Ligand-dependent synergy of thyroid hormone and retinoid X receptors.

The binding of thyroid hormone receptors to DNA is enhanced by heterodimerization with nuclear proteins. One such heterodimerization partner has recently been characterized as the retinoid X receptor. 9-cis-Retinoic acid has been identified as a natural ligand for retinoid X receptors, suggesting a potential receptor-mediated interaction between thyroid hormone and 9-cis-retinoic acid in the regulation of thyroid hormone-responsive genes. A transient cotransfection assay was used to test for such an interaction. When a complex thyroid hormone response element composed of both direct and inverted repeat hexamers was tested, these two ligands activated gene expression synergistically. In contrast, when the response element consisted only of directly repeated hexamers, unliganded retinoid X receptors enhanced thyroid hormone responsiveness, but 9-cis-retinoic acid induced no additional activation. The results suggest a unique mechanism to achieve differential suggest a unique mechanism to achieve differential thyroid hormone sensitivity of thyroid hormone-responsive genes within a cell. Genes with appropriate response elements will show amplification of the thyroid hormone response by 9-cis-retinoic acid in the presence of retinoid X receptors; other thyroid hormone-responsive genes will be influenced by retinoid X receptors, but not 9-cis-retinoic acid.     

A distinct thyroid hormone response element mediates repression of the human cholesterol 7alpha-hydroxylase (CYP7A1) gene promoter.

We examined the molecular basis by which T3 regulates the human cholesterol 7alpha-hydroxylase gene (CYP7A1) promoter. L-T3 decreased chloramphenicol acetyltransferase activity in hepatoma cells cotransfected with a plasmid encoding the T3 receptor (TR) alpha [NR1a1] and a chimeric gene containing nucleotides -372 to +61 of the human CYP7A1 gene fused to the chloramphenicol acetyltransferase structural gene. Deoxyribonuclease I footprinting revealed that recombinant TRalpha protected two regions in this segment of the human CYP7A1 gene promoter. In EMSAs, TRalpha bound to both regions. The binding was competed by oligonucleotides bearing an idealized TRalpha binding motif and abolished by mutation of these elements. In assays of promoter function, mutation of only one of the TRalpha binding sites blocked repression by T3. The results indicate that T3-dependent repression of human CYP7A1 gene expression is mediated via a novel site in the human CYP7A1 gene promoter.     

A novel second myostatin gene is present in teleost fish.

We report on the isolation and characterisation of the complete cDNA sequence encoding a novel bone morphogenetic protein-like protein (sbMSTN-b) in the teleost fish Sparus aurata. The encoded protein is 68% identical to S. aurata MSTN at the amino acid level, and homologues were also found in Umbrina cirrosa and Tetraodon nigroviridis. Phylogenetic analysis suggests that the MSTN-b gene may be present in most, perhaps all, teleost fish species. RT-PCR on different tissues/stages indicates that MSTN-b is expressed almost exclusively in the central nervous system, starting from late larval stages. Quantitative analyses indicate an increase of sbMSTN-b expression in the brain associated with metamorphosis, at the same time as completion of nervous system differentiation.     

A detailed functional and structural analysis of a major thyroid hormone inhibitory element in the human thyrotropin beta-subunit gene.

The first exon of the human thyrotropin-beta (hTSH beta) gene has been demonstrated in our laboratory to contain a major thyroid hormone inhibitory element. In order to characterize fully this element, we have performed a detailed functional and structural scanning mutational analysis of this element. Various -1192 to +37 (base pairs) bp fragments of the hTSH beta gene containing consecutive five deoxythymidine substitution mutations of the first exon were inserted into a luciferase reporter plasmid and transiently transfected into human embryonal cells (293) and stably transfected into rat pituitary cells (GH3). Two domains (domain 1 and 2) were identified by scanning mutations that were essential for function of the thyroid hormone inhibitory element: +3 to +13 bp and +28 to +37 bp. Biotinylated DNA fragments containing -12 to +43 bp of the hTSH beta gene and the identical scanning mutations demonstrate that in vitro synthesized c-erbA-beta binding is disrupted as much as 95% by mutations from -3 to +17 bp and to a lesser extent (20-30%) by mutations from +23 to +27 bp and from +33 to +43 bp. Domain 1 displayed a higher affinity for c-erbA-beta than domain 2 in avidin-biotin complex DNA-binding and gel-mobility assays. Using increasing amounts of in vitro synthesized c-erbA-beta, we were unable to demonstrate more than one protein-DNA complex in gel-mobility assays. However, using the avidin-biotin complex DNA-binding assay and the cross-linking reagent, 1,6-bismaleimidohexane, we were able to demonstrate thyroid hormone receptor dimer formation on domain 1 but not to any significant extent on domain 2. In conclusion, functional and DNA-binding studies suggest that the thyroid hormone receptor binds to two distinct regions in the first exon of the hTSH beta gene. The upstream site (domain 1) binds c-erbA-beta with higher affinity and is capable of binding c-erbA-beta as a dimer under some conditions, while the downstream site (domain 2) appears to bind a single molecule of c-erbA-beta with lower affinity. These results suggest that thyroid hormone receptor, binding to at least two sites in the first exon, act in conjunction to mediate T3 inhibition of hTSH beta expression.     

Ligand-dependent and -independent transactivation by thyroid hormone receptor beta 2 is determined by the structure of the hormone response element.

Chicken thyroid hormone receptor beta 2 (cTR beta 2) is likely to serve specific functions in gene regulation since it possesses a unique N-terminal domain and is expressed in very few tissues. We demonstrate here that TR beta 2 exhibits distinct transactivation properties which are dependent on the availability of ligand and on the structure of the hormone response element. First, a strong ligand-independent transactivation was observed with hormone response elements composed of direct repeats and everted repeats. Second, TR beta 2 was induced by triiodothyronine to transactivate more efficiently than TR beta 0 on palindromic and everted-repeat types of hormone response elements. However, coexpression of the retinoid X receptor reduced the strong transactivation by TR beta 2 but not by TR beta 0 via palindromic response elements, suggesting that TR beta 2 can transactivate as a homodimer. Finally, the N terminus of TR beta 2 contains two distinct transactivation regions rich in tyrosines, which are essential for transactivation. Our results thus show that the activity of the novel transactivating region of TR beta 2 is dependent on the organization of the half-sites in the response element.     

Early expression of thyroid hormone receptor beta and retinoid X receptor gamma in the Xenopus embryo

The role of thyroid hormone in Xenopus metamorphosis is particularly well understood as it plays an essential role in that process. However, recent evidence suggests that thyroid hormone may play an earlier role in amphibian embryogenesis. We demonstrate that Xenopus thyroid hormone receptor beta (XTR beta) is expressed shortly after neural fold closure, and that its expression is localized to the developing retina. Retinoid X receptor gamma (RXR gamma), a potential dimerization partner for XTR beta, was also found to be expressed in the retina at early stages, and at later stages RXR gamma was also expressed in the liver diverticulum. Addition of either thyroid hormone or 9-cis retinoic acid, the ligands for XTR beta and RXR gamma, respectively, did not alter the expression of their receptors. However, the addition of thyroid hormone and 9-cis retinoic acid did alter rhodopsin mRNA expression. Addition of thyroid hormone generates a small expansion of the rhodopsin expression domain. When 9-cis retinoic acid or a combination of thyroid hormone and 9-cis retinoic acid was administered, there was a decrease in the expression domain of rhodopsin in the developing retina. These results provide evidence for an early role for XTR beta and RXR gamma in the developing Xenopus retina.