Thyroid Response Element Research Articles

Differential responses to ligands of overexpressed thyroid hormone and retinoid X receptors in a Xenopus cell line and in vivo

In an attempt to explain the contrasting patterns of expression of Xenopus thyroid hormone (xTR) and retinoid X (xRXR) receptor genes and to extend our understanding of the role of heterodimerization of these receptors during amphibian metamorphosis, we have investigated the response to their respective ligands of cells in which xTR and xRXR were overexpressed. Results obtained with two separate approaches are now described. In the first, 3,3′,5-triiodothyronine (T 3) was found to strongly upregulate xTR β mRNA in XTC-2 cells, but not of xTR α or xRXR α mRNAs, while xRXR γ transcripts could not be detected. 9- cis-retinoic acid (9- cis-RA) did not substantially influence the expression of any of these four receptor genes. When transcription from three different thyroid response elements (TREs) (a palindromic TREpal, an inverted repeat +6 [F2] and a direct repeat +4 [DR+4] as present in the promoter of xTR β gene) was measured in XTC-2 cells in which xTR β and xRXR α were overexpressed, only T 3 upregulated transcription while 9- cis-RA, alone or together with T 3, was ineffective. 9- cis-RA however enhanced transcription from an RXR responsive element (RXR-RE). The second approach involved overexpression of xTR β and xRXR α in premetamorphic Xenopus tadpole tail muscle followed by measuring the response of the tails to T 3 in organ culture. After validating the microinjection/culture procedure histochemically, we found that T 3 enhanced transcription from the xTR β DR+4 TRE in tails in which xTR β was overexpressed but the overexpression of xRXR α failed to modify this response. It is concluded that in both XTC-2 cells and tadpole tails, overexpressed xRXR fails to modify the enhanced transcriptional response of endogenous and overexpressed xTR β to T 3 and that exogenous 9- cis-RA is ineffective.

Direct modulation of simian virus 40 late gene expression by thyroid hormone and its receptor.

Transcription of the late genes of simian virus 40 (SV40) is repressed during the early phase of the lytic cycle of infection of primate cells by the binding of cellular factors, called IBP-s, to the SV40 late promoter; repression is relieved after the onset of viral DNA replication by titration of these repressors (S. R. Wiley, R. J. Kraus, F. R. Zuo, E. E. Murray, K. Loritz, and J. E. Mertz, Genes Dev. 7:2206-2219, 1993). Recently, we showed that IBP-s consists of several members of the steroid/thyroid hormone receptor superfamily (F. Zuo and J. E. Mertz, Proc. Natl. Acad. Sci. USA 92:8586-8590, 1995). Here, we show that the thyroid hormone receptor TRalpha1, in combination with retinoid X receptor alpha (RXRalpha), is specifically bound at the transcriptional initiation site of the major late promoter of SV40. This binding repressed transcription from the SV40 late promoter by preventing the formation of pre-initiation complexes. Addition of the thyroid hormone 3,5,3'-L-triiodothyronine (T3) resulted in reversal of this repression in cotransfected CV-1 cells. Interestingly, repression did not occur when this thyroid response element (TRE) was translocated to 50 bp upstream of the major late initiation site. Binding of TRalpha1/RXRalpha heterodimers to this TRE induced bending of the promoter DNA. We conclude that hormones and their receptors can directly affect the expression of SV40, probably by affecting protein-protein and protein-DNA interactions involved in the formation of functional preinitiation complexes.

An artificial thyroid hormone receptor mutant without DNA binding can have dominant negative effect

