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Single strand DNA-binding proteins and thyroid transcription factor-1 conjointly regulate thyrotropin receptor gene
H Shimura1, Y Shimura, M Ohmori
1Section on Cell Regulation, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Molecular Endocrinology (Baltimore, Md.)
|May 1, 1995
Summary
Thyroid transcription factor-1 (TTF-1) and single-strand binding proteins (SSBPs) bind adjacent sites on the TSH receptor promoter, crucial for gene expression and TSH/cAMP-induced regulation in thyroid cells.
Area of Science:
- Molecular Biology
- Endocrinology
- Genetics
Background:
- The TSH receptor (TSHR) is critical for thyroid function and its expression is tightly regulated.
- Thyroid transcription factor-1 (TTF-1) is a known regulator of thyroid-specific gene expression.
- The precise mechanisms of TSHR gene regulation, particularly autoregulation, require further elucidation.
Purpose of the Study:
- To identify and characterize novel regulatory elements in the TSHR promoter.
- To investigate the roles of TTF-1 and associated factors in TSHR gene expression and autoregulation.
- To determine the functional significance of these elements in both thyroid and non-thyroid cells.
Main Methods:
- Gel shift assays to assess protein-DNA interactions.
- Site-directed mutagenesis to probe the function of specific DNA elements.
- Reporter gene transfection assays in FRTL-5 thyroid cells and rat liver cells.
- Analysis of TSHR RNA levels under various treatment conditions.
Main Results:
- A novel single-strand binding protein (SSBP) element, located adjacent to a TTF-1 binding site (-186 to -176 bp) in the TSHR promoter, was identified.
- Mutation of either the TTF-1 or SSBP element reduced constitutive TSHR promoter activity by approximately 50% in FRTL-5 cells.
- Simultaneous mutation of both elements drastically reduced promoter activity, indicating their cooperative role in constitutive expression.
- TSH/cAMP treatment led to decreased binding of both TTF-1 and SSBPs to their respective sites, correlating with reduced TSHR gene expression.
- The SSBP site also contributed to TSHR expression in non-thyroid cells, suggesting broader regulatory roles.
Conclusions:
- Both TTF-1 and SSBP binding elements are essential for full constitutive TSHR gene expression in thyroid cells.
- These elements function conjointly in the TSH/cAMP-induced negative autoregulation of the TSHR.
- The findings reveal a complex, additive regulatory mechanism involving distinct but contiguous DNA elements for TSHR gene control.