HIGH-AFFINITY AND SPECIFICITY OF DIMERIC BINDING OF THYROID-HORMONE RECEPTORS TO DNA AND THEIR LIGAND-DEPENDENT DISSOCIATION

The thyroid hormone receptors are ligand-dependent, DNA binding, trans-acting transcriptional factors belonging to the erbA-related steroid/thyroid hormone receptor superfamily. We report here the high affinity and specificity of dimeric DNA binding of human thyroid hormone receptor-alpha1 (hTRalpha1) and hTRbeta1 and the effect of T3 on its DNA binding. Gel mobility shift assay showed that hTRa1 and -beta1 bind to the rat GH-thyroid hormone response element (TRE) and rat malic enzyme (rME)-TRE as a monomer, dimer, and oligomer in the absence of T3 and bind to an irrelevant DNA sequence from the adenovirus 5 promoter only as a monomer. In competition studies using unlabeled TRE, dimer binding was displaced by lower concentrations of TRE than was the monomer, indicating that the dimeric binding has higher affinity than the monomer binding. These results suggest that the formation of dimers of TR increases the specificity and affinity for the response element. Surprisingly, T3 disrupted dimer binding and increased the intensity of monomer binding to rME-TRE in a dose-dependent manner. This does not occur with the rat GH-TRE. We also demonstrate that this disruption of dimeric binding by T3 occurred on a TRE formed by a direct repeat and not on a palindromic TRE. Furthermore, a mutant hTRbeta (Mf), which has no detectable ligand-binding activity because of a glycine to arginine substitution at amino acid 340 in the hormone-binding domain, does not show dissociation from rME-TRE after the addition of T3. This suggests that a comformational change in TR induced by T3 binding may result in dimer-specific dissociation from rME-TRE or other sites containing a direct repeat, and could theoretically lead to increased formation of heterodimer with binding enhancer proteins on the TREs.