Single strand DNA-binding proteins and thyroid transcription factor-1 conjointly regulate thyrotropin receptor gene expression.

Abstract

An element, -186 to -176 base pairs (bp), in the minimal TSH receptor (TSHR) promoter binds thyroid transcription factor-1 (TTF-1) and is important for both constitutive expression and TSH/cAMP-induced negative autoregulation of the TSHR in thyroid cells. An element on the noncoding strand of the TSHR, contiguous with the 5'-end of the TTF-1 element, has single strand binding activity. It is distinct from the TTF-1 site, as evidenced by competition experiments using gel shift assays; but the association of the two elements is not random. Thus, the single strand binding protein (SSBP) element also exists contiguous to the 5'-end of an upstream TTF-1 site, -881 to -866 bp; mutation of two conserved nucleotides in each SSBP element results in the loss of SSBP binding and cross-competition. Transfection experiments indicate that full, constitutive TSHR gene expression in FRTL-5 thyroid cells requires the binding of both SSBPs and TTF-1, since mutation of either element halves thyroid-specific promoter activity, whereas mutation of both decreases promoter activity to values near those of a control vector. Transfection experiments with rat liver cells support their independent activities and show that the SSBP site contributes to TSHR gene expression in non-thyroid tissue. The SSBPs function conjointly with TTF-1 in thyroid-specific, TSH/cAMP-induced negative autoregulation of the TSHR. Thus, TSH or forskolin-treated FRTL-5 cells coordinately decrease TSHR RNA levels and TSHR DNA binding to both the SSBPs and TTF-1; also the maximal TSH/cAMP-induced decrease in gene expression requires both elements. The TSH-induced effect in each case is inhibited by cycloheximide; the TSH-induced decrease in SSBP/DNA complex formation requires the presence of insulin or calf serum, exactly as does TSH-induced down-regulation of TSHR RNA levels. In sum, full, constitutive expression of the TSHR in thyroid cells requires TTF-1 and the SSBPs to bind separate, contiguous elements on the TSHR promoter. TSH/cAMP decreases the binding of each factor to its respective site, thereby decreasing TSHR gene expression. The role of the SSBP and TTF-1 sites in constitutive TSHR expression and in TSH/cAMP-induced negative regulation of the TSHR is, therefore, additive and independent.