Regulation of Clara cell secretory protein gene transcription by thyroid transcription factor-1.
CCSP is a 16 kDa protein expressed selectively in the Clara cells of the lung. Cis-acting elements conferring Clara cell-specific expression of rat CCSP gene were contained within the sequences -320 to +57 of the rCCSP promoter. DNA-TTF-1 protein interactions were identified within the sequences -302 to -278 and -90 to -66 from the transcriptional start site of the rat CCSP gene promoter. In electrophoretic mobility shift assays, recombinant TTF-1 homeodomain protein bound to each site. Nuclear extracts from H441 adenocarcinoma cells bound to the TTF-1 binding sites, were supershifted by anti-TTF-1 antibody, and were competed by other TTF-1 DNA binding motifs. Mutations that replaced two nucleotides in each of the TTF-1 binding motifs disrupted TTF-1 binding to the rCCSP promoter. In HeLa cells, mutagenesis of both TTF-1 sites reduced transactivation by TTF-1, while the activities of single-site mutants were similar to that of the wild-type plasmid. In H441 cells, transactivation by TTF-1 was inhibited by mutations of each or both of the TTF-1 binding sites. TTF-1 activates transcription of the rCCSP gene, binding to distinct but interacting cis-acting elements in the 5'-flanking region of the gene.
Zhang
L
L
Children's Hospital Medical Center, Division of Pulmonary Biology, TCHRF, Cincinnati, OH 45229-3039, USA.
Whitsett
J A
JA
Stripp
B R
BR
D000230:Adenocarcinoma; D000818:Animals; D002457:Cell Extracts; D002467:Cell Nucleus; D004247:DNA / Q000378:metabolism; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008168:Lung / Q000378:metabolism; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011506:Proteins / Q000235:genetics*; D051381:Rats; D011993:Recombinant Fusion Proteins; D013961:Thyroid Gland; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics; D014407:Tumor Cells, Cultured; D014598:Uteroglobin
D002457:Cell Extracts; D018398:Homeodomain Proteins; C000614412:NKX2-1 protein, human; C000614416:Nkx2-1 protein, rat; D009687:Nuclear Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C556389:SCGB1A1 protein, human; C556391:Scgb1a1 protein, rat; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors; D004247:DNA; D014598:Uteroglobin