The syndrome of resistance to thyroid hormone (RTH) encompasses a heterogenous group of conditions which are caused by mutations of thyroid hormone receptor β1 (TRβ1). Mutations usually cluster in two regions of the ligand-binding domain. The mutant receptors can inhibit normal receptor activity in a dominant negative manner, consistent with the dominant mode of inheritance of RTH. Recent evidence suggested that this dominant negative effect (DNE) of the RTH mutants involves competition for DNA binding and emphasized the essential role of intact DNA binding activity for mutants in order to exert DNE. However, we found that a Cys73Ser substitution in the DNA-binding domain (DBD) of wild-type produces a TR which can inhibit the transcriptional activation by TRα1, either in the presence or absence of T 3, on three different TRE-containing reporter genes, in transient co-transfection studies. Co-expression of TRvα2, a TRα splicing variant, can enhance this DNE. However, DNE was not observed on the negatively-regulated TSHα Luc reporter gene when wild-type and DBD mutant were co-transfected at equimolar ratios. The DNE of DBD mutant is not reversed by co-transfection with excess retinoid X receptor α. DBD mutant alone can also inhibit the transactivation from a TK-luciferase reporter gene either linked with rat malic enzyme thyroid response element, or not. These observations parallel those we previously observed using TRvα2. Our results indicate that a DBD mutant can have DNE, possibly through a mechanism similar to that of TRvα2, which may involve interference with basal transcription factors. The clinical significance of these DBD mutants is currently unclear, but it is logical to expect such mutants do occur in nature.

Structure of the growth hormone-encoding gene and its promoter in mice

The isolation and nucleotide sequence determination of the 5′ flanking region of the mouse growth hormone (mGH)-encoding gene (mGH) is described. The mGH gene consists of five exons and four introns, as is observed in other mammalian species. The second intron in mGH is much smaller than its rat counterpart, thus being similar in size to human, bovine and porcine GH. The transcription start point was determined to be a C residue 62 bp upstream from the start codon, ATG. Analysis of 1767 bp of the 5' flanking region, with respect to putative regulatory elements, revealed a TATA box, two binding sites for growth hormone factor (GHF1), a GC box (SP1), a thyroid-response element ( TRE) and a silencer (SiL) sequence motif. As expected, the mGH promoter shows a higher degree of homology with rat, as compared to the other mammalian species like pig, cattle and human, where an overall homology exists only at the proximal promoter region

Distinct sexual dimorphism in the effect of hypothyroidism on the expression of the growth hormone receptor and growth hormone-binding protein gene in rat liver.

Impairment of growth is a hallmark of hypothyroidism in animals. The ability of the thyroid hormone-thyroid hormone receptor complex to regulate gene transcription may be relevant to the growth impairment associated with hypothyroidism. To study the role of thyroid hormone in the expression of the GH receptor (GHR) and GH-binding protein (GHBP) gene, we examined the serum and liver tissue of female and male hypothyroid (thyroidectomized), thyroxine-treated thyroidectomized and euthyroid control rats. Compared to the control and to the thyroxine-treated group, the hypothyroid rats had significantly lower serum levels of thyroxine, increased levels of TSH, and decreased rates of weight gain. GHR and GHBP mRNA levels in liver were estimated by ribonuclease protection assays. In female rats, the levels of hepatic GHR and GHBP mRNA were increased in the hypothyroid group compared to euthyroid controls (p < 0.001 for GHR and p < 0.05 for GHBP). In contrast, in males the hypothyroid state was associated with decreased levels of GHR (p < 0.001) and GHBP (p < 0.001) mRNA levels compared to euthyroid controls. In both females and males, administration of thyroxine for a period of 2 weeks to the thyroidectomized rats prevented these changes in GHR and GHBP mRNA levels in liver. The differences observed between females and males could not be attributed to differences in the circulating levels of GH at sacrifice (female vs. male. 9.9 +/- 1.3 vs. 13.9 +/- 6.5 ng/ml). We conclude that (1) thyroid hormone affects the transcription of the GHR/GHBP gene; (2) there is a distinct sexual dimorphism in the effect of hypothyroidism on the expression of the GHR/GHBP gene, and (3) this effect is reversible following amelioration of the hypothyroid state. We speculate that regulation of expression of the GHR/GHBP gene by thyroid hormones involves multiple thyroid response elements that have opposite effects depending on the status of other factors such as sex hormones.

An in vivo analysis of transcriptional elements in the mouse alpha-myosin heavy chain gene promoter.

