The rat TSHβ gene contains distinct response elements for regulation by retinoids and thyroid hormone

Molecular and Cellular Endocrinology 131 (1997) 137 – 146

The rat TSHb gene contains distinct response elements for regulation by retinoids and thyroid hormone Joseph J. Breen a,*, Noreen J. Hickok 1,c, James A. Gurr 2,a,b a Department of Biochemistry, Temple Uni6ersity School of Medicine, Philadelphia, PA 19140, USA Fels Institute for Cancer Research and Molecular Biology, Temple Uni6ersity School of Medicine, Philadelphia, PA 19140, USA c Department of Dermatology and Department of Biochemistry and Molecular Biology, Thomas Jefferson Uni6ersity, Philadelphia, PA 19107, USA

b

Received 22 November 1996; accepted 9 May 1997

Abstract We have previously shown that thyroid stimulating hormone-b (TSHb) mRNA levels are modulated by vitamin A status in vivo and using transient transfection, that suppression of rat TSHb gene promoter activity by all-trans retinoic acid (RA) requires RA receptor (RAR) and retinoid X receptor (RXR). In this paper we have used deletion analysis to delineate the sequences of the rTSHb gene involved in RA regulation, their relationship to the rTSHb gene negative thyroid hormone response elements and the retinoid receptor species that interact with these sequences. Using transient transfection in CV-1 cells, we found that the −204/+9 region of the rat TSHb gene, when fused to a luciferase reporter, was sufficient for suppression by all-trans-RA in the presence of RAR/RXR. Thus, regulation by RA did not involve the major rTSHb negative TRE located between + 15 and +43. Mutational analysis also showed that the minor rTSHb negative TRE between −11 and + 5 was not required by suppression by RA. However, in a heterologous promoter this sequence element acted as a strong positive RARE. The combination of RA and T3 treatment caused synergistic inhibition of rat TSHb gene expression in the presence of RAR/RXR and TR. EMSA analysis demonstrated that the −204/−79 sequence binds RAR/RXR heterodimer. Therefore, we conclude that there are separate response elements for RA and T3 on the rat TSHb gene, that the RARE binds RAR/RXR heterodimer and that RA and T3 interact functionally via these elements in the negative regulation of rat TSHb gene expression. © 1997 Elsevier Science Ireland Ltd. Keywords: Thyroid hormone; Retinoid; Thyrotropin b-subunit; Gene expression; Response element

1. Introduction The pituitary hormone thyrotropin (thyroid stimulating hormone, TSH) is a heterodimer consisting of two

* Corresponding author. Present address: Building 6B Room 4B420, Laboratory of Molecular Genetics National Institute of Child Health and Human Development, Bethesda, MD 20892, USA. Tel.: +1 301 4969689; fax: +1 301 4960243; e-mail: [email protected] 1 Present address: Department of Orthopaedic Research, Thomas Jefferson University, Philadelphia, PA 19107, USA. 2 Present address: Wyeth Ayerst Research, Radnor, PA 19087, USA.

noncovalently bound, glycosylated subunits, a and b, and is a member of the glycoprotein hormone family, which also includes luteinizing hormone, follicle stimulating hormone, and chorionic gonadotropin (Pierce and Parsons, 1981; Shupnik et al., 1989; Magner, 1990). The a subunit is common to each of these hormones whereas the b subunits are unique and confer biological specificity to the dimers. Thyroid hormones (L-triiodothyronine and tetraiodothyronine) are the major regulators of TSH production by the pituitary (Shupnik et al., 1989) and inhibit transcription of the a and TSHb genes (Gurr and Kourides, 1985; Shupnik et al., 1985; Shupnik and Ridgway, 1987).

