©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Coordinated Regulation of and Transcriptional Activation by Xenopus Thyroid Hormone and Retinoid X Receptors (*)

(Received for publication, March 29, 1995; and in revised form, May 18, 1995)

Jiemin Wong Yun-Bo Shi (§)

From the Laboratory of Molecular Embryology, National Institutes of Child Health and Human Development/National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Thyroid hormone (T(3)) plays a causative role in amphibian metamorphosis. This regulation is thought to be mediated by heterodimers of T(3) receptors (TRs) and retinoid X receptors (RXRs). We report here that Xenopus TRs can indeed form strong heterodimers with Xenopus RXRs on the T(3) response element (TRE) present in Xenopus TRbeta genes. Using a T(3)-responsive in vivo transcription system established by introducing TRs and RXRs into Xenopus oocytes, we demonstrated that TR-RXR heterodimers repressed TRbeta gene promoter in the absence of T(3) and activated the promoter in the presence of the hormone. Furthermore, by analyzing the expression of TR and RXR genes, we showed that TR and RXR genes were coordinately regulated in different tissues during metamorphosis. Thus high levels of their mRNAs are present in the limb during early stages of limb development when morphogenesis occurs and in the tail toward the end of metamorphosis when it is being resorbed. Such correlations coupled with our TRE-binding and in vivo transcriptional activation experiments provide strong evidence that TRs and RXRs function together to mediate the effects of T(3) during metamorphosis. These results further suggest a possible molecular basis for the temporal regulation of tissue-specific metamorphosis.


INTRODUCTION

Thyroid hormone influences biological processes by regulating gene expression through its nuclear receptors, the thyroid hormone receptors (TRs). (^1)TRs belong to the superfamily of steroid hormone receptors(1, 2, 3) . These receptors contain a highly conserved DNA binding domain consisting of two Zn-fingers that mediate the specific recognition of their respective hormone response elements (e.g. TREs for TRs) located in their target genes. The hormone binding domain is located at the carboxyl-half of the proteins and is unique to each hormone. In the presence of their cognate hormones, these receptors can either activate or repress the transcription of their target genes, depending on the promoter context. Studies in mammals and birds have shown that while TRs can bind to TREs as homodimers and more weakly as monomers, they form strong heterodimers with several other members of the receptor superfamily(3, 4) . In particular, the strongest heterodimers formed are those with receptors for 9-cis-retinoic acid, or retinoid X receptors (RXRs). Furthermore, the heterodimers between TRs and RXRs confer specificity for gene regulation by thyroid hormone in cotransfection experiments in tissue culture cells. However, it remains to be determined whether TR-RXR heterodimers are the active complexes mediating the effect of thyroid hormone in vivo.

We are studying the molecular regulation of amphibian metamorphosis, a process that systematically transforms every single tissue of a tadpole (5, 6) . Although different tissues undergo very different changes, such as the complete resorption of the tail and intestinal remodeling, the entire process is controlled by thyroid hormone. The hormone presumably regulates a cascade of gene expression in each metamorphosing tissues through TRs. Indeed, numerous genes that are regulated directly or indirectly by thyroid hormone have been identified in different tissues of metamorphosing Xenopuslaevis tadpoles(7, 8) . Among these are the two TRalpha and two TRbeta genes themselves. In addition, both TRalpha and TRbeta genes are highly expressed during metamorphosis, suggesting that they participate in the regulation of the process.

We provide here evidence that TRs and RXRs function together to mediate the regulatory effect of thyroid hormone on metamorphosis. First of all, we demonstrate that the Xenopus RXR genes are also expressed during metamorphosis. Furthermore, expression of both TR and RXR genes is highly tissue specific and its regulation in different tissues correlates strongly with tissue-specific transformation. In addition, we show that Xenopus TR-RXR heterodimers can bind to the TRE present in Xenopus TRbeta genes with high specificity and affinity and activate the TRbeta promoter in a hormone-dependent manner.


MATERIALS AND METHODS

RNA Isolation and Analysis

RNA was isolated from whole tadpoles or individually dissected tadpole organs at different developmental stages as described(9) . Total RNA was electrophoresed on a 1% agarose formaldehyde gel and analyzed by Northern blot hybridization. To show that equivalent amounts of total RNA were present, the blots were stained with methylene blue (10) and the same or duplicated blots were hybridized with a cDNA probe for ribosomal protein L8 (rpL8), a gene whose mRNA is maintained at relatively constant levels during development(11) .

