Thyroid-specific enhancer-binding protein (T/EBP): cDNA cloning, functional characterization, and structural identity with thyroid transcription factor TTF-1.
A cDNA clone encoding a thyroid-specific enhancer-binding protein (T/EBP) was isolated from a rat thyroid-derived FRTL-5 cell lambda gt 11 expression library, using a double-stranded oligonucleotide probe. This oligonucleotide was previously demonstrated to have the strongest binding affinity among three cis-acting DNA elements within the thyroid-specific enhancer region located 5.5 kbp upstream of the human thyroid peroxidase gene transcription start site. Nucleotide and deduced amino acid sequences of the cDNA revealed that T/EBP is identical to the previously reported thyroid-specific transcription factor 1 (TTF-1), which binds to the promoter of the rat thyroglobulin gene and controls its thyroid-specific expression. Expression of the T/EBP cDNA under control of the human cytomegalovirus major immediate-early gene promoter conferred thyroid-specific enhancer activity of as high as 26-fold to nonpermissive human hepatoma HepG2 cells when cotransfected with a vector containing 6.3 kbp of upstream sequence of the human thyroid peroxidase gene connected to a luciferase reporter gene. T/EBP was further expressed in HepG2 cells by using the vaccinia virus expression system. The expressed protein was partially purified by using sequence-specific affinity column chromatography and was further shown, by gel mobility shift experiments, to specifically bind to the enhancer-derived double-stranded oligonucleotide. These results clearly indicate that the binding of T/EBP (TTF-1) to the specific cis-acting enhancer element is largely responsible for thyroid-specific enhancer activity.
Mizuno
K
K
Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892.
Gonzalez
F J
FJ
Kimura
S
S
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular; D004742:Enhancer Elements, Genetic / Q000502:physiology; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D007453:Iodide Peroxidase / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012689:Sequence Homology, Nucleic Acid; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014616:Vaccinia virus / Q000235:genetics
C000614412:NKX2-1 protein, human; C000614416:Nkx2-1 protein, rat; D009687:Nuclear Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors; D007453:Iodide Peroxidase; D008156:Luciferases