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Thyroid hormone (T3) acts by binding to nuclear thyroid hormone receptors (TRs). TRs are members of the steroid/thyroid hormone receptor superfamily of ligand-inducible transcription factors which exert their actions by binding to specific hormone response elements (HREs) on target genes (Evans, 1988; Mangelsdorf et al., 1995). There are two families of TRs, a and b, which are derived from separate genes and are expressed in a tissue-specific manner (Yen and Chin, 1994). The consensus DNA target sequences for TRs are closely related to those for the retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which mediate the actions of all-trans-retinoic acid (RA) and 9-cis-retinoic acid (9CRA), respectively (Leid et al., 1992a); both RA and 9CRA are biologically active derivatives of vitamin A. The receptors for RA and 9CRA are each encoded by a family of three closely-related genes and several isoforms are generated in addition by the use of multiple transcription start-sites and alternative RNA splicing (Leid et al., 1992b; Glass, 1994; Gigurere, 1994). The consensus sequences for the binding sites of TRs, RARs, and RXRs consist of 5%-AGGTCA-3% hexamer direct repeats separated by 4, 3 and 5, and 1 nucleotides, respectively (Umesono et al., 1991; Mangelsdorf et al., 1991). However, the receptor specificity of these recognition elements is not absolute and many naturally occurring HREs are complex and contain several consensus-like elements arranged as direct, inverted or everted repeats (Mangelsdorf et al., 1995; Yen and Chin, 1994; Glass, 1994; Gigurere, 1994). In addition, both TRs and RARs form heterodimers with RXRs and these heterodimers very often exhibit a stronger activation of a given TRE or RARE than the corresponding receptor homodimers (Mangelsdorf and Evans, 1995). Together, this degeneracy and the potential for heterodimerization allow an HRE to bind more than one receptor species and facilitates the coordinate regulation of overlapping gene networks. Several examples of natural HREs that are responsive to both T3 and RA have been described (Williams et al., 1992). In current models for the activation of transcription by T3 and RA, interaction of the ligand with TR or RAR in the TR/RXR or RAR/RXR heterodimer bound to the HRE induces the dissociation of a corepressor and facilitates the binding of co-activators, thus stimulating transcription (Horlein et al., 1995; Chen and Evans, 1995). In contrast, the mechanism of negative regulation by T3 and RA remains unclear. Several studies have shown that suppression of TSHb gene transcription by T3 is mediated by a negative TRE (nTRE) that consists of a core of two TRE half-sites surrounding the transcription start-site (Wood et al., 1989; Bodenner et al., 1991; Carr et al., 1992). The

relative importance of each of these core half-sites to inhibition by T3 appears to vary between the rat, mouse, and human genes, possibly due to differences in their sequence context. Thus, in the rat TSHb (rTSHb) gene the downstream TRE (TRE2) predominates over the upstream TRE (TRE1) (Carr et al., 1992) whereas in the human gene the reverse is true (Bodenner et al., 1991). Existing data suggest that the suppression of TSHb gene transcription by T3 is mediated by the binding of TR monomer to TRE half-sites and protein–protein interaction of this monomer with the basal transcription machinery, probably via other cofactors (Fondell et al., 1993; Cohen et al., 1995). We have previously shown that TSHb mRNA levels are elevated in the pituitaries of vitamin A-deficient rats and that rTSHb mRNA levels are rapidly returned to normal by administration of RA (Breen et al., 1995). We also showed, using transient transfection, that the suppression of TSHb gene transcription by RA required both RAR and RXR (Breen et al., 1995). Because of the close similarity between TREs and RAREs, this raised the possibility of a multifunctional HRE on the rTSHb gene. In the present study, we therefore asked whether the rTSHb gene RARE overlaps the TREs which have been previously identified. We have found that, in fact, the rTSHb gene contains a distinct RARE that is located upstream of the known TREs and that binds RAR/RXR heterodimer. However, there is a functional interaction between the RARE and the TREs in that there is synergistic suppression of TSTb promoter activity by RA and T3 in combination in the regulation of TSHb gene transcription.

2. Materials and methods

2.1. Materials Restriction enzymes and DNA-modifying enzymes were purchased from Promega, US Biochemicals, and New England Biolabs and used as recommended by the suppliers. T3 and all-trans-RA were from Sigma. [a32 P]dATP and [ring,3,5-3H]chloramphenicol were obtained from DuPont-New England Nuclear-, [35S]methionine was from Amersham.

2.2. Oligonucleotides Oligonucleotides were synthesized by the Oligonucleotide Synthesis Laboratory at Temple University School of Medicine. To facilitate subcloning and/or labeling, oligonucleotides were synthesized with either BamHI or HindIII cohesive termini.

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2.3. Plasmids The plasmid pTSHb luciferase (pTSHbLUC) was constructed by inserting the EcoRI – PstI fragment of the rTSHb gene (Croyle et al., 1986); (kindly supplied by R.A. Maurer), which contains approximately 0.8 kb of 5%-flanking DNA, exon 1, and 150 bp of intron 1, into the HincII site in the promoterless reporter plasmid pLUCLINK (d’Emden et al., 1992) to direct luciferase expression. The 5% deletion plasmid p( − 520/I150)TSHbLUC was obtained by inserting the HincII–PstI fragment of the rTSHb gene into the HincII site of pLUCLINK. The plasmids p(− 204/ I150)TSHbLUC and p( − 204/ +6)TSHbLUC were constructed by polymerase chain reaction (PCR) amplification of the appropriate gene fragments, using primers containing a 5% BamHI and a 3% HindIII restriction site, followed by insertion of the PCR product between the BamHI and HindIII sites of pLUCLINK. The plasmid p(− 204/ +6tt)TSHbLUC was constructed using overlap extension PCR with mutant primers which introduced a TT mutation into the TRE half-site between − 15 and +9 (see Fig. 1). The expression plasmid pDMTVLUC (kindly supplied by R.M. Evans) contains a portion of the mouse mammary tumor virus (MMTV) promoter with a deletion of five of six glucocorticoid response elements

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(Hollenberg and Evans, 1988). This plasmid contains a unique HindIII site within the MMTV promoter for insertion of heterologous test sequences. Derivatives of pDMTVLUC containing rTSHb gene sequences were obtained by insertion of the appropriate doublestranded oligonucleotide, containing HindIII ends, into the unique HindIII site in pDMTVLUC. The following receptor expression vectors were the kind gift of M.G. Rosenfeld: pRSVRARa and pRSVTRb which express hRARa and hTRb, respectively, under the control of the RSV promoter (Glass et al., 1989); pCMVRXRb, which expresses rRXRb under the control of the CMV promoter (Yu et al., 1991).