Overproduction of TRs and RXRs in Oocytes and Gel Mobility Shift Assays

Although there are two TRalpha (alphaA and alphaB) and two TRbeta (betaA and betaB) genes in Xenopus and each of the TRbeta genes gives rise to two receptor isoforms, the members in each subfamily are highly homologous(13) . Therefore, we chose only TRalphaA and TRbetaA II for the analysis below. pSP64(A)-TRalphaA and pSP64(A)-TRbetaA II for in vitro synthesis of TR mRNAs were kind gifts from Dr. A. Kanamori (Carnegie Institution of Washington, also see (13) ). The cDNAs for the entire coding regions of RXRalpha and RXR (12) were polymerase chain reaction-amplified and cloned into a modified pSP64poly(A) vector (Promega) (^2)using the restriction sites introduced in the polymerase chain reaction primers to generate pSP64(A)-RXRalpha and pSP64(A)-RXR.

The pSP64(A) plasmids containing TR and RXR cDNAs were linearized and transcribed in vitro using a SP6 kit (Ambion). The resulting capped mRNAs were purified and resuspended in water. Each mRNA was injected at a concentration of 100 ng/µl into the cytoplasm of about 30 stage VI Xenopus oocytes (27.6 nl/oocyte) as described (14) . After overnight incubation at 18 °C, the oocytes were collected, rinsed once with modified Barth's medium(14) , and then homogenized in 600 µl of 20 mM Hepes, pH 7.5, 60 mM KCl, 5 mM MgCl(2), 5 mM dithiothreitol, 10% glycerol, 0.1% Nonidet P-40, and 1 mM phenylmethylsulfonyl fluoride. The extracts were centrifuged twice at 12,000 rpm at 4 °C for 15 min to remove yolk proteins and debris.

To determine the relative levels of TRs and RXRs in the oocytes, mRNA for each receptor was coinjected with [S]methionine (1 µCi/µl mRNA) into oocytes. The protein extracts were analyzed by 12% SDS-polyacrylamide gel electrophoresis. The relative levels of unlabeled receptors were analyzed by Western blotting using a chemiluminescence kit from Amersham and polyclonal antibodies against recombinant TRbetaA II and RXRalpha(15) .

To determine the DNA binding activity of TR-RXR heterodimers, 0.5 µl of TR and RXR extracts were first mixed on ice for 10 min. The mixture or individual extracts were then used in the DNA binding and competition experiments as described previously(15) .

Transcriptional Activation of the TRbeta Promoter by TRs and RXRs in Oocytes

A 1.9-kilobase EcoRI fragment from the EcoRI site at -1.6 kilobase of TRbetaA promoter to the EcoRI site at +300 of the CAT reporter gene in pCAT-WT (15) was cloned into pBluescript KS(-) to generate pKS-TRbetaAe. The single-stranded pKS-TRbetaAe was then prepared as described(16) . The single-stranded DNA was injected into the nuclei of the stage VI oocytes at a concentration of 50 ng/µl (23 nl/oocyte). When mRNAs for TRs and/or RXRs were used, they were injected into the cytoplasm (23 nl/oocyte at a total concentration of 10 ng/µl) 2-3 h before the injection of DNA, and the oocytes were incubated overnight in modified Barth's medium with 100 units of ampicilin and streptomycin in the presence or absence of 50 nM thyroid hormone T(3) (3,5,3`-L-triiodothyronine). The injected oocytes(15, 16, 17, 18, 19, 20) were homogenized in 0.25 M Tris-HCl, pH 7.5 (10 µl/oocyte). Half of the extract was transferred into a tube containing 500 µl of RNAzol TM reagent (TEL-TEST, Inc., Friendswood, TX) for RNA isolation and the remaining half was used to isolate DNA(14) . Primer extension with a primer located in the CAT gene was used to analyze the transcripts from the TRbetaA promoter as described(14) . The DNA isolated from the other half of the oocytes was analyzed by slot-blot hybridization to verify the amounts of injected DNA.


RESULTS

Both RXR and TR Genes Are Regulated in a Tissue-dependent Manner during Metamorphosis

The expression of Xenopus RXRalpha and RXR genes was initially examined using RNA from whole animals at various embryonic and metamorphosing stages. The mRNA level for RXRalpha gradually increased as zygotic transcription began after midblastula transition (stage 9) and reached highest values by stage 40, shortly after tadpole hatching at stages 35/36 (Fig. 1). The RXRalpha mRNA was then maintained at relatively constant levels throughout tadpole development and metamorphosis, resembling that of TRalpha mRNA(18) . In contrast, the RXR mRNA levels, which were relatively high in early embryos due to maternal storage(12) , gradually decreased to a minimum around neurula stages (stages 16/17) and was elevated slightly in pre- as well as metamorphosing tadpoles (Fig. 1).


