Characterization of a thyroid-specific enhancer located 5.5 kilobase pairs upstream of the human thyroid peroxidase gene.
A 6.3-kbp segment of DNA, upstream of the human thyroid peroxidase gene, and various deletions thereof were linked to a promoterless bacterial chloramphenicol acetyltransferase reporter gene. These constructs were analyzed by transfection and expression in rat FRTL-5 thyroid cells and in human hepatoma HepG2 cells to localize sequences that are important for thyroid cell-specific expression of the thyroid peroxidase gene. A thyroid-specific enhancer element, capable of activating enhancerless simian virus 40 promoter expression in FRTL-5 cells, was localized to a 230-bp region approximately 5.5 kbp upstream of the human thyroid peroxidase gene transcription start site. DNase I footprinting, using nuclear extracts prepared from FRTL-5 cells, revealed three regions within the 230-bp fragment; none of these regions were protected by nuclear extracts from HepG2 cells. Gel mobility shift assays, using double-stranded oligonucleotides corresponding to the three protected regions, further confirmed the existence of factors in FRTL-5 cells, but not HepG2 cells, able to specifically bind to the enhancer sequences. These results suggest the presence of three cis-acting DNA elements in the human thyroid peroxidase gene enhancer that interact with thyroid-specific trans-acting factors.
Kikkawa
F
F
Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892.
Gonzalez
F J
FJ
Kimura
S
S
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D002872:Chromosome Deletion; D004742:Enhancer Elements, Genetic; D005796:Genes; D006801:Humans; D007453:Iodide Peroxidase / Q000235:genetics*; D008113:Liver Neoplasms; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D013961:Thyroid Gland; D014162:Transfection
D015345:Oligonucleotide Probes; D011993:Recombinant Fusion Proteins; D007453:Iodide Peroxidase; D015500:Chloramphenicol O-Acetyltransferase