2.4. Cell culture and transfection CV-1 cells were obtained from the American Type Culture Collection and were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Cellgro) supplemented with 10% fetal bovine serum (FBS, Sigma), 2 mM glutamine, 100 mg/ml penicillin, and 50 mg/ml streptomycin. Where appropriate, FBS was stripped of thyroid hormones and retinoids by treatment with anion-exchange resin and/or activated charcoal, respectively (Samuels et al., 1979). Forty-eight hours before transfection, cells were replated at a density of 3 × 105 cells per 60 mm plate in DMEM containing complete serum; 24 h before transfection this medium was replaced with DMEM containing stripped serum. Cells were transfected using the calcium-phosphate method (Graham and van der Eb, 1973). Each 60 mm plate received a total of 11 mg of DNA, consisting of 3 mg luciferase reporter plasmid, either 1 mg pRSVRAR, 1 mg pCMVRXR, or both, 3 mg pBlueScriptKSII(+ ) as carrier, and 3 mg pRSVCAT to monitor transfection efficiency. The amount of each viral promoter added to each plate was kept constant by the addition of pRSVneo and/or pCMV-1, as appropriate. After exposure to the precipitate for 18 h, cells were washed and incubated in fresh medium containing stripped serum for a further 30 h. All-transRA, 5×10 − 7 M in ethanol, was added for the final 24 h of the incubation period. In some experiments 100 nM T3 (Sigma), in PBS/10 mM potassium hydroxide, was added for the final 24 h. Control cultures received the appropriate vehicle alone.

2.5. Luciferase and chloramphenicol-acetyl transferase assays Fig. 1. Thyroid hormone response elements in the rat TSHb gene. A, The − 30 to +45 region of the rat TSHb gene. Arrows indicate TRE half-sites, bent arrow indicates the transcription start-site, exon 1 is boxed. B, Oligonucleotide sequences. Arrows denote TRE/RARE half-sites. Lowercase letters show the HindIII restriction sites used to facilitate cloning. − 15/+9, contains sequences − 15 to + 9 of the rat TSHb gene; − 15/+9tt contains a ‘tt’ mutation at positions −9 and − 8;. DR5, synthetic RARE. TREpal, palindromic TRE/RARE.

Extracts from transfected cells were prepared in 500 ml of lysis buffer containing 1% (v/v) Triton X-100, 25 mM glycylglycine, pH 7.8, 15 mM MgSO4, 4 mM EGTA, and 1 mM DTT. Following clarification by centrifugation at 12 000×g for 5 min at 4°C, the supernatant was assayed for luciferase activity (de Wet

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et al., 1987) either immediately or after storage at − 70°C. An aliquot of each extract was also assayed for chloramphenicol-acetyl transferase (CAT) activity using the phase extraction assay (Seed and Sheen, 1988) after incubation at 65°C for 15 min. Luciferase activity was then expressed relative to CAT activity to correct for differences in transfection efficiency between plates.

2.6. Polymerase chain reaction PCR was used to obtain rTSHb gene fragments for subcloning into reporter plasmids. The reaction mixture contained 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 0.2 mM dATP, 1.5 mM MgCl2, 50 pmol each oligonucleotide primer, 10 mM Tris – Cl, pH 8.3, 50 mM KCl, 0.1–1.0 mg substrate DNA, and 2.5 U Amplitaq enzyme (Perkin-Elmer) in a reaction volume of 100 ml. The reaction mixture was covered with an equal volume of mineral oil and the reaction was carried out in a PTC-100 programmable thermal cycler (MJ Research). After one cycle of denaturation at 92°C for 3 min, amplification consisted of 39 cycles of annealing at 55°C for 45 s, extension at 72°C for 45 s and denaturation at 92°C for 20 s. Reactions were kept at 4°C until analysis.

