Transcription of the lung-specific surfactant protein C gene is mediated by thyroid transcription factor 1.
Surfactant protein C (SP-C) is expressed in alveolar Type II epithelial cells of the lung. In order to determine the mechanism(s) that regulate gene transcription, we have analyzed the activation of the murine SP-C promoter in mouse lung epithelial cells (MLE cells) and in HeLa cells after co-transfection with a vector expressing rat thyroid transcription factor-1 (TTF-1). TTF-1 transactivated SP-C-chloramphenicol acetyltransferase constructs containing -13 kilobase pairs to -320 base pairs (bp) of the 5 flanking region of the SP-C gene. Essential cis-acting elements were functionally localized to between -320 and -180 bp from the start of transcription by transfection analysis. Five DNase-protected regions, indicating multiple protein-DNA interactions within the -320 bp TTF-1-responsive region of the SP-C gene, were identified by DNase footprint analysis. A 40-bp segment of SP-C DNA from -197 to -158 linked to a heterologous promoter-chloramphenicol acetyltransferase construct activated expression after co-transfection with CMV-TTF-1 in HeLa and MLE cells. The -197 to -158 segment contained two consensus TTF-1 sites, which were specifically identified as TTF-1 binding sites by gel retardation and antibody supershift with MLE cell nuclear extracts and purified TTF-1 homeodomain protein. Site-specific mutagenesis of either of the TTF-1 binding sites completely blocked activation by TTF-1, indicating both sites are required for TTF stimulation of SP-C transcription.
Kelly
S E
SE
Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039, USA.
Bachurski
C J
CJ
Burhans
M S
MS
Glasser
S W
SW
D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011510:Proteolipids / Q000235:genetics*; D011663:Pulmonary Surfactants / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
C000614412:NKX2-1 protein, human; C000614414:Nkx2-1 protein, mouse; D009687:Nuclear Proteins; D011510:Proteolipids; D011663:Pulmonary Surfactants; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D013937:Thymidine Kinase