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Human thyroxine-binding globulin gene: complete sequence and transcriptional regulation
Y Hayashi1, Y Mori, O E Janssen
1Department of Medicine, University of Chicago, Illinois 60637-1470.
Molecular Endocrinology (Baltimore, Md.)
|August 1, 1993
Summary
Researchers characterized the human T4-binding globulin (TBG) gene, identifying its genomic structure and promoter region. They found a key regulatory element essential for liver-specific gene expression and TBG synthesis.
Area of Science:
- Genetics
- Molecular Biology
- Biochemistry
Background:
- T4-binding globulin (TBG) is a serum glycoprotein produced in the liver, responsible for transporting thyroid hormone.
- Understanding the genetic regulation of TBG is crucial for comprehending thyroid hormone homeostasis.
Purpose of the Study:
- To elucidate the genomic organization, promoter activity, and regulatory mechanisms of the human TBG (hTBG) gene.
- To identify key DNA elements and transcription factors involved in hTBG gene expression.
Main Methods:
- Isolation of a full-length hTBG cDNA clone and localization of the transcription start site (TSS).
- Analysis of genomic clones to determine exon-intron structure.
- Reporter gene assays using chloramphenicol acetyltransferase (CAT) constructs with varying lengths of the 5'-flanking region in HepG2 and other cell lines.
- Site-directed mutagenesis of putative regulatory elements.
Main Results:
- The hTBG gene comprises five exons with an organization similar to serine protease inhibitors.
- A 3.2 kbp 5'-flanking region, including a noncoding exon 0, demonstrated significant promoter activity in HepG2 cells.
- The phorbol ester 12-O-tetradecanoylphorbol-13-acetate markedly downregulated promoter activity and hTBG synthesis.
- Thyroid hormone, glucocorticoids, estrogen, and nicotinic acid had minimal impact on promoter activity.
- The minimal promoter region (-218 to +4) exhibited the highest activity, indicating liver-specific enhancer elements between -218 and -102.
- A hepatocyte nuclear factor-1 (HNF-1) binding site at -65 bp relative to the TSS was essential for promoter activity in all tested cell lines.
Conclusions:
- The hTBG gene structure is conserved within the serine protease inhibitor family.
- The promoter region contains essential elements for liver-specific transcription, including an HNF-1 binding site.
- Transcriptional regulation of hTBG is significantly influenced by specific signaling pathways, notably down-regulated by phorbol esters.