2.7. Electrophoretic mobility shift assay In vitro transcribed and translated receptors were synthesized for use in electrophoretic mobility shift assay (EMSA) using the TNT Coupled Wheat Germ Extract Transcription/Translation system (Promega). The substrate plasmids pKSIITRb (Hodin et al., 1989), pSG5RARa (Zelent et al., 1989), and pKSIIRXRb, were the kind gifts of M. Lazar, P. Chambon, and N. Wong, respectively. To monitor receptor synthesis, reactions containing [35S]methionine (10 mCi/ml, Amersham) were run in parallel with the unlabeled preparative reactions and analyzed by SDS-polyacrylamide gel electrophoresis. Purified recombinant hRARa and hRXRb were obtained from M.G. Rosenfeld. These proteins were produced in E. coli as glutathione-S-transferase (GST) fusion proteins and cleaved with thrombin (Kurokawa et al., 1994) to release RARa and RXRb. Double-stranded oligonucleotide probes, which contained HindIII sites at their 5% and 3% ends, were labeled with [a-32P]dATP using the Klenow fragment of DNA polymerase I, and purified by polyacrylamide gel electrophoresis. The binding reaction was carried out in a volume of 30 ml and contained 3×l05 cpm of oligonucleotide probe, 0.05% Triton X-100, 25 mM HEPES– Cl, pH 7.9, 0.5 mM EDTA, 1 mM DTT, 10% glycerol, 100 mg/ml of poly (dI-dC) (Pharmacia), 2 – 4 ml of in vitro synthesized receptors or 1 – 3 ml of purified E. coli-derived receptors. Reactions were incubated at

room temperature for 30 min and then loaded onto pre-run 5% polyacrylamide/bisacrylamide (29:1) gels in 0.5× TBE (TBE: 90 mM Tris–borate, 2 mM EDTA, pH 8.0) at 4°C. Electrophoresis was at 200 V for 2 h at 4°C. Dried gels were either exposed to Fuji RX X-ray film at − 70°C with 1 intensifying screen for 12–48 h or were exposed to a Fuji PhosphoImager screen for 4–6 h and analyzed using a Fuji BAS2000 PhosphoImager.

3. Results

3.1. The rat TSHb gene RARE is located between − 204 and +9 We have previously shown that a fragment of the rTSHb gene containing 0.8 kb of 5%-flanking DNA, exon 1, and 150 bp of intron 1 is sufficient to mediate suppression of rTSHb promoter activity by RA. To further define the region of the rTSHb gene required for negative regulation by RA, chimeric plasmids were constructed containing portions of this rTSHb gene fragment fused to the luciferase reporter gene. These plasmids were then transiently transfected into CV-1 cells together with plasmids expressing RARa and RXRb and treated with RA (5× l0 − 7 M), or vehicle, for 24 h. The data (Fig. 2) show, firstly, that rTSHb promoter activity was increased by cotransfected RARa and RXRb in the absence of RA with each of the 5%-flanking region deletion plasmids tested. Secondly, RA treatment suppressed expression of p(−800/ I150)TSHbLUC, p(− 520/I150)TSHbLUC, and p(− 204/I150)TSHbLUC by similar amounts (48.2, 41.0, and 31.4%, respectively) compared to expression with unliganded receptors, demonstrating that the region of the rTSHb gene between −800 and − 204 was not required for regulation by RA. In order to determine whether the major rTSHb gene negative TRE (TRE2), located between + 9 and +43 (Fig. 1), was required for regulation by RA, we also tested the response of a construct in which the sequences + 10 to Il50 had been deleted. This construct, p(−204/+ 9)TSHbLUC, was positively regulated by cotransfected, unliganded RARa and RXRb (Fig. 2) and its expression was decreased by 42% by RA treatment (Fig. 2), suggesting that TRE2 is not required for RA regulation. These data are consistent with the presence of a negative RARE in the rTSHb gene between − 204 and +9 that is distinct from the major TRE.

3.2. The negati6e rTSHb gene TRE between − 10 and − 5 can mediate a positi6e response to RA Because of the similarity between RARE and TRE consensus half-site motifs, it was also possible that the

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Fig. 2. Deletion analysis of the RA-responsive region of the rat TSHb gene. DNA constructs, at left, are shown with sequence numbered relative to the rTSHb gene start-site of transcription. Filled triangle, TRE2; open triangle, TRE1; I150, 150 base pairs of intron 1 (exon 1 contains 27 base pairs); LUC, luciferase reporter. CV-1 cells were transfected as described in Section 2 with 3.0 mg of the indicated reporter construct and 1.0 mg each of pRSVRARa plus pCMVRXRb and treated with 5 × 10 − 7 M all-trans RA. All cultures also received pRSVCAT. Luciferase activity in cell extracts was then expressed relative to CAT activity to control for differences in transfection efficiency between plates. The activities of each deletant is expressed relative to basal activity in the absence of RA as 1.0. Values shown are mean9S.E.M. from three to six independent experiments with duplicate transfections per treatment.