During development in the murine ventricle, there is a switch in myosin heavy chain gene (MyHC) transcription. The beta-MyHC is expressed in the ventricles during foetal development, but is shut down at or around birth, at which time alpha-MyHC transcription is activated. This antithetical switch is thought to be mediated by circulating levels of thyroid hormone (TH) and both low and high affinity thyroid response elements (TREs) have been identified in the proximal promoter region of the murine alpha-MyHC. Myosin gene expression in the atria is relatively unaffected by the TH status. Previously, we used site-directed mutagenesis of the promoter in a transgenic analysis to define those elements responsible for high levels of transcription in vivo. These analyses focused on the role(s) of two cis elements, TRE1 and TRE2 that are located at -129 to -149 and -102 to -120, respectively, on the alpha-MyHC promoter. Although the elements' ablation had differential effects on transgene expression, neither single mutation abolished transgene expression completely. Here, we show that mutating both elements results in a complete inactivation of the transgene in both ventricles and atria under euthyroid conditions. However, expression still can be detected in the hyperthyroid state, implying that, although the TRE1 and TRE2 elements are critical elements for high levels of alpha-MyHC transcription in vivo, other promoter sites can mediate at least some degree of transcriptional activation.

Effect of 3,5,3'-Triiodothyronine (T3) administration on dio1 gene expression and T3 metabolism in normal and type 1 deiodinase-deficient mice.

The type 1 deiodinase (D1) catalyzes the monodeiodination of T4 to produce T3, the active thyroid hormone. In the C3H mouse, hepatic D1 and the dio1 messenger RNA (mRNA) are only 10% that in the C57 strain, the common phenotype. Low activity cosegregated with a series of five GCT repeats located in the 5'-flanking region of the C3H dio1 gene that impaired C3H promoter potency and provided a partial explanation for the lower D1. The present studies were performed to search for additional explanations for low D1 activity in C3H mice. Previous studies have shown that T3 up-regulates the dio1 gene. Therefore, loss of the capacity to respond to endogenous T3 is a possible additional cause of the lower D1 levels in the C3H mice. The hepatic C3H dio1 mRNA increases 10- to 20 fold after T3 administration. The t3 effect occurs at a transplantation level and T3 does not alter the dio1 mRNA half-life. Despite the transcriptional response to T3, no functional thyroid response elements were identified in the 1.5-kilobase 5'-flanking region of either the C57 or C3H dio1 gene. After the same dose of exogenous T3, both dio1 mRNA and D1 of the C3H mouse respond to a greater extent than those of the C57 strain. This can be explained in part by the reduction in T3 clearance due to the lower D1 levels in C3H mice in which higher concentrations of circulating T3 are maintained. The decrease in serum T3 levels and T3 production observed in fasting and systemic illness in both human and experimental animals has been attributed in part to a decrease in hepatic D1. In contrast, despite markedly lower hepatic and renal D1 levels, serum T3 concentrations remain normal in C3H mice. The present studies suggest that the absence of stress-induced hypothalamic-pituitary suppression that allows T4 production to be maintained together with the reduced clearance of T3 and T4 via inner ring deiodination compensate for the D1 deficiency.

Effect of 3,5,3'-Triiodothyronine (T3) administration on dio1 gene expression and T3 metabolism in normal and type 1 deiodinase-deficient mice

