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.