rTSHb nTRE between −10 and − 5 (TRE1, Fig. 1) was involved in the suppression of gene activity by RA. To test this possibility, we asked whether this sequence element would mediate suppression by RA when placed in the context of the heterologous MMTV promoter in the plasmid pDMTVLUC. This plasmid contains the promoter region of MMTV from which five of the six glucocorticoid response elements have been removed and a unique HindIII site into which test putative hormone response elements can be inserted (Hollenberg and Evans, 1988). The activity of this plasmid is thus no longer responsive to glucocorticoids. The data in Fig. 3 show that the expression of the parent plasmid pDMTVLUC was not affected by the presence of either unliganded RARa and RXRb or by concomitant RA treatment when transfected into CV-1 cells. However, with pDMTV(−15/ +9)LUC, which contained the TSHb TRE half-site − 10/ − 5, there was a 2.5-fold increase in luciferase activity in the presence of unliganded receptors and a 9-fold increase after RA treatment. To confirm that TRE 1 was mediating the response to RA, the − 10GGGTCA − 5 half-site motif was mutated to − 10GTTTCA − 5 to give the plasmid pDMTV(− 15/+9tt)LUC (see Fig. 1); this mutation has been shown to abolish TR binding to this motif within the growth hormone gene TRE (Glass et al., 1988). As expected, the mutated TRE1 sequence element did not mediate a response to RA (Fig. 3).

The majority of RAREs characterized thus far consist of tandem repeats of a core hexamer motif and bind a receptor dimer. To test the possibility that the −15/ + 9 oligo contained a cryptic half-site downstream of the consensus half-site, we examined the RA-responsiveness of an oligonucleotide containing the truncated TSHb sequence element − 15/+ 1 in the context of the MMTV promoter. Expression of this mutant plasmid, pDMTV(− 15/+ l)LUC, was unresponsive to both unliganded receptors and RA treatment (Fig. 3), suggesting that the + 1 to + 9 sequence contained a binding site for a retinoid receptor or another cofactor involved in RA action. To confirm the conclusion from the data in Fig. 2 that TRE2 did not mediate suppression of rTSHb promoter activity by RA, the response of this sequence element to RA was also tested in the context of the MMTV promoter. As shown in Fig. 3, expression of pDMTV(+ 15/+43)LUC was not affected by either unliganded receptors or RA treatment. In contrast, in other experiments (not shown) we have found that T3 suppresses pDMTV(+ 15/+ 43)LUC expression by 23 and 53% in the presence of TR and TR + RXR, respectively, demonstrating that this sequence element is an effective TRE in our experimental system. Taken together, these data demonstrated that the negative rTSHb TRE, TRE1, acts as a positive RARE in the context of a heterologous promoter and, therefore, suggested that this sequence element did not medi-

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ate, alone, negative regulation of the rTSHb gene by RA.

3.3. The − 15 / +9 sequence does not bind RAR/RXR To determine whether the retinoid responsiveness of the rTSHb − 15/+ 9 sequence element was mediated by binding of RAR and/or RAR/RXR, we used EMSA with in vitro transcribed and translated RARa and RXRb. As a positive control we used an oligonucleotide containing the sequence DR-5, a direct repeat of the consensus 5%-AGGTCA-3% half-site separated by five nucleotides (Fig. 1), that has been shown previously to bind RAR/RXR heterodimer with high affinity (Gigurere, 1994; Umesono et al., 1991; Na¨a¨r et al., 1991). The data in Fig. 4 show, firstly, that the DR-5 probe formed a retarded complex when incubated with in vitro synthesized RARa and RXRb (lane 12) that was effectively competed by a 100-fold molar excess of unlabeled DR-5 oligonucleotide (lane 13) but was not competed by a 100-fold molar excess of chicken b-actin gene oligonucleotide (lane 14). Presumably, this complex arose from binding of RAR/RXR heterodimer to DR5. The complex seen with RAR alone (lane 11) was apparently not receptor-related since it was also obtained when the DR-5 probe was incubated with unprogrammed extract (lane 10). Interestingly, no specific Fig. 4. EMSA analysis of RAR, RXR, and TR binding to rat TSHb −15/ +9 sequence. ln vitro translated RARa, TRb, and/or RXRb (3 ml each), or unprogrammed wheat germ lysate (6 ml) were incubated with 30 000 cpm of either 32P-labeled DR5 probe or 32P-labeled − 15/ + 9 probe and the products were analysed by electrophoresis and autoradiography as described in Section 2. Shown is an autoradiogram that was produced by exposure to Fuji RX X-ray film for 16 h at −70°C with one intensifying screen. Incubations included the receptors indicated above each lane. LYS, unprogrammed lysate; COM, unlabeled competitor: lane 3, 100-fold molar excess of − 15/ +9; lane 4, 100-fold molar excess of b-actin; lane 13, 100-fold molar excess of DR5; lane 14, 100-fold molar excess of b-actin.