The type 1 deiodinase (D1) catalyzes the monodeiodination of T4 to produce T3, the active thyroid hormone. In the C3H mouse, hepatic D1 and the dio1 messenger RNA (mRNA) are only 10% that in the C57 strain, the common phenotype. Low activity cosegregated with a series of five GCT repeats located in the 5'-flanking region of the C3H dio1 gene that impaired C3H promoter potency and provided a partial explanation for the lower D1. The present studies were performed to search for additional explanations for low D1 activity in C3H mice. Previous studies have shown that T3 up-regulates the dio1 gene. Therefore, loss of the capacity to respond to endogenous T3 is a possible additional cause of the lower D1 levels in the C3H mice. The hepatic C3H dio1 mRNA increases 10- to 20 fold after T3 administration. The t3 effect occurs at a transplantation level and T3 does not alter the dio1 mRNA half-life. Despite the transcriptional response to T3, no functional thyroid response elements were identified in the 1.5-kilobase 5'-flanking region of either the C57 or C3H dio1 gene. After the same dose of exogenous T3, both dio1 mRNA and D1 of the C3H mouse respond to a greater extent than those of the C57 strain. This can be explained in part by the reduction in T3 clearance due to the lower D1 levels in C3H mice in which higher concentrations of circulating T3 are maintained. The decrease in serum T3 levels and T3 production observed in fasting and systemic illness in both human and experimental animals has been attributed in part to a decrease in hepatic D1. In contrast, despite markedly lower hepatic and renal D1 levels, serum T3 concentrations remain normal in C3H mice. The present studies suggest that the absence of stress-induced hypothalamic-pituitary suppression that allows T4 production to be maintained together with the reduced clearance of T3 and T4 via inner ring deiodination compensate for the D1 deficiency.

Control of beta-myosin heavy chain expression in systemic hypertension and caloric restriction in the rat heart

In the rat left ventricle, both pressure overload induced by abdominal aortic constriction (Abcon) and caloric restriction (CR) induce an increase in the steady-state level of the beta-myosin heavy chain (MHC) protein and mRNA. Both models also induce a concomitant decrease in the alpha-MHC protein and mRNA. The goals of this study were to 1) determine if the changes in MHC expression in the models are due to altered transcription and 2) identify the relative levels of some key factors interacting with the regulatory regions of these genes. Female Sprague-Dawley rats were randomly assigned to the following groups: 1) normal control (NC), 2) Abcon, and 3) CR. After 5 wk of experimental manipulations, myocardial nuclei were isolated. These nuclei were used for 1) nuclear run-on assays or 2) nuclear extract, which was prepared and used for gel mobility shift assays (GMSAs). Nuclear run-on assays demonstrated that the increase in beta-MHC mRNA and protein expression in both Abcon and CR can be at least partially attributed to increased transcription. The concomitant decrease in alpha-MHC content can similarly be attributed to a decrease in transcription of this gene. Furthermore, GMSAs demonstrate that nuclear extract from each group interact differently with certain elements known to be important for expression in vitro. CR nuclear extracts have a 25.6 +/- 7.2% decrease (P < 0.05 vs. NC) in interaction with a thyroid-responsive element, a potential repressor of beta-MHC transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural determinants of nuclear receptor assembly on DNA direct repeats.

Nuclear receptor heterodimers recognize response elements composed of two direct repeats of the consensus sequence 5'-AGGTCA-3' separated by one to five base pairs. The 1.9 A crystal structure of the complex formed by the DNA-binding domains of the 9-cis retinoic acid receptor and thyroid hormone receptor bound to a thyroid-response element shows that the subunits interact through a DNA-supported interface involving the carboxy-terminal extension of the DNA-binding domain of the thyroid hormone receptor. The stereochemistry suggests a mechanism by which heterodimers recognize the inter-half-site spacing between direct repeats.

Cooperative interaction of oestrogen receptor ‘zinc finger’ domain polypeptides on DNA binding

The consensus oestrogen response element (ERE) contains two inverted copies of an AGGTCA consensus hexameric half-site, spaced by three base pairs. It differs from many other hormone response elements, such as consensus thyroid (TREp) and retinoic acid (DR-5 RARE) response elements, only in the relative spacing and orientation of these sequences. In the present study we report values for cooperativity (omega) of an oestrogen receptor DNA-binding domain polypeptide upon binding to these sequences. The polypeptide binds with negative cooperativity, or without cooperativity to retinoic acid and thyroid response elements respectively, but with high cooperativity to the ERE. We have also examined cooperativity upon binding of the polypeptide to an ERE variant. Since naturally occurring EREs commonly contain one hexamer which is considerably more degenerate than the other, we designed a hybrid response element in which one hexamer is a consensus ERE, while specific mutations were introduced into the other. We chose to mutate the second half-site to a glucocorticoid response element (GRE) half-site sequence (AGAACA), since normally no binding of the DNA-binding domain polypeptide to a GRE hexamer alone can be detected. In the hybrid response element, however, the GRE half-site is recognized with relatively high affinity, although binding to this sequence is dependent on the previous binding of a polypeptide to the ERE hexamer. Thus, cooperative interactions are capable of mediating the recognition of ERE sequence degeneracy. The ability of protein-protein interactions to mediate recognition of DNA sequence degeneracy may also have implications for transcription factors in general.