Figure 1: Northern blots showing that Xenopus RXRalpha and RXR are differentially regulated during development. Each lane contained 10 µg of total RNA from whole animals at different stages. Equal loading was confirmed by staining the membrane for total RNA with methylene blue (not shown). The positions of 28 S and 18 S rRNA are indicated.



Although metamorphosis is initiated by the rising concentrations of endogenous thyroid hormone around stage 54, different tissues undergo their specific transformations at very different stages(17) . In particular, hindlimb development begins around stage 54 while tail resorption takes place mostly around stage 62 and later. Reflecting this stage dependence, the levels of at least the TRalpha mRNA correlate with metamorphosis in these different tissues(18, 19, 20, 21) . To investigate whether such regulation exist for other receptors, mRNA levels for RXRalpha, RXR, and TRbeta genes were determined in the tail, hindlimb, and intestine at different stages ( Fig. 2and 3). Like the TRalpha genes, the RXRalpha gene was found to be highly expressed in the tail around stages 62 to 64 when rapid resorption occurred, and in the hindlimb around stages 54 to 56 when limb morphogenesis took place. Very low levels of its mRNA were present in either the tail or the hindlimb at other stages. In the intestine, the RXRalpha mRNA was maintained at moderate levels throughout metamorphosis, again resembling the expression pattern of the TRalpha genes.


Figure 2: Northern blots showing tissue-dependent regulation of RXRalpha gene during metamorphosis. Two µg of total RNA from different organs at the indicated stages per lane were analyzed. The probe rpL8 served as a loading control.



Similarly, both RXR and TRbeta genes were expressed at relatively high levels during tail resorption (stages 62-64, Fig. 3). Unlike the other receptor genes, TRbeta mRNA levels in the hindlimb were low even during morphogenesis (around stage 56). In the intestine, both TRbeta and RXR expression was elevated during remodeling, in contrast to that of the TRalpha and RXRalpha genes. On the other hand, it is worth pointing out that the absolute levels of TRalpha and RXRalpha mRNAs were higher than the corresponding values for TRbeta and RXR mRNAs (data not shown; also see (20) ). However, the overall correlation of the mRNA levels of these genes with tissue-specific transformations strongly suggest that they all participate in metamorphosis.


Figure 3: Coordinated regulation of TR and RXR genes during tissue remodeling. The mRNA levels for TRs and RXRs in tail (A), hindlimb (B), and intestine (C) were determined using a PhosphorImager to quantify the signals from Northern blots similar to those shown in Fig. 2and normalized against rpL8 signals. The mRNA levels for TRalpha and RXRalpha were determined using 2 µg of total RNA. Due to the lower levels of their expression, 10 µg of total RNA per sample was used for the analysis of TRbeta and RXR mRNAs. This made it difficult to determine their expression in the hindlimb at stage 54 when the organ is very small. The plasma T(3) levels were from Leloup and Buscaglia(28) . Low levels of T(3) are likely present around stage 54, as measurable amounts of T(4), the precursor for T(3), is present by this stage(28) .



TRs and RXRs Form Strong Heterodimeric Complexes on the TRE in the TRbeta Genes

We have recently identified a TRE (xTRE) consisting of two near perfect direct repeat of AGGTCA separated by 4 base pairs in both TRbetaA and TRbetaB genes of X. laevis(15) . While little binding of the TRE by TRbeta or RXRalpha produced in E. coli was observed, strong specific complexes were formed in the presence of both TRbeta and RXRalpha. However, most of the proteins made in E. coli were insoluble, making it difficult to determine the relative binding affinity of different heterodimers. We, therefore, turned to Xenopus oocytes to generate functional receptors. When the receptor mRNAs were injected into oocytes, they were efficiently translated. By a combination of in vivo labeling with [S]methionine and Western blotting with anti-Xenopus TR and RXR antibodies, we concluded that within a few folds, the amounts of receptors made were the same when equal amounts of mRNAs were injected into oocytes (Fig. 4). These experiments also showed that there were little endogenous TRs and RXRs in the oocytes even though their mRNAs appeared to be present during oogenesis(12, 19) . Such a conclusion is in agreement with recent results on TR levels in oocytes by Eliceiri and Brown (22) and is also consistent with the DNA binding and transcription experiments described below.


