. Cell. 1988; 54: 313-323Abstract Full Text PDF PubMed Scopus (462) Google Scholar), βRARE was identified in the promoter of the human RARβ2 gene(39de Thé H. del Mar Vivanco-Ruiz M. Tiollais P. Stunneberg H. Dejean A. Nature. 1990; 343: 177-180Crossref PubMed Scopus (840) Google Scholar), CRBPI-RARE was identified in the mouse cellular retinol binding protein I gene(40Smith W.C. Nakshatri H. Leroy P. Rees J. Chambon P. EMBO J. 1991; 10: 2223-2230Crossref PubMed Scopus (189) Google Scholar). B, radiolabeled γF-HRE was incubated with reticulocyte lysate (2 μl) programmed with human RORα1 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next compared the transcriptional activity of RORα1 on γF-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 γF-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 γF-HRE reporter could be activated by transfected RORα1.Figure 4:RORα1 is transcriptionally inactive on TREpal. Reporter plasmids (2 μg/plate) containing either the γF-HRE or TREpal in front of the thymidine kinase promoter linked to the luciferase reporter gene were transfected into P19 cells together with RORα1 expression vector (0.5 μg/plate) and plasmid RSV-βgal. 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Spacer Region and Downstream Half-site of γF-HRE Are Sufficient for High Affinity Binding of RORα1To determine which half-site of γF-HRE is occupied by RORα1 we tested two oligonucleotides (m1 and m2) containing either the 5′ or 3′ half-site and spacer sequences for binding of RORα1 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 RORα. This conclusion is supported by transfection studies indicating that reporter plasmids containing two copies of either the intact γF-HRE or the 3′ half-site and spacer sequences are transactivated with the same efficiency by RORα1 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 RORα1. 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 RORα1 expression vectors (500 ng/plate). The mean values of three independent experiments are plotted with the standard error indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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 RORα1. As expected from previous analysis of the RORα1 binding properties(13Giguère V. Tini M. Flock G. Ong E.S. Evans R.M. Otulakowski G. Genes & Dev. 1994; 8: 538-553Crossref PubMed Scopus (448) Google Scholar), 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(13Giguère V. Tini M. Flock G. Ong E.S. Evans R.M. Otulakowski G. Genes & Dev. 1994; 8: 538-553Crossref PubMed Scopus (448) Google Scholar, 14Giguère V. McBroom L.D.B. Flock G. Mol. Cell. Biol. 1995; 15: 2517-2526Crossref PubMed Google Scholar), 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 RORα1. Mutant oligonucleotides (A) were used as competitors in binding reactions (B and C). Each composite was derived from the same experiment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)RAR/RXR Heterodimers Repress RORα-dependent Activation by Competing for Occupancy of the γF-HREWe have previously shown that primary cultures of chick lens cells are responsive to RA indicating the presence of endogenous retinoid receptors(7Tini M. Otulakowski G. Breitman M.L. Tsui L.-T. Giguère V. Genes & Dev. 1993; 7: 295-307Crossref PubMed Scopus (128) Google Scholar). Furthermore, mice disrupted for RAR α and γ genes display extensive ocular defects including lens fiber abnormalities (23Lohnes D. Mark M. Mendelsohn C. Dollé P. Dierich A. Gorry P. Gansmuller A. Chambon P. Development. 1994; 120: 2723-2748Crossref PubMed Google Scholar). These studies provide evidence for a role of RARs in lens and eye development. In P19 cells, combined transfection of RORα1 and RA treatment results in greater levels of transactivation of the γF-HRE suggesting that retinoid receptors and RORα cooperate in transcriptional regulation (Fig. 4).To investigate the functional relationship between retinoid receptors and RORα 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 γF-HRE was activated approximately 20-fold by transfected RORα1 (Fig. 7A). Additional RA-dependent stimulation was not observed unless RAR expression vector was cotransfected. In the absence of RA, transfected RAR represses RORα mediated activation in a dose-dependent manner. Thus, RAR can either repress RORα 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 