Fig. 3. The response of rat TSHb gene sequence to RA in a heterologous promoter context. CV-1 cells were transfected as described in Section 2 with 3.0 mg of either pDMTVLUC, pDMTV(− 15/ +9)LUC, pDMTV( −15/+ 9tt)LUC, pDMTV(− 15/+ l)LUC, or pDMTV( +1 5/+ 43)LUC and 1.0 mg each of pRSVRARa plus pCMVRXRb and treated with 5 × 10 − 7 M all-trans RA, as indicated. Luciferase acitivity in cell extracts was expressed relative to CAT activity from co-transfected pRSVCAT. Activities are expressed relative to each reporter plasmid in the absence of RA as 1.0. Values shown as mean9S.E.M. from three independent experiments each with duplicate transfections per treatment.

complex was formed when the −15/+ 9 sequence was incubated with RARa and RXRb (lane 2), when compared to unprogrammed extract (lane 1). Furthermore, no specific complex was formed when TRb was incubated with the −15/+ 9 sequence either alone (lane 5) or in the presence of RAR (lane 6) or RXR (lane 7). These results suggest that the mechanism by which the − 15/+ 9 sequence of the rTSHb gene acts as a positive RARE in the context of the heterologous MMTV promoter does not involve a simple binding of RAR/ RXR heterodimer to this sequence element.

3.4. The − 10 /− 5 TRE half-site is not essential for the response of the nati6e rTSHb gene to RA Although TRE1 acted as a positive RARE in the context of the heterologous MMTV promoter, it was

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possible that it mediated a negative response to RA when in the context of the native TSHb gene. We therefore examined the effect of mutation of this motif on negative regulation of native rTSHb promoter activity by RA. We compared the effects of RA treatment with cotransfected RARa and RXRb on the expression of the plasmids p( −204/ + 43)TSHbLUC and p(−204/+43tt)TSHbLUC; the latter contained the − 10GGGTCA − 5 to − 10GTTTCA − 5 mutation tested above. The data in Fig. 5 show, firstly, that luciferase expression was increased to a very similar extent in the presence of unliganded RARa and RXRb with both p(− 204/ + 43)TSHbLUC and p(− 204/ + 43tt)TSHbLUC (2.9- and 3.1-fold, respectively). Secondly, the activity of the mutated TSHb promoter was suppressed by RA treatment to a similar extent as the wild-type promoter (18 and 27%, respectively). These data therefore demonstrate that the − 10/− 5 half-site sequence is not essential for regulation of the native rTSHb gene promoter by RA. In these experiments, we also assessed the effects of this mutation on negative regulation by T3. We found that, similar to RA, both stimulation by unliganded TR/RXR (2- and 2.25-fold, respectively) and suppression by T3 (55 and 55%, respectively) were equivalent with p(−204/+43)TSHbLUC and p( − 204/+

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Fig. 6. RA and T3 interact to suppress TSHb gene expression. CV-1 cells were transiently transfected as described in Section 2 with 3.0 mg of p( − 520/I150)TSHbLUC and 1.0 mg each of either pRSVRARa and pCMVRXRb, pRSVTRb and pCMVRXRb, or pRSVRARa, pCMVRXRb and pRSVTRb and treated uith 5 ×10 − 7 M RA and/or 100 nM T3, as indicated. All cultures also received pRSVCAT and luciferase activity in cell extracts was then expressed relative to CAT activity. Activities are expressed relative to p( − 520/ I150)TSHbLUC in the absence of hormone treatment and within each group of transfection conditions as 100%. Values shown are mean9 S.E.M. from three independent experiments with duplicate transfections per treatment.

43tt)TSHbLUC. These data are consistent with those of others suggesting a relatively minor role for TRE1 compared with TRE2 in the negative regulation of rTSHb gene expression by T3 (Carr et al., 1992, 1989).

3.5. Interaction between RA and T3 in suppression of the rTSHb gene

Fig. 5. The − 10/− 5 TRE is not required for response to RA. CV-1 cells were transfected as described in Section 2 with 3.0 mg of either p(− 204/+ 43)TSHbLUC or p(−204/+ 43tt)TSHbLUC and 1.0 mg each of either pRSVRARa and pCMVRXRb or pRSVTRb and pCMVRXRb and treated with 5×10 − 7 M RA or 100 nM T3, as indicated. All cultures also received pRSVCAT and luciferase activity in cell extracts was expressed relative to CAT activity. Activites are expressed relative to the activity of each reporter in the absence of RA or T3 as 1.0. Values are shown as mean9S.E.M. from three independent experiments each with duplicate transfections per treatment.

We next tested whether there was an interaction between regulation by RA and T3 when present in combination or whether they acted independently. We therefore tested the response of p(−520/I1 50)TSHbLUC to RA and T3 alone and in combination in the presence of RARa and RXRb, TRb and RXRb, or RARa, TRb and RXRb. We found, firstly, that luciferase expression was increased in the presence of cotransfected RAR/RXR, TR/RXR, or TR/RAR/ RXR in the absence of ligands but that the increase was smallest when all three receptors were present (2.6-, 2.3-, and 1.9-fold, respectively). Secondly, as before, the activity of the rTSHb promoter was suppressed by treatment with RA (34%) and T3 (32%) individually in the presence of cotransfected RAR/RXR and TR/ RXR, respectively (Fig. 6). Thirdly, suppression by RA in the presence of RAR/RXR was blunted by the

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addition of unliganded TR (34% reduction with RAR/ RXR vs. 12% reduction with TR/RAR/RXR; Fig. 6). In contrast, the action of T3 was unaffected by the presence of unliganded RAR (32% suppression with both TR/RXR and TR/RAR/RXR; (Fig. 6). Finally, the 57% suppression of luciferase expression obtained by treatment with both RA and T3 in the presence of TR/RAR/RXR was greater than the sum of the decreases seen with RA and T3 alone (12 and 32%, respectively; Fig. 6).