Nuclear factors specifically favor thyroid hormone binding to c-ErbAα1 protein (thyroid hormone receptor α) over-expressed in E. coli

A recombinant rat thyroid hormone receptor α (TRα or c-ErbAα1) was produced in E. coli as a non-mutated,non-fusioned protein and obtained as an efficient DNA and T3 binding protein that could be easily handled in a buffer-soluble state (rec-TRα). It was found that nuclear extracts (NE) added to rec-TRα markedly amplified not only DNA binding, which has been well documented, but also T3 binding (increased binding site concentration), which has not yet been reported. This T3 binding amplifying effect on rec-TRα occurs at low NE protein concentrations that produce no or minimal endogenous TR with respect to rec-TR, while similar concentrations of other proteins (e.g. ovalbumin or cytosol) only moderately enhanced T3 binding. The T3 binding amplifying nuclear factors, which are partly heat-labile, appeared as necessary auxiliaries in the analyses of partially purified rec-TRα. A protective effect of NE against a loss of affinity for T3 under the action of antibodies directed to certain sequences in the TRα D domain suggests that nuclear factors help rec-TRα to acquire and/or stabilize a conformation that allows the high affinity T3 binding. The nature of this nuclear amplifying factor is still unknown: RXRα which, produced in vitro, could amplify binding of the rec-TRα to a DNA thyroid response element, was unable to display such a rescue of high affinity binding sites.

The developmental switch in ventricular myosin expression in vivo is not triggered by an increase in cyclic AMP.

Ventricular myosin heavy chain (HC) expression undergoes a rapid change from the beta to the alpha isoform shortly after birth. Thyroid hormone is required for this transition to occur, but the time course of developmental changes in circulating thyroid hormone levels suggests that it cannot be the trigger for this event. In this study, the possibility was examined that cyclic AMP (cAMP), either acting separately or as a mediator of the permissive actions of thyroid hormone, triggers the developmental transition in ventricular myosin HC expression. One-day-old euthyroid or propylthiouracil-hypothyroid rat pups were treated with a membrane-permeable cAMP analogue, 8-bromo-cAMP (8-Br-cAMP) or triiodothyronine (T3). Two and four hours after acute treatment, the relative synthetic rates of alpha and beta myosin HC were measured using an in vivo pulse labelling technique. Myosin HC isoforms were separated electrophoretically and then quantitated by densitometry. Acute treatment in vivo with 8-Br-cAMP did not alter the relative rate of alpha myosin HC synthesis in euthyroid animals at either 2 or 4 h. Hypothyroidism significantly reduced the relative rate of alpha HC synthesis and obturated the transition from beta to alpha HC. The effect on synthesis was rapidly reversed by acute treatment with T3, but not with 8-Br-cAMP. Thus, an increase in cAMP does not appear to trigger the developmental change in myosin isoform expression, nor does it appear to mediate the stimulatory effect of thyroid hormone on alpha myosin HC synthesis. Instead, thyroid hormone is likely to be acting directly on the alpha HC gene by binding to the thyroid response element. The identity of stimuli that mediate the increased responsiveness to thyroid hormone during development remains to be determined, but it is not due to an increase in the levels of cAMP in perinatal cardiocytes in vivo.

The dominant negative effect of thyroid hormone receptor splicing variant alpha 2 does not require binding to a thyroid response element.

