Negative regulation of the thyroid-stimulating hormone alpha gene by thyroid hormone: receptor interaction adjacent to the TATA box.

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Abstract
Thyroid-stimulating hormone .alpha. and .beta. subunit genes are negatively regulated by thyroid hormone at the transcriptional level. Transient gene expression studies were used to demonstrate that the erbA.beta. form of the thyroid hormone receptor mediates negative regulation of the .alpha.-subunit promoter in a hormone-dependent manner. In JEG-3 choriocarcinoma cells, which are deficient in thyroid hormone receptors, coexpression of erbA.beta. with .alpha.CAT reporter genes markedly suppressed .alpha.CAT expression after treatment with thyroid hormone, whereas a reporter gene containing a known positive thyroid response element was induced. Thus, a single form of thyroid hormone receptor mediates both positive and negative responses to thyroid hormone in this system. Transient expression analyses of .alpha. gene 5'' flanking sequence deletion mutants localized the negative thyroid response element to the proximal region of the promoter between -100 and +4 base pairs. The location of the negative thyroid response element in the .alpha. gene is therefore distinct from that of previously identified regulatory elements including the tissue-specific upstream regulatory elements, the cAMP response elements, and the glucocorticoid response elements. Overlapping segments of the .alpha. promoter were examined for potential thyroid hormone receptor binding sites by using gel shift assays and biotinylated DNA-binding studies. A specific thyroid hormone receptor binding site was identified between -22 and -7 base pairs, which is immediately downstream from the TATA box. This region of the .alpha. promoter interacts with erbA.beta. receptor synthesized in vitro as well as with endogenous nuclear thyroid hormone receptors, and it completes for receptor binding to a known positive thyroid response element. These studies suggest a mechanism for negative regulation in which the thyroid hormone receptor interacts with the .alpha. gene promoter immediately downstream of the TATA box to inhibit transcription.