Cloning of the single strand DNA-binding protein important for maximal expression and thyrotropin (TSH)-induced negative regulation of the TSH receptor.
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.
Ohmori
M
M
Cell Regulation Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Ohta
M
M
Shimura
H
H
Shimurat
Y
Y
Suzuki
K
K
Kohn
L D
LD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D005786:Gene Expression Regulation; D005802:Genes, MHC Class II / Q000235:genetics; D006706:Homeostasis; D008099:Liver / Q000166:cytology / Q000378:metabolism; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D051381:Rats; D011989:Receptors, Thyrotropin / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013961:Thyroid Gland / Q000166:cytology / Q000378:metabolism; D000074482:Thyroid Nuclear Factor 1; D013972:Thyrotropin / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; C000614416:Nkx2-1 protein, rat; D009687:Nuclear Proteins; D011989:Receptors, Thyrotropin; C104272:Ssbp1 protein, rat; D000074482:Thyroid Nuclear Factor 1; D015534:Trans-Activators; D014157:Transcription Factors; D013972:Thyrotropin; D000242:Cyclic AMP