The functionally inactive thyroid hormone receptor splicing variant-alpha 2 (TRv alpha 2) can inhibit transcriptional activation by TR alpha 1 or beta 1, demonstrating a dominant negative effect (DNE). We examine here the three commonly proposed mechanisms, namely, competition for binding to thyroid hormone response elements (TREs), formation of inactive heterodimers, and squelching. A mutation introduced into the DNA-binding domain (DBD) of the TRv alpha 2 was designed to prevent its binding to TREs. In transient cotransfection studies, the DBD mutant has nearly the same DNE as does TRv alpha 2 on three different TRE-containing reporter genes. The DNE of TRv alpha 2 is also not reversed by cotransfection with excess retinoid X receptor-alpha. Extracts of COS cells cotransfected with TR alpha 1 and either TRv alpha 2 or DBD mutant at different ratios were analyzed by gel shift assays. Neither TRv alpha 2 or the mutant altered binding of TR alpha 1 to four radiolabeled TREs. TRv alpha 2 itself can inhibit constitutive transactivation by a thymidine kinase promoter-driven reporter construct. Our results suggest that TRv alpha 2 can function in a dominant negative manner without binding to a TRE, at least for certain TREs. It is concluded that the DNE of TRv alpha 2 may occur through another unrecognized mechanism, perhaps by binding to basal transcription factors.

The dominant negative effect of thyroid hormone receptor splicing variant alpha 2 does not require binding to a thyroid response element

The dominant negative effect of thyroid hormone receptor splicing variant alpha 2 does not require binding to a thyroid response element

Mechanisms for synergistic activation of thyroid hormone receptor and retinoid X receptor on different response elements.

The thyroid hormone receptors (TR) form heterodimers with the retinoid X receptors (RXR) and activate target genes through thyroid-responsive elements (TRE). Heterodimerization elevates the DNA binding efficiency and thus can result in functional synergism between TR and RXR. Here we demonstrate that DNA sequences dictate the cooperative activation between TR and RXR despite the high affinity binding of the heterodimer to those TREs. We provide evidence that the C-terminal activation domain of RXR can modulate the triiodothyronine (T3) responsiveness of TR/RXR heterodimers on reporter genes without altering the DNA binding properties of the heterodimers. The modulation function of this relatively small region is under the control of specific TRE sequences and promoter context. These data indicate that this C-terminal region of RXR is likely involved in receptor-cellular factor(s) interactions. Finally, we propose that the synergistic activation by TR and RXR is achieved through elevated DNA binding and, dependent on the DNA sequence, the interaction of RXR with other transcription factors.

Cardiac Myocyte Differentiation Induced by 3,5,3′-Triiodo-L-Thyronine (T 3) in P19-Teratocarcinoma Cells Is Accompanied by Preferential Binding of RGG(T/A)CA Direct Repeats Spaced by Four Base Pairs in the DNA

When treated with T 3, P19 cells differentiate into ultrastructurally proven cardiac myocytes and express the cardiac ventricular specific marker ventricular myosin light chain 2V. This differentiation is irreversibly induced in culture during the first 48 hrs of exposure to T 3. We studied the binding of P19-indigenous transcription factors of the Steroid-Thyroid-Retinoic superfamily of nuclear receptors to oligonucleotide response elements bearing direct, inverted and palindromic repeats of the consensus sequence RGG(T/A)CA. Electrophoretic mobility shift assays showed a preference in T 3-treated P19 cells for binding RGG(T/A)CA "half sites" in direct repeat orientation separated by 4 base pairs. The specificity of binding was confirmed in competition experiments. This finding suggests that target genes bearing thyroid response elements spaced by 4 base pairs in their promoter regions play an important role in the cardiac differentiation induced by T 3 in P19 teratocarcinoma cells.

