3,5,3'-triiodothyronine receptor auxiliary protein (TRAP) enhances receptor binding by interactions within the thyroid hormone response element.

We have previously demonstrated that binding of in vitro synthesized thyroid hormone receptor (TR) to thyroid hormone response elements (TREs) is enhanced by the addition of nuclear extracts from several different cell types, suggesting that binding of TR is partially dependent on a T3 receptor auxiliary protein (TRAP). We have used the avidin-biotin complex DNA-binding assay to discriminate between regions of TREs that bind TR alone and sites that are influenced by interactions with TRAP. Mutations in the TREs from rat GH and glycoprotein hormone alpha-subunit genes show that a specific DNA sequence is required for TRAP-mediated enhancement of TR binding. Mutations in the B half-site of the rat GH TRE or in similar sequences [(T/A)GGGA] in the alpha-subunit TRE ablate the enhancement of TR binding by TRAP. Furthermore, binding of TR to a natural half-site in the TSH beta-subunit gene (bases -16 to 6), which lacks an additional AGGGA-like sequence, is not enhanced by the addition of TRAP. Binding of TR to TREs was also tested at physiological salt concentrations in the avidin-biotin complex DNA-binding assay. Binding of human TR beta to TREs decreases dramatically at 140 mM KCl compared to binding at 50 mM KCl; however, the addition of TRAP enhances the binding to almost 4-fold of basal binding, suggesting that TRAP may be important for stabilization of TR binding to TREs in the cell.     

A nuclear factor that enhances binding of thyroid hormone receptors to thyroid hormone response elements

Recent studies from this laboratory have demonstrated the presence of thyroid hormone response elements (TREs) in the 5'-flanking region of the rat alpha and TSH beta subunit genes. Using an avidin-biotin complex DNA binding assay, we have shown that these TREs bind the thyroid hormone (T3) receptor present in nuclear extracts of GH3 cells, as well as the in vitro synthesized Hc-erbA beta, which has been identified as a member of the family of T3 receptors. The binding of Hc-erbA beta to the alpha subunit TRE can be enhanced 3-4-fold by including GH3 nuclear extract in the binding assay. Binding to the TRE present in the TSH beta gene or the rat growth hormone gene was similarly enhanced, although to a lesser degree. The enhanced binding activity is trypsin-sensitive and heat labile, and is not reproduced by the addition of histones, bovine serum albumin, or cytosol instead of nuclear extract. Gel exclusion chromatography suggests a molecular size of approximately 65,000 Da. This protein, which is present in several different cell types, is also able to complement binding of the rat erbA alpha-1 and the pituitary-specific erbA beta-2 forms of the receptor. These data suggest that the binding of the T3 receptor to a TRE is augmented by another nuclear protein, which may be involved in the mechanism of action of thyroid hormone.     

A novel retinoid X receptor-independent thyroid hormone response element is present in the human type 1 deiodinase gene.

We identified two thyroid hormone response elements (TREs) in the 2.5-kb, 5'-flanking region of the human gene encoding type 1 iodothyronine deiodinase (hdio1), an enzyme which catalyses the activation of thyroxine to 3,5,3'-triiodothyronine (T3). Both TREs contribute equally to T3 induction of the homologous promoter in transient expression assays. The proximal TRE (TRE1), which is located at bp -100, has an unusual structure, a direct repeat of the octamer YYRGGTCA hexamer that is spaced by 10 bp. The pyrimidines in the -2 position relative to the core hexamer are both essential to function. In vitro binding studies of TRE1 showed no heterodimer formation with retinoid X receptor (RXR) beta or JEG nuclear extracts (containing RXR alpha) and bacterially expressed chicken T3 receptor alpha 1 (TR alpha) can occupy both half-sites although the 3' half-site is dominant. T3 causes dissociation of TR alpha from the 5' half-site but increases binding to the 3' half-site. Binding of a second TR to TRE1 is minimally cooperative; however, no cooperativity was noted for a functional mutant in which the half-sites are separated by 15 bp, implying that TRs bind as independent monomers. Nonetheless, T3 still causes TR dissociation from the DR+15, indicating that dissociation occurs independently of TR-TR contact and that rebinding of a T3-TR complex to the 3' half-site occurs because of its slightly higher affinity. A distal TRE (TRE2) is found at bp -700 and is a direct repeat of a PuGGTCA hexamer spaced by 4 bp. It has typical TR homodimer and TR-RXR heterodimer binding properties. The TRE1 of hdio1 is the first example of a naturally occurring TRE consisting of two relatively independent octamer sequences which do not require the RXR family of proteins for function.     

Cell type-dependent modulation of the dominant negative action of human mutant thyroid hormone beta 1 receptors.

