Thyroid-hormone receptors (TRs) form heterodimers with retinoid-X receptors (RXRs) on thyroid-hormone-response elements (TREs). However, it is not known whether the formation of liganded TR/RXR heterodimer on a TRE alone is sufficient to dictate transcriptional activity. We designed several mutated DR4s (half-sites arranged as direct repeats with a nucleotide gap of 4) that bound TR/RXR heterodimers preferentially, and employed them to characterize functional and biochemical properties of the heterodimers on DNA. Although TR/RXR heterodimer binding was similar on some of the mutated DR4s, transient transfection assays showed that TRalpha failed to support triiodothyronine (T3)-stimulated transcription on "inactive" DR4s but mediated basal repression on both "active" and inactive mutated DR4. T3 binding assays showed that the mutated DR4s did not affect T3 binding to the heterodimer. Finally, partial proteolysis studies revealed that binding of active DR4 elements and T3 to the heterodimer synergistically enhanced heterodimerization-induced protease resistance of TR, but not RXR, in the heterodimer. These results suggest that: 1) liganded TR/RXR heterodimer binding to a DR4 is not sufficient for transcriptional activation of the target gene, and 2) DNA sequences in specific TREs may modify T3-mediated transcription by affecting the conformation of the liganded heterodimer.

Thyroid hormone receptors (TRs) and steroid hormone receptors belong to a large superfamily of nuclear hormone receptors. The interactions between these receptor subfamilies are poorly understood. In this study, cotransfection assays were used to examine the effects of estrogen and glucocorticoid receptors on TR-mediated repression of basal transcription by unliganded TR and transcriptional activation by liganded TR with two different thyroid hormone response element-containing reporter plasmids. Surprisingly, it was found that steroid hormone receptors blocked T3-mediated transcriptional activation with little or no effect on basal repression by unliganded TR. The mechanism for blocking TR-mediated transcriptional activation does not require steroid hormone receptor binding to the thyroid hormone response element but, rather, may involve titration of a critical coactivator(s) required for T3-mediated transcriptional activation. These studies strongly suggest divergent pathways for transcriptional activation and basal repression by TRs. Additionally, these studies raise the potential for nuclear hormone receptors to modulate TR-mediated transcriptional activation in steroid hormone-responsive tissues.

Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that regulate target gene transcription. Interestingly, in the absence of ligand, TRs also can repress basal transcription of positively regulated target genes, suggesting that unliganded TR may have a distinct role in gene regulation. In this paper, DNA binding, truncation, and natural human TR beta mutants were used in cotransfection and electrophoretic mobility shift assays to study various aspects of TR-mediated basal repression. Presently, little is known about the role(s) of natural human TR beta mutants on basal repression. These results show that: 1) TR binding to DNA likely is required for basal repression; 2) the amino-terminal region of TR is not required for basal repression; 3) TR homodimer binding is not absolutely required for basal repression, as TR mutants that selectively form TR-retinoid X receptor heterodimers can mediate basal repression; and 4) TR mutants with poor T3-binding affinity likely have constitutive basal repression, even in the presence of ligand. These findings provide new insight on the mechanism of basal repression by unliganded TRs.

Thyroid hormone receptors bind to thyroid hormone response elements (TREs) as heterodimers with 3,5,3'-L-triiodothyronine (T3) receptor auxiliary protein (TRAP) and retinoid X receptors (RXRs). Currently, it is not known whether TR/TRAP or TR/RXR heterodimers need to bind to both TRE half-sites and whether there is a preferred orientation for TR/RXR heterodimer binding to TREs or transcriptional activation. Accordingly, we created a mutant TR alpha (TR-P box) by changing 3 amino acids in the P box region of the first zinc finger of the DNA-binding domain to that of the glucocorticoid receptor (GR), and we examined wild-type TR alpha and TR-P box complex binding to hybrid response elements containing TRE and glucocorticoid receptor element (GRE) half-sites arranged as a direct repeat with a four-nucleotide gap. TR-P box/RXR heterodimers selectively bound to the hybrid response elements in which GRE half-site was the downstream half-site, whereas TR alpha/RXR bound to hybrid response elements in which GREs were in either position. Additionally, TR/TRAP or TR/RXR heterodimer required two half-sites for binding to DNA, with strong binding to at least one of the half-sites. Last, co-transfection assays and methylation interference studies using the hybrid response elements suggest that the sequential arrangement of strong and weak half-sites in the TRE may be a critical determinant of TR/RXR heterodimer binding and transcriptional activation.