RORα for occupancy of γF-HRE. To demonstrate this we performed EMSA with a limiting concentration of radiolabeled γF-HRE, a constant amount of RAR and RXR and increasing amounts of RORα. An RORα mutant with truncated carboxyl terminus (RORαΔ235) 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 RORαΔ235 in the binding reaction causes a dose-dependent decrease in the formation of RAR•RXR complexes. Consistent with these results, co-transfection of RORαΔ235 which is transcriptionally inactive blocks both RA- and RORα-dependent activation on γF-HRE reporter plasmids (data not shown).Figure 7:Retinoid receptors and ROR compete for binding to γF-HRE. A, a constant amount of RORα1 expression vector (1 μg) was transfected with the indicated amounts of RARα expression vector. A luciferase reporter plasmid was used containing three copies of the γF-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 (RORΔ235). 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 RORΔ235 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONDuring lens development, differentiation of epithelial cells into fiber cells is regulated by factors secreted from the retina(24Coulombre J. Coulombre A. Science. 1963; 142: 1489-1494Crossref PubMed Scopus (171) Google Scholar, 25Reyer R.W. Exp. Eye Res. 1977; 24: 501-509Crossref PubMed Scopus (7) Google Scholar, 26Yamamoto Y. Dev. Growth Diff. 1976; 18: 273-278Crossref Scopus (54) Google Scholar). The γF-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 T3 activate γF-crystallin gene expression through a novel HRE (γF-HRE) located in the upstream enhancer element (−210 to −185) of the γF-crystallin promoter (7Tini M. Otulakowski G. Breitman M.L. Tsui L.-T. Giguère V. Genes & Dev. 1993; 7: 295-307Crossref PubMed Scopus (128) Google Scholar, 8Tini M. Tsui L.-C. Giguère V. Mol. Endocrinol. 1994; 8: 1494-1506PubMed Google Scholar) and proposed that RA may serve as a regulatory signal in lens development. This suggestion is supported by the analysis of mice bearing disrupted RARα and γ genes which display severe ocular defects including abnormal lens fibers and agenesis of the lens(23Lohnes D. Mark M. Mendelsohn C. Dollé P. Dierich A. Gorry P. Gansmuller A. Chambon P. Development. 1994; 120: 2723-2748Crossref PubMed Google Scholar). In the present study, we report the regulation of the γF-crystallin gene by RORα1, an orphan member of the superfamily of steroid-thyroid-retinoid receptors, via interactions with the γF-HRE. We further demonstrate that constitutive activation of the γF-HRE by RORα1 is repressed by the competitive binding of RAR/RXR heterodimeric complexes to the element in the absence of RA.The γF-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, T3R, and RXR and these binding activities require that both half-sites be intact(7Tini M. Otulakowski G. Breitman M.L. Tsui L.-T. Giguère V. Genes & Dev. 1993; 7: 295-307Crossref PubMed Scopus (128) Google Scholar, 8Tini M. Tsui L.-C. Giguère V. Mol. Endocrinol. 1994; 8: 1494-1506PubMed Google Scholar). In contrast, the 3′ half-site and six adjacent nucleotides (TTAACCAGGTCA) of the γF-HRE are sufficient for proper recognition by RORα1. Since transcriptional activation by transfected RORα1 is constitutive, these findings are consistent with the mutational analyses of the γF-crystallin promoter. Specifically, previous experiments indicated that alterations of the 5′ half-site of the γF-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(7Tini M. Otulakowski G. Breitman M.L. Tsui L.-T. Giguère V. Genes & Dev. 1993; 7: 295-307Crossref PubMed Scopus (128) Google Scholar, 16Lok S. Stevens W. Breitman M.L. Tsui L.-C. Nucleic Acids Res. 1989; 17: 3563-3582Crossref PubMed Scopus (41) Google Scholar). 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 RORα binding sites may allow local distortion of DNA upon binding which may optimize protein:DNA interactions. Consistent with this notion, RORα1 has been shown recently to induce a large DNA bend (∼130°) upon binding to a consensus ROR binding site(15McBroom L.D.B. Flock G. Giguère V. Mol. Cell. Biol. 1995; 15: 796-808Crossref PubMed Google Scholar).The orphan receptor RORα1 activates the γF-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 γ-crystallin genes and lens development but probably in many developmental processes since ROR is widely expressed(27Becker-André M. André E. DeLamarter J.F. Biochem. Biophys. Res. Commun. 