3.6. Recombinant RAR/RXR forms a complex with the − 204 / +79 rTSHb gene sequence The − 204/+9 sequence was sufficient to mediate negative regulation of the rTSHb gene promoter by RA but retinoid receptors did not appear to bind to the putative RARE within −15/ +9. This suggested that retinoid receptor binding sites existed upstream of this sequence element. We therefore examined the binding of recombinant RAR and RXR (a kind gift of M.G. Rosenfeld) to the − 204/ +9 sequence by EMSA. As shown in Fig. 7, a DNA-protein complex was formed when labeled −204/ + 9 probe was incubated with RAR and RXR (lane 4) that was competed effectively with an excess of unlabeled probe (lanes 5, 6) but not by actin (lanes 7, 8). Consistent with the idea that this

Fig. 7. Recombinant RAR/RXR forms a complex with the TSHb −204/ − 79 gene sequence. Recombinant RARa and RXRb were produced in E. coli and incubated in the indicated combinations with either 32P-labeled − 204/ +9 or −204/−79 oligonucleotides. EMSA was carried out as descibed in Section 2 and products were analysed by electrophoresis and autoradiography. Lanes 1–8: 32P-labeled − 204/ + 9; lanes 9 and 10: 32P-labeled −204/− 79. COM, unlabeled competitors: lane 5, 10-fold molar excess of − 204/+ 9; lane 6, 50-fold molar excess of − 204/+ 9; lane 7, 10-fold molar excess of b-actin; lane 8, 50-fold molar excess of b-actin.

complex was formed by binding of an RAR/RXR heterodimer to the − 204/+ 9 sequence, no retarded bands were seen following incubation with either RAR (lane 2) or RXR (lane 3) alone. To further localize the putative RARE, the rTSHb sequence element − 204/− 79 was also labeled and incubated with recombinant RAR and RXR. A retarded band consistent with the formation of a receptor/DNA complex was also obtained under these conditions (lane 10), strongly suggesting that the − 204/− 79 sequence contained one or more binding sites for the RAR/RXR heterodimer.

4. Discussion In this paper we have shown that the rTSHb gene contains a negative RARE within its 5%-flanking region that is distinct from the TRE that has previously been identified on this gene. This RARE appears to mediate the suppression of rTSHb gene expression by RA via interaction with RAR/RXR heterodimers. Our data also demonstrate a functional interaction between the RARE and TRE in regulating rTSHb gene expression. We had previously shown using transient transfection that sequences between − 800 and + I150 in the rTSHb gene were essential for inhibition of rTSHb gene transcription by RA (Breen et al., 1995), consistent with the possibility that the major TRE on the rTSHb gene, TRE2, located between + 15 and +43, was also involved in the response to RA. Confirming the assignment of TRE2 as the predominant rTSHb TRE (Carr et al., 1992, 1989), we have found in our experimental system that a construct lacking these sequences, p(− 204/+ 9)TSHbLUC, is unresponsive to T3 and that the + 15/+ 43 sequence element mediates a 53% suppression of luciferase expression when inserted into pDMTVLUC (unpublished results). However, deletion analysis demonstrated that negative regulation by RA in the presence of RAR and RXR was maintained using the sequences − 204/+ 9 (Fig. 2) and that the − 15/+ 43 sequence element was unresponsive to RA in the heterologous MMTV promoter (Fig. 3). Thus, the response of the rTSHb promoter to RA does not appear to require the major rTSHb TRE. This is in contrast to the functional overlap between these two hormone response elements that has been reported for several other genes regulated by both T3 and RA (Williams et al., 1992). Examination of the possible involvement of the upstream rTSHb TRE half-site (TRE1), in RA regulation led to a similar conclusion; since p(−204/+ 43tt)TSHbLUC maintained responsiveness to RA (Fig. 5) and the −15/+ 9 sequence element did not confer a negative RA response on the MMTV promoter (Fig. 3), there was no evidence that the − 15/+ 9 motif func-