Figure 4: TRs and RXRs are efficiently synthesized when their mRNAs are injected into oocytes. A, equal amounts of TR or RXR mRNA were coinjected with [S]methionine into oocytes and the resulting labeled proteins were analyzed by SDS-polyacrylamide gel electrophoresis. The arrowheads point to the positions of the receptors. B and C, Western blot analysis of S-labeled or unlabeled TR (B) and RXR (C) produced in oocytes. The anti-TRbeta antibody also recognize TRalpha and the anti-RXRalpha antibody does not recognize RXR. The stars in B and C indicate the same samples as used in A. By comparing the S signals and the signals from Western blots, it was concluded that roughly equal amounts of TRs were present in the unlabeled extracts, which were used in the DNA binding experiments ( Fig. 5and Fig. 6).




Figure 5: TRE binding requires the presence of both TRs and RXRs. Extracts from oocytes preinjected with mRNAs for the indicated receptors were tested for their ability to bind the TRE (xTRE) present in the TRbetaA promoter by the gel mobility shift assay. The binding was carried with individual extracts or mixtures of different combinations of TR and RXR extracts.




Figure 6: TRE made of two direct repeats of AGGTCA separated by 4 bp (xTRE) is the preferred binding site by Xenopus TRalpha (upper) or TRbeta (lower) heterodimers with RXRs. Mixtures of oocyte extracts containing the indicated receptors were analyzed for TRE binding. The xTRE were end-labeled and binding was performed in the presence of the indicated amounts (ng) of unlabeled competitors. The unlabeled xTRE itself competed effectively while TREp and TREgh were weaker competitors. The mTRE failed to compete.



When the extracts from the oocytes injected with individual receptor mRNAs were tested for binding to xTRE, no complexes were detected (Fig. 5). However, when the extracts containing TRalpha or TRbeta were mixed with those containing RXRalpha or RXR, the resulting extracts formed strong complexes with xTRE. Different combinations of receptors produced roughly equal binding. The addition of thyroid hormone T(3) had little effect on the amount of complexes formed (not shown). These results indicate that heterodimers formed between any TR and RXR can bind to xTRE with similar affinities.

To test the DNA-binding specificity by TR-RXR heterodimers, competition was performed with various known TREs. All complexes formed between xTRE and different combinations of TR-RXR heterodimers showed identical competition profiles (Fig. 6). The unlabeled xTRE itself competed very efficiently while its mutated version (mTRE), the mutations in which abolish the T(3) dependence of the TRbeta promoter in Xenopus tissue culture cells(15) , failed to compete. In addition, a palindromic TRE made of two inverted repeat of AGGTCA (TREp) and the TRE present in the human growth hormone gene (TREgh), which differs from both TREp and xTRE, competed less efficiently for the complex formation. This same TRE sequence preference has also been found for mammalian TRs(23, 24) , consistent with the high sequence conservation among the mammalian and frog receptors.

Transcriptional Regulation by TRs in Xenopus Oocytes

The fact that there is little TRs and RXRs in oocytes also made it possible to study the function of TRs. We took the advantage that when single-stranded plasmid DNA containing a reporter promoter is injected into oocytes, they undergo one round of replication and the resulting double-stranded DNA is quickly chromatinized to form templates with low levels of basal transcription(14) . Thus, mRNAs of TRs or RXRs were individually or co-injected into oocytes first. Following a few hours of incubation to allow the synthesis of the receptors, single-stranded plasmid DNA containing the T(3)-inducible promoter of TRbetaA gene was injected and the promoter activity was determined by primer extension analysis of the transcribed RNA. The TRbetaA promoter exhibited a weak activity in oocytes in the absence of T(3) and the addition of T(3) had little effect on this activity (Fig. 7A). Identical results were found with oocytes that were preinjected with only RXRalpha and RXR mRNAs. Similarly, when the promoter DNA was injected into oocytes preinjected with only TRalpha or TRbeta mRNA, little or very low levels of activation was observed in the presence of T(3) (Fig. 7A). In contrast, preinjection of various combinations of TR and RXR mRNAs resulted in suppression of the basal promoter expression and the addition of T(3) relieved the suppression and led to strong activation. These results indicate that TR alone is either insufficient to activate the TRE-containing promoter or can only weakly activate the promoter. On the other hand TR-RXR heterodimers are strong activators of the TRE-containing promoter, in agreement with the TRE binding results discussed above.