3,5,3′-Triiodo-L-Thyronine Induces Cardiac Myocyte Differentiation but Not Neuronal Differentiation in P19 Teratocarcinoma Cells in a Dose-Dependent Manner

P19 teratocarcinoma cells differentiate into neurons or muscle when treated with varying doses of retinoic acid, dimethylsulfoxide, thioguanine or butyrate. We induced cardiac differentiation in P19 cells by treating them with 3,5,3′-Triiodo-L-Thyronine (T3). P19 cells received doses of T3 ranging from 30 pM to 300 nM. The beating colonies were counted, and a dose response curve showed that the optimal concentration of T3 was 30 nM. The colonies beat rhythmically for 4-6 weeks, and the cardiac myocytes showed clearly evident cardiac-specific organelles such as nexuses and atrial granules. No evidence of neuronal or skeletal muscle differentiation was seen with any of the concentrations of T3 used. Reverse transcription-polymerase chain reaction showed these cells to express the cardiac ventricular specific marker myosin light chain 2V. T3 is capable of inducing P19 cells to differentiate, in a dose related manner, into spontaneously beating cardiac myocytes identified as such on the basis of ultrastructural criteria. The induction of differentiation is accompanied by expression of cardiac-specific genes. These findings suggest that perhaps genes bearing thyroid response elements in their promoter regions play an important role in the cardiac differentiation induced by T3 in P19 teratocarcinoma cells,

Spectrum of transcriptional, dimerization, and dominant negative properties of twenty different mutant thyroid hormone beta-receptors in thyroid hormone resistance syndrome.

Resistance to thyroid hormone (RTH) is usually dominantly inherited and characterized by elevated thyroid hormone levels, impaired feedback inhibition of pituitary TSH production, and variable hormonal responsiveness in peripheral tissues. We have identified 20 different mutations in the thyroid hormone beta-receptor (TR beta) gene in RTH and assayed mutant receptor properties using the TSH alpha subunit gene promoter or promoters containing three different types of positive thyroid response element (TRE). Dominant negative inhibition of wild type TR beta action by mutant receptors was also tested. The mutant receptors exhibited differing transcriptional inhibitory properties and dominant negative potential with the TSH alpha promoter that correlated with their impaired hormone binding, whereas transactivation and dominant negative effects with promoters containing positive TREs varied depending on their configuration. Heterodimeric mutant receptor-retinoid X receptor (RXR) interactions, either in cultured cells or as TRE-bound complexes in gel retardation assays, were uniformly preserved, whereas homodimeric receptor interactions could not be detected in vivo, and in vitro homodimer formation on TREs was variably reduced or absent for some mutant proteins. We correlate these findings with the distribution of receptor mutations that cluster in two areas within the hormone binding domain outside putative dimerization regions and show that artificial mutations that impaired heterodimerization abrogated dominant negative activity. Therefore, we suggest that the dominant negative effect of mutant receptors in the pituitary-thyroid axis generates the characteristic biochemical abnormality of RTH and that variable resistance in other tissues may be due to response element-dependent differences in their dominant negative potential.

Dimerization characteristics of the DNA- and steroid-binding domains of the androgen receptor

The DNA-binding domain (DBD) of the androgen, mineralocorticoid, and glucocorticoid receptors and the steroid-binding domain (SBD) of the androgen receptor (AR) were expressed separately as fusion proteins with glutathione-S-transferase (GST) in Escherichia coli. Native polyacrylamide gel electrophoresis and gel exclusion HPLC demonstrated that the GST-ARDBD fusion protein was present as a dimer. On the other hand, the GST-ARSBD fusion protein formed a high-molecular weight oligomer, which seemed to be formed by two separate interactions, i.e. GST-GST and ARSBD-ARSBD between the fusion molecules. These findings strongly suggest that ARSBD has a potent ability to form a homodimer and that ARDBD does not. GST-ARDBD specifically interacted with the glucocorticoid response elements of the mouse mammary tumor virus long terminal repeat (GREMMTV). Cleavage of the fusion protein by thrombin abolished the binding, while the nonspecific DNA-cellulose binding ability was retained. Therefore, the dimeric configuration of GST-ARDBD, even if accomplished through the interaction with the GST moiety, is needed for high-affinity binding to the response element. The binding of GST-ARDBD to GREMMTV was strongly competed by the glucocorticoid response element of rat tyrosine aminotransferase gene, followed by the androgen response element of the rat probasin gene. A palindromic thyroid response element showed no competition. Unexpectedly, no apparent different in the binding affinity to these response elements was observed among the DBDs of androgen, mineralocorticoid and glucocorticoid receptors.