Thyroid hormone receptor DNA binding is required for both positive and negative gene regulation

The β isoform of thyroid hormone receptor (TR-β) has a key role in the feedback regulation of the hypothalamic-pituitary-thyroid (H-P-T) axis. The mechanism of trans-repression of the hypothalamic thyrotropin-releasing hormone (TRH) and pituitary thyroid-stimulating hormone (TSH) subunit genes, however, remains poorly understood. A number of distinct mechanisms for TR-β-mediated negative regulation by thyroid hormone have been proposed, including those that require and do not require DNA binding. To clarify the importance of DNA binding in negative regulation, we constructed a DNA-binding mutant of TR-β in which two amino acids within the P box were altered (GSG for EGG) to resemble that found in the glucocorticoid receptor (GR). We termed this mutant GS125, and as expected, it displayed low binding affinities for positive and negative thyroid hormone-response element (pTRE and nTRE, respectively) in gel-mobility shift assays. In transient transfection assays, the GS125 mutant abolished transactivation on three classic pTREs (DR+4, LAP, and PAL) and all negatively regulated promoters in the H-P-T axis (TRH, TSH-β, and TSH-α). However, GS125 TR-β bound to a composite TR/GR-response element and was fully functional on this hybrid TR/GR-response element. Moreover, the GS125 TR-β mutant displayed normal interactions with transcriptional cofactors in mammalian two-hybrid assays. These data do not support a DNA-binding independent mechanism for thyroid hormone negative regulation in the H-P-T axis.