BACKGROUND: Mutations in the ligand-binding domain of the thyroid hormone receptor beta (TR beta) gene cause the syndrome of resistance to thyroid hormone (RTH). The clinical phenotype results from the antagonism of the normal TR alpha and the non-mutated TR beta alleles by the TR beta 1 mutants, via a dominant negative effect. There is, however, marked heterogeneity of organ resistance within and among kindreds with RTH. This study examines the potential role of cell type in modulating the dominant negative potency of human TR beta 1 (h-TR beta 1) mutants. MATERIALS AND METHODS: Transient transfections were performed in HeLa and NIH3T3 cells, using a wild type (WT) and three naturally occurring mutant h-TR beta 1 constructs, and three natural thyroid hormone response elements (TREs). Immunocytochemistry was performed to detect levels of TR beta 1 expression in these two cell types. In order to determine how TR beta 1 interacts with other cellular partners, gel-shift analyses using HeLa and NIH3T3 nuclear extracts were performed. RESULTS: Transfection studies using WT h-TR beta 1 in HeLa and NIH3T3 cells, showed that the 3,3',5-triiodothyronine (T3)-induced transactivation of the different TREs varied between cell types. Unlike the non-T3-binding h-TR beta 1 mutant, PV, mutants ED and OK displayed the expected T3-induced dose responsiveness in these two cell types. For each TRE examined, the magnitude of the dominant negative effect varied between the cell types. The levels of receptor expression in HeLa and NIH3T3 cells were identical, as determined by immunocytochemistry. Gel-shift analyses showed differences in the formation of hetero- and homodimers depending on both the cell type and TRE motif. CONCLUSIONS: The cell type in which a mutant receptor operates affects the relative amounts of hetero- and homodimers. Together with the nature of the mutation and the TRE-motif, this could modulate the dominant negative action of mutant receptors in different tissues, which, in turn, could contribute to the variable phenotypic characteristics of RTH.     

Isolation and characterization of a cDNA encoding a chicken beta thyroid hormone receptor.

We have isolated and characterized a cDNA encoding a chicken beta homolog of c-erbA, or thyroid hormone receptor (TR). Chicken liver cDNA libraries were screened with a rat TR beta-1 cDNA probe, and several cDNA inserts were isolated and characterized. The sequence of one cDNA predicts a 369-amino-acid open reading frame (ORF), with a protein sequence that possesses 96% identity with that of rat TR beta-1, but only 88% identity with chicken TR alpha. These data indicate that the cDNA likely encodes a beta form of TR that has the expected putative DNA and T3 binding domains. The chicken TR beta (chTR beta) in vitro translated protein binds T3 with high affinity, and binds both the thyroid hormone response element (TRE) from the rat growth hormone gene and the Xenopus vitellogenin A2 gene estrogen response element (ERE), similarly to that of the rat TR beta-1. Northern blot analysis revealed the expression of a 7.0-kb RNA in several tissues including cerebellum, pituitary, kidney, and liver. This chicken liver TR beta cDNA sequence varies in both the 5' and 3' untranslated regions from the chicken kidney TR beta cDNA sequence recently reported (Forrest et al., 1990). The 5' untranslated cDNA sequence divergence occurs near a potential splice site junction of the human TR beta gene, suggesting that this chicken liver cDNA may represent an alternatively spliced RNA product of the chicken TR beta gene.     

Differential expression of thyroid hormone receptor isoforms dictates the dominant negative activity of mutant Beta receptor

Mutations in the thyroid hormone receptor beta gene (TRbeta) cause resistance to thyroid hormone (RTH). Genetic analyses indicate that phenotypic manifestation of RTH is due to the dominant negative action of mutant TRbeta. However, the molecular mechanisms underlying the dominant negative action of mutants and how the same mutation results in marked variability of resistance in different tissues in vivo are not clear. Here we used a knock-in mouse (TRbetaPV mouse) that faithfully reproduces human RTH to address these questions. We demonstrated directly that TRbeta1 protein was approximately 3-fold higher than TRalpha1 in the liver of TRbeta(+/+) mice but was not detectable in the heart of wild-type and TRbetaPV mice. The abundance of PV in the liver of TRbeta(PV/PV) was more than TRbeta(PV/+) mice but not detectable in the heart. TRalpha1 in the liver was approximately 6-fold higher than that in the heart of wild-type and TRbetaPV mice. Using TR isoforms and PV-specific antibodies in gel shift assays, we found that in vivo, PV competed not only with TR isoforms for binding to thyroid hormone response elements (TRE) but also competed with TR for the retinoid X receptors in binding to TRE. These competitions led to the inhibition of the thyroid hormone (T(3))-positive regulated genes in the liver. In the heart, however, PV was significantly lower and thus could not effectively compete with TRalpha1 for binding to TRE, resulting in activation of the T(3)-target genes by higher levels of circulating thyroid hormones. These results indicate that in vivo, differential expression of TR isoforms in tissues dictates the dominant negative activity of mutant beta receptor, thereby resulting in variable phenotypic expression in RTH.