1993; 194: 1371-1379Crossref PubMed Scopus (230) Google Scholar, 28Retnakaran R. Flock G. Giguère V. Mol. Endocrinol. 1994; 8: 1234-1244Crossref PubMed Scopus (84) Google Scholar). Recently, it has been reported that human RZRα and RZRβ (isoforms of ROR) can be activated by melatonin and a synthetic ligand, CGP 52608(29Wiesenberg I. Missbach M. Kahlen J.P. Schrader M. Carlberg C. Nucleic Acids Res. 1995; 23: 327-333Crossref PubMed Scopus (239) Google Scholar, 30Becker-André M. Wiesenberg I. Schaeren-Wiemers N. André E. Missbach M. Saurat J.-H. Carlberg C. J. Biol. Chem. 1994; 269: 28531-28534Abstract Full Text PDF PubMed Google Scholar). Numerous attempts to demonstrate activation of RORα1 by melatonin on γF-HRE or consensus RORα1 binding sites reporter plasmids have failed. 2M. Tini, I. Dussault, and V. Giguère, unpublished data. It is clear from our results that additional experiments are needed in order to ascribe a possible role for melatonin in the activation of RORα. Nonetheless, the regulation of the γF-crystallin promoter by ligand activated transcription factors (7Tini M. Otulakowski G. Breitman M.L. Tsui L.-T. Giguère V. Genes & Dev. 1993; 7: 295-307Crossref PubMed Scopus (128) Google Scholar, 8Tini M. Tsui L.-C. Giguère V. Mol. Endocrinol. 1994; 8: 1494-1506PubMed Google Scholar) 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 RARα blocks RORα1 activation of γF-HRE reporter genes in a dose-dependent manner. These results are similar to those obtained in our study of the activation of the γF-crystallin promoter by T3R/RXR heterodimers(8Tini M. Tsui L.-C. Giguère V. Mol. Endocrinol. 1994; 8: 1494-1506PubMed Google Scholar). Transfected RARα potently represses T3 response from γF-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 γF-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(31Ben-Shushan E. Sharir H. Pikarski E. Bergman Y. Mol. Cell. Biol. 1995; 15: 1034-1048Crossref PubMed Google Scholar, 32Sylvester I. Schöler H.R. Nucleic Acids Res. 1994; 22: 901-911Crossref PubMed Scopus (73) Google Scholar, 33Schoorlemmer J. van Puijenbroek A. van den Eijnden M. Jonk L. Pals C. Kruijer W. Mol. Cell. Biol. 1994; 14: 1122-1136Crossref PubMed Scopus (91) Google Scholar). 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 RORα can regulate transcription by binding as monomers to target sequences consisting of a single half-site and a few adjoining bases(14Giguère V. McBroom L.D.B. Flock G. Mol. Cell. Biol. 1995; 15: 2517-2526Crossref PubMed Google Scholar, 28Retnakaran R. Flock G. Giguère V. Mol. Endocrinol. 1994; 8: 1234-1244Crossref PubMed Scopus (84) Google Scholar, 34Wilson T.E. Fahrner T.J. Johnson M. Milbrandt J. Science. 1991; 252: 1296-1300Crossref PubMed Scopus (479) Google Scholar, 35Wilson T.E. Fahrner T.J. Milbrandt J. Mol. Cell. Biol. 1993; 13: 5794-5804Crossref PubMed Scopus (356) Google Scholar, 36Harding H.P. Lazar M.A. Mol. Cell. Biol. 1993; 13: 3113-3121Crossref PubMed Google Scholar). Since response elements for RA, T3, and vitamin D3 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(37Carter M.E. Gulick T. Raisher B.D. Caira T. Ladias J.A.A. Moore D.D. Kelly D.P. J. Biol. Chem. 1993; 268: 13805-13810Abstract Full Text PDF PubMed Google Scholar). The combined action of RORα and related nuclear receptors in the regulation of the γF-crystallin gene is likely to be of primary importance in the developmental expression in the lens. The identification of putative RORα binding sites in other γ-crystallin genes 3M. Tini, unpublished observations. suggests a comprehensive role of this orphan receptor in the regulation of γ-crystallin gene family. INTRODUCTIONThe murine γ-crystallin gene locus contains six closely related but differentially regulated genes encoding lens structural proteins that are expressed concomitantly with lens cell differentiation(1Murer-Orlando M. Paterson R.C. Lok S. Tsui L.-C. Breitman M.L. Dev. Biol. 1987; 119: 260-267Crossref PubMed Scopus (37) Google Scholar, 2Goring D.R. Breitman M.L. Tsui L.-C. Exp. Eye Res. 1992; 54: 785-795Crossref PubMed Scopus (46) Google Scholar, 3Breitman M.L. Lok S. Wistow G. Piatigorsky J. Tréton J.A. Gold R.J.M. Tsui L.-C. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7762-7766Crossref PubMed Scopus (68) Google Scholar, 4McAvoy J.W. Differentiation. 1980; 17: 137-149Crossref PubMed Scopus (97) Google Scholar). Lens-specific expression of the γF-crystallin gene is directed by proximal promoter sequences located immediately upstream of the TATA box which are conserved in all γ-crystallin genes, while upstream enhancer sequences are necessary for proper spatial expression within the lens(5Liu Q. Tini M. Tsui L.-C. Breitman M.L. Mol. Cell. Biol. 1991; 11: 1531-1537Crossref PubMed Google Scholar, 6Goring D.R. Bryce D.M. Tsui L.-C. Breitman M.L. Liu Q. Dev. Dyn. 1993; 196: 143-152Crossref PubMed Scopus (30) Google Scholar). We have previously shown that the γF-crystallin promoter is activated by retinoic acid (RA) 1The abbreviations used are: RAretinoic acidRARretinoic acid receptorRXRretinoid X receptorRORαretinoic acid receptor-related orphan nuclear receptorγF-HREγF-crystallin hormone response elementT3triiodothyronineT3RT3 receptorPCRpolymerase chain reactionRSVRous sarcoma virusβgalβ-galactosidaseEMSAelectrophoretic mobility shift assayCATchloramphenicol acetyltransferasebpbase pair(s)LUCluciferaseTKthymidine kinaseRAREretinoic acid response elementCRBPcellular retinol binding protein I. treatment and have characterized a complex hormone response element (γF-HRE) located within the upstream enhancer region(7Tini M. Otulakowski G. Breitman M.L. Tsui L.-T. Giguère V. Genes & Dev. 1993; 7: 295-307Crossref PubMed Scopus (128) Google Scholar). The γF-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(7Tini M. Otulakowski G. Breitman M.L. Tsui L.-T. Giguère V. Genes & Dev. 1993; 7: 295-307Crossref PubMed Scopus (128) Google Scholar). The γF-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 γF-HRE also mediates thyroid hormone (T3) responsiveness of the γF-crystallin promoter(8Tini M. Tsui L.-C. Giguère V. Mol. Endocrinol. 1994; 8: 1494-1506PubMed Google Scholar). Although the γF-HRE is bound by the T3 receptor (T3R) in the form of heterodimers with either RXR or RAR, only T3R•RXR heterodimeric complexes appear to be transcriptionally active. These results show that RARα exerts a dominant role in the regulation of transcription of the γF-crystallin gene and underscore the complexity of the retinoid signal at the level of gene expression (for review, see (9Giguère V. Endocr. Rev. 1994; 15: 61-79Crossref PubMed Google Scholar, 10Chambon P. Semin. Cell Biol. 1994; 5: 115-125Crossref PubMed Scopus (499) Google Scholar, 11Mangelsdorf D.J. Umesono K. Evans R.M. Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids. 2nd Ed. Raven Press, New York1994: 319-349Google Scholar)).The retinoid and T3 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(12Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6290) Google Scholar). These proteins are referred to as orphan receptors. We have recently identified a novel family of orphan receptors (RORα, 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 (13Giguère V. Tini M. Flock G. Ong E.S. Evans R.M. Otulakowski G. Genes & Dev. 1994; 8: 538-553Crossref PubMed Scopus (448) Google Scholar). RORα 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(13Giguère V. Tini M. Flock G. Ong E.S. Evans R.M. Otulakowski G. Genes & Dev. 1994; 8: 538-553Crossref PubMed Scopus (448) Google Scholar, 14Giguère V. McBroom L.D.B. Flock G. Mol. Cell. Biol. 1995; 15: 2517-2526Crossref PubMed Google Scholar, 15McBroom L.D.B. Flock G. Giguère V. Mol. Cell. Biol. 1995; 15: 796-808Crossref PubMed Google Scholar). We have shown that the γF-HRE is bound by RORα1 and acts as a strong HRE for this orphan receptor(13Giguère V. Tini M. Flock G. Ong E.S. Evans R.M. Otulakowski G. Genes & Dev. 1994; 8: 538-553Crossref PubMed Scopus (448) Google Scholar). In this report, we have investigated more closely the interaction of RORα with the γF-HRE and have established that RORα can activate the γF-crystallin promoter in primary chick lens cell cultures. We demonstrate that RAR/RXR heterodimers compete with RORα for occupancy of the γF-HRE and that, in the absence of RA, the retinoid receptor complexes block RORα activation. Since the retinoid receptors and RORα are expressed in the lens, we suggest that both classes of receptors are likely to play important roles in the regulation of the γF-crystallin gene.