Figure 7: Transcriptional repression by unliganded TR/RXR heterodimers and activation in the presence of T(3). A, oocytes were preinjected with mRNAs for the individual receptors or combinations of TRs and RXRs. Subsequently, they were injected with a single-stranded plasmid containing the T(3)-inducible promoter of TRbetaA gene and the activity of the promoter in the presence or absence of T(3) was determined by primer extension. B, equal amounts of plasmid DNA were injected into each oocyte sample. DNA was recovered from the same batch of oocytes used for RNA analysis and subjected to slot-blot analysis using P-labeled plasmid probe.



As a control, our Western blot and [S]methionine labeling showed that the amounts of TRs and RXRs synthesized were similar (above). Furthermore, when the injected DNA from the same batch of oocytes used for RNA analysis were extracted and analyzed by hybridization, equal amounts of the plasmid DNA were present in all samples (Fig. 7B). As only the single-stranded plasmid injected into the nucleus would be replicated and protected from degradation, these results indicate that equal amounts of the promoter plasmid were present in all samples. Thus, the results above reflect the true functional difference between the TR-RXR heterodimers and TR or RXR homodimers.


DISCUSSION

Xenopus TRs Form Strong Heterodimers with RXRs and Regulate Transcription in Oocytes

Four TR (two TRalpha and two TRbeta) and two RXR (RXRalpha and RXR) genes have been cloned in X. laevis. We and others have shown that the TRbeta genes are themselves directly regulated by T(3)(15, 25, 26, 29) . Our current work clearly demonstrate that both TRalpha and TRbeta can form strong TRE-binding heterodimers with either RXRalpha or RXR. Furthermore, the TRE sequence preference is identical among different heterodimers and is the same as observed in mammals and birds, i.e. the direct repeats of AGGTCA separated by 4 base pairs being the stronger TRE than either TREp or TREgh.

Using a tissue culture transfection assay, we have shown recently that the TRE in the TRbetaA gene appears to confer the repression of the promoter in the absence of T(3) and the addition of T(3) relieves this repression(15) . Our results here demonstrate that such repression is mediated by TR/RXR heterodimers. Furthermore, the addition of T(3) leads to a strong activation of the promoter. In contrast, TRs alone have little or very small effects on TRbeta promoter activity. This appears to differ from previous reports where cotransfection of a reporter plasmid together with a plasmid to overproduce only TR leads to T(3)-dependent transcription (e.g. see (30) ). However, RXRs and/or other heterodimerization partners for TR seem to be present in tissue culture cells(15, 30) . In addition, our transcription results are consistent with the TRE-binding property of TR homo- and heterodimers. However, it is worth mentioning that although we failed to detect DNA binding by TR monomers or homodimers, such weaker binding might be detectable under different conditions (e.g. see (30) ). The low level of transcriptional activity when TR mRNAs alone were injected into oocytes may be due to either this weaker binding by TRs alone or heterodimers formed by the TRs with the low levels of endogenous RXRs that eluded our Western blot and DNA binding assays. In any case, our results suggest that in developing tadpoles, unliganded TRs can repress TRE-bearing promoters in the absence of T(3) and activate them in the presence of T(3).

Coordinated Regulation of TR and RXR Genes as a Means to Control Temporal Regulation of Tissue-specific Metamorphosis

The interesting regulation of amphibian metamorphosis by thyroid hormone has led to the cloning and analysis of the expression of TR genes in both X. laevis and Rana catesbeiana in several laboratories(18, 19, 20, 21, 31, 32) . We have demonstrated here that the regulation of not only TR but also RXR genes is highly tissue specific. In general, TR and RXR genes are coordinately regulated in different tissues. Currently it is unknown whether mRNA levels reflect protein levels. A recent quantification of TRalpha and TRbeta levels in Xenopus suggest that in general, higher levels of mRNAs result in higher levels of the receptors(22) . The exception is that TRalpha protein levels did not correlate well with the mRNA levels during development in the tail and head, suggesting the existence of mechanisms to regulate translation and/or protein stability. Alternatively, TRalpha could undergo post-translational modifications during development which altered their ability to be immunoprecipitated and detected by Western blot. Thus, it is likely that TR and RXR protein levels are also coordinately regulated.

