Identification of a functional thyroid hormone response element in the upstream flanking region of the human Na,K-ATPase beta 1 gene.
The human Na,K-ATPase beta 1 subunit gene promoter activity is stimulated by thyroid hormone (T3) in the human intestinal Caco-2 cells. To identify potential cis-acting transcriptional regulatory elements involved in this process, chimeric plasmids containing varying lengths of the 5' flanking region of the human beta 1 Na,K-ATPase gene linked to the firefly luciferase reporter gene were introduced into Caco-2 cells by transient transfection. Analysis of T3-regulated luciferase activity of cells carrying these plasmids, and subsequent use of site-directed mutagenesis revealed that a region from -459 to -438 (relative to the transcriptional start site) is required for the induction of the beta 1 Na,K-ATPase gene by T3. An oligonucleotide containing this sequence from -465 to -433 confers T3 responsiveness to a heterologous promoter. Gel mobility shift assays showed specific binding of nuclear proteins of Caco-2 cells to this region and immunoreactive T3 receptor was identified in one of these complexes. These data demonstrate that there is a cis-acting thyroid hormone responsive element in the 5' flanking region of the human beta 1 Na,K-ATPase gene and induction of transcription of this gene by T3 involves specific binding of the thyroid hormone receptor to the TRE located at position -459 to -438.
Feng
J
J
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, OH 45267-0524.
Orlowski
J
J
Lingrel
J B
JB
D000230:Adenocarcinoma; D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D001517:Coleoptera / Q000201:enzymology / Q000235:genetics; D003110:Colonic Neoplasms; D004247:DNA / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics; D014158:Transcription, Genetic; D014284:Triiodothyronine / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D011993:Recombinant Fusion Proteins; D014284:Triiodothyronine; D004247:DNA; D008156:Luciferases; D013937:Thymidine Kinase; D000254:Sodium-Potassium-Exchanging ATPase