Human SP-C gene sequences that confer lung epithelium-specific expression in transgenic mice

Abstract

We used transgenic mice to identify cis-active regions of the human pulmonary surfactant protein C ( SP-C) gene that impart tissue- and cell-specific expression in vivo in the lung. Approximately 3.7 kb of genomic SP-C DNA upstream of the transcription start site was sufficient to direct chloramphenicol acetyltransferase ( CAT) reporter gene expression specifically in bronchiolar and alveolar epithelial cells of the lung. To further define cis-active regulatory elements that mediate cell-specific expression, we tested deletions of