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tioned as the TSHb negative RARE. In fact, our data showed that the −15/ + 9 sequence element acted as a strong positive RARE in the context of the MMTV promoter and point mutations implicated the + 1/+ 9 half-site in this response (Fig. 3). Recently, Cohen et al. (1995) have presented evidence that the equivalent halfsite in the human and mouse TSHb promoters acts as a negative TRE by interaction with TR monomer. In contrast, our finding that the − 15/ + 1 truncation mutant does not respond to RA (Fig. 3) suggests that binding of a dimeric receptor species is involved in the positive RA response. Thus, we propose that this nTRE acts as a positive RARE because it interacts with a different form of the different receptors, i.e. dimeric retinoid receptors versus monomeric thyroid hormone receptors. However, the identity of the retinoid receptor species mediating induction by RA is not clear. Although both RXR and RAR were required in the functional assay, we were unable to demonstrate binding of RAR or RXR homodimers or heterodimers to the −15/+ 9 sequence element by EMSA (Fig. 4). One mechanism accounting for this discrepancy would include a co-activator in CV-1 cells that facilitates the binding of RAR/RXR heterodimer to the −15/+ 9 sequence, for example, similar to p140 and p160 (Horlein et al., 1995; Chen and Evans, 1995). Consistent with this idea, preliminary data (not shown) showed that RAR/RXR can form a complex with the − 204/ + 9 sequence in the presence of CV-1 nuclear extracts. It should be noted that the data in Fig. 5 imply that TRE1 mediates less than 10% of suppression of rTSHb promoter activity by T3, consistent with the fact that binding of TR monomer to this motif was not observed using EMSA (Fig. 4). In contrast, this sequence element in the context of the human TSHb gene is a strong nTRE that clearly interacts with TR monomer (Bodenner et al., 1991; Cohen et al., 1995). This difference may be due to differences in the sequences surrounding the core half-site motif in the two genes. In addition, the data in Fig. 5 show that the downstream rTSHb TRE in the context of the rat gene responds to T3 in the presence of both TR and RXR; other experiments showed that suppression of p(−15/ +43)DMTVCAT expression by T3 in the presence of TR was unaffected by addition of RXR. These data contrast with those of Carr and Wong (1994) who found that cotransfection with RXR completely abrogated the suppression of rTSHb promoter activity by T3 and TR. Our findings with the rat gene mirror the situation with the human gene, where RXR alone also has no effect on suppression by T3 and TR; with the human gene, abrogation by RXR of the response to T3 and TR requires the addition of 9CRA (Cohen et al., 1995). The reason for the difference in the effect of unliganded RXR on expression of the rat gene between the two groups is unclear. One possibility is the effect of endogenous

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9CRA in the culture medium. Our medium contained serum which had been charcoal-stripped unlike medium used by Carr and co-workers, where the serum was apparently not treated to remove endogenous retinoids. Neither the functional nor the EMSA data support a role for either TRE1 or TRE2 in suppression of rTSHb promoter activity by RA. However, the − 204/+9 fragment was clearly responsive to RA (Fig. 2), implying that upstream sequences contained the rTSHb gene RARE. In addition, EMSA showed that the − 204/+9 native gene fragment formed a complex when incubated with RAR and RXR but not with either receptor alone (Fig. 7). Computer analysis of the − 204/+ 9 sequence for matches with the consensus half-site sequence 5%AGGTCA-3% identified three potential RARE half-sites in this region, − 191ATGTCA − 186, − 130GGTTCA − 125, and 56AGATCA − 51, in addition to TRE1. Since a DNA/protein complex was also formed between the − 204/− 79 gene fragment and RAR/RXR (Fig. 7), our data implicate half-sites − 186/− 191 and/or − 130/−125 in mediating suppression by RA, although we cannot rule out a role for the − 56/− 51 sequence element. Notably, although suppression by RA and complex formation required both RAR and RXR, no typical heterodimer binding sites consisting of closely spaced half-sites were identified in the − 204/+9 fragment. This suggests either that the putative half-sites form heterodimer binding sites with adjacent cryptic half-sites or that the two half-sites are able to form a heterodimer binding site by being brought together by DNA topology. Our data show that the pathways for the regulation of TSHb gene expression by T3 and RA overlap in several ways. Firstly, they both involve RXR, heterodimer formation by TR and RAR will thus both be dependent on RXR levels, which will in turn respond to their own regulatory signals. Secondly, there is an interaction between the unliganded receptors. The data in Fig. 6 show that unliganded TR abrogated suppression by RA, although unliganded RAR did not appear to affect suppression by T3. Interestingly, these data reflect our earlier findings on regulation of TSHb gene expression by RA in vivo, where we showed that TSHb mRNA levels were not affected by RA treatment in vitamin A-deficient, hypothyroid rats but were suppressed by RA in vitamin A-deficient, euthyroid animals. The data in Fig. 6, when expressed relative to baseline activity in the absence of receptors and ligands, also show that the stimulation of TSHb promoter activity by unliganded TR+ unliganded RAR is significantly reduced compared with that seen with either unliganded receptor individually. Since our results do not support the idea that TR and RXR compete for overlapping binding sites, the interaction between the receptors in modulating TSHb gene transcription seems

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likely to be occurring via protein-protein interactions. Either the receptors contact each other directly or alter each others interaction with one or more components of the transcriptional complex.

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