Document Detail

Thyroid transcription factor-1 activates the promoter activity of rat thyroid Na+/I- symporter gene.
MedLine Citation:
PMID:  9328356     Owner:  NLM     Status:  MEDLINE    
We have cloned 15 kbp of rat thyroid Na+/I- symporter gene from liver genomic library, which contains 6 kbp upstream sequence from the translation initiation site. Southern blot analysis of the genomic DNA from the liver has revealed that thyroid Na+/I- symporter gene is the single gene in the rat. To study the tissue-selective expression mechanism of the gene, we at first determined the transcriptional start site of the gene. Results of a rapid amplification of cDNA end procedure as well as that of primer extension analysis indicated that the transcriptional start sites clustered between -96, -95, and -93 bp of the gene (A in ATG is designated as +1). Chimeras containing 1.9 kbp (-1967 to -46 bp) of the 5'-flanking sequence of the Na+/I- symporter gene and luciferase gene expressed significant enzyme activity when transfected into a rat thyroid cell line, FRTL-5, but little activity was observed in BRL-3A rat liver cells. Deletion analysis of the constructs indicated that a minimal region, exhibiting promoter activity and cell specificity, is located between -291 and -134 bp of the gene. Deoxyribonuclease I footprinting shows that nuclear extracts from FRTL-5, but not BRL-3A, cells protect a region between -245 and -230 bp. Electrophoretic mobility shift assays have demonstrated that nuclear extracts from FRTL-5 cells formed a specific DNA-protein complex with an oligonucleotide probe corresponding to -250 to -211 bp of the gene, but that from BRL-3A cells did not, suggesting that thyrocyte-selective nuclear factors bind to the region. When the nuclear extracts from FRTL-5 cells were preincubated with antibody against thyroid transcription factor-1 (TTF-1), homeodomain containing nuclear protein, formation of the complex was abolished and the band was supershifted. We also found that the probe formed a DNA-protein complex with the recombinant TTF-1 homeodomain, and mutations of the binding site eliminated factor binding. When pRc/CMV-TTF-1 was cotransfected with the minimal promoter fragment of thyroid Na+/I- symporter gene into FRT cells, which express no TTF-1, it caused a significant increase in the transcriptional activity of the reporter construct, but not of the construct having mutated TTF-1-binding element. These results suggest that TTF-1 confers the cell-selective expression of Na+/I- symporter gene in thyrocytes.
T Endo; M Kaneshige; M Nakazato; M Ohmori; N Harii; T Onaya
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Molecular endocrinology (Baltimore, Md.)     Volume:  11     ISSN:  0888-8809     ISO Abbreviation:  Mol. Endocrinol.     Publication Date:  1997 Oct 
Date Detail:
Created Date:  1998-01-12     Completed Date:  1998-01-12     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  8801431     Medline TA:  Mol Endocrinol     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  1747-55     Citation Subset:  IM    
Third Department of Internal Medicine, Yamanashi Medical University, Tamaho, Japan.
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MeSH Terms
Base Sequence
Carrier Proteins / genetics*
Cell Line
Cloning, Molecular
DNA Footprinting
Gene Expression Regulation*
Gene Library
Genes, Reporter
Liver / chemistry
Membrane Proteins / genetics*
Molecular Sequence Data
Nuclear Proteins / physiology*
Promoter Regions, Genetic*
Protein Binding
Rats, Sprague-Dawley
Recombinant Fusion Proteins / biosynthesis,  genetics
Sequence Deletion
Thyroid Gland / metabolism*
Transcription Factors / physiology*
Transcription, Genetic
Reg. No./Substance:
0/Carrier Proteins; 0/Membrane Proteins; 0/Nuclear Proteins; 0/Recombinant Fusion Proteins; 0/Symporters; 0/Transcription Factors; 0/sodium-iodide symporter; 0/thyroid nuclear factor 1

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