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Contiguous with the 5'-end of the thyroid transcription factor-1 (TTF-1) element upstream of the minimal TSH receptor (TSHR) promoter and within it, there is an element on the noncoding strand with single strand- binding activity. Mutation analyses indicate that it is functionally distinct from the TTF-1 element and is important for the constitutive expression and TSH/cAMP-induced negative autoregulation of the TSHR in thyroid cells but only constitutive expression in nonthyroid cells. In this report we identify a cDNA encoding a single strand-binding protein (SSBP) that forms a specific complex with the noncoding strand of the TSHR, contiguous with the 5'-end of both TTF-1 elements; we term it SSBP-1. SSBP-1 increases promoter activity when contransfected with heterologous SV40 promoter-chloramphenicol acetyltransferase (CAT) chimeras containing the upstream SSBP-binding element from the TSHR promoter or with TSHR promoter-CAT chimeras containing both or only the downstream SSBP element. Mutational analyses reveal that a GXXXXG motif is important for the binding and enhancer function of SSBP-1. TSH/cAMP decreases SSBP-1 RNA levels, as well as SSBP-1/TSHR DNA complex formation, in functioning rat FRTL-5 thyroid cells but not nonfunctioning FRT thyroid or Buffalo rat liver cells that have no TTF-1. SSBP-1 RNA is present ubiquitously; however, its levels are higher in FRTL-5 cells and are increased by overexpression of TTF-1 in cells treated with TSH. This reverses TSH-induced negative regulation of the TSHR. SSBP-1 is, therefore, a positive regulator of TSHR gene expression that contributes to TSHR maximal expression by binding to the SSBP elements. It is a ubiquitous, single-strand transcription factor whose expression in FRTL-5 thyroid cells is, however, regulated by a thyroid-specific gene, TTF-1. TSH/cAMP induces negative autoregulation of the TSHR, in part, by decreasing maximal expression resultant from SSBP-1 binding to the SSBP elements. Like Y-box proteins, which are involved in negative regulation of the TSHR, SSBP-1 also interacts with the major histocompatibility class II promoter S-box; the interaction is single strand-specific. This supports the hypothesis that common transcription factors regulate TSHR and major histocompatibility gene expression. Of additional interest and again like Y-box proteins, SSBP-1 is a member of a family of SSBPs that interact with RNA and are important in RNA processing, can interact with the promoter of retroviruses, and can interact with a gene linked to growth and DNA replication, c-myc.

molecular endocrinology

Cloning of the single strand DNA-binding protein important for maximal expression and thyrotropin (TSH)-induced negative regulation of the TSH receptor.