Amphibian metamorphosis involves systematic transformation of every single organ of a tadpole. Different tissues metamorphose at very different developmental stages. For example, hindlimb morphogenesis takes place very early, around stages 54-56, and tail resorption takes place very late, after stage 60, while intestinal remodeling occurs between stages 58 and 66. Such stage-dependence can be controlled at several levels. First of all, the plasma T(3) concentration peaks around stages 60 to 62 when drastic tissue transformation takes place. Second, we have shown recently that the expression a cytosolic T(3)-binding protein is inversely correlated with tissue metamorphosis, i.e. low levels in the hindlimb during morphogenesis and in the tail during resorption but high levels at other stages(21) . This suggests that the levels of free intracellular T(3) could be regulated in a tissue dependent manner. Related to this, the Xenopus type III iodothyronine 5-deiodinase, which converts T(3) into an inactive form, has also been suggested to play a role in regulating T(3) levels(27) .

Finally the coordinated, tissue-specific temporal regulation of TR and RXR genes are likely to be an important factor in the timing of organ specific metamorphosis. Thus, in the hindlimb around stages 54-56, even though the plasma T(3) levels are low, the high levels of TR and RXR expression could allow the activation of genes important for limb morphogenesis. Subsequently, as the limb undergoes growth and the expression of these genes are no longer required, the reduced expression of TRs and RXRs will lead to the down-regulation of these genes although the plasma T(3) concentrations are even higher. On the other hand, intestinal remodeling takes place from stage 58, when its length begins to shorten, to stage 66, when secondary epithelial cell differentiation is complete. This is the period when high levels of T(3) are present. Furthermore, TRbeta and RXR expression are elevated even though the mRNA levels for TRalpha and RXRalpha do not change appreciably. Finally, in the tail before stage 50, TR and RXR expression is low, this coupled with factors that could reduce free intracellular T(3) concentration, can effectively prevent metamorphosis to occur. After stage 60, the elevated expression of TRs and RXRs then activate the tail resorption process.

Currently, it is unknown how these receptor genes are regulated. Clearly, factors other than the receptors themselves or T(3) levels are involved. This is especially true in premetamorphic tadpoles when there is little T(3). The high levels of TR and RXR mRNAs in the limb but not the tail or intestine in those tadpoles strongly suggest the importance of tissue-specific expression of yet unknown genes in receptor gene regulation. Whatever the underlying mechanism is, the tissue-specific regulation of the receptors and free cellular T(3) levels also provides a molecular explanation for TRbeta gene expression, which is directly regulated by T(3). As TRalpha and RXRalpha are likely the predominant forms of TRs and RXRs at least in premetamorphic tadpoles based on absolute mRNA levels and protein quantification ( (20) and (22) and data not shown), the activation of TRbeta genes are probably mediated mostly by TRalpha-RXRalpha heterodimers. In the intestine, TRalpha and RXRalpha levels appear to be similar at all stages, thus TRbeta expression would be expected to follow the levels of endogenous T(3) as we have observed. In the tail, for the reasons discussed above, the activation of TRbeta genes would be limited to the later period of metamorphosis. Finally, the relatively low levels of TRbeta expression in the hindlimb is due first to insufficient amounts of T(3) and later the down-regulation of TR and RXR genes.

In conclusion, biochemical and tissue culture transfection studies have suggested that TR/RXR heterodimers mediate the effects of T(3)in vivo. However, it has been difficult to prove such a hypothesis as RXRs are likely involved in gene regulation by several different types of hormone receptors. On the other hand, thyroid hormone is the single most important hormone that controls the drastic organ remodeling during amphibian metamorphosis. Our demonstration of coordinated regulation of TR and RXR genes coupled with the functional studies, therefore, provide strong evidence for the importance of TR/RXR heterodimers in vivo.


FOOTNOTES

*
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.

§
To whom correspondence should be addressed. Tel.: 301-402-1004; Fax: 301-402-0078.

^1
The abbreviations used are: TR, thyroid hormone receptor; TRE, thyroid hormone response element; mTRE, mutated TRE; TREgh, TRE present in the human growth hormone gene; TREp, palindromic TRE; RXR, retinoid X receptor or receptor for 9-cis-retinoic acid; T(3), 3,5,3`-L-triiodothyronine or thyroid hormone; CAT, chloramphenicol acetyltransferase.

^2
M. Stolow and Y.-B. Shi, unpublished data.


ACKNOWLEDGEMENTS

We thank Drs. E. De Robertis for the gifts of Xenopus RXR clones and A. Kanamori and D. D. Brown for the gifts of TR cDNA clones. We are also grateful to Dr. A. Wolffe for helpful comments on the manuscript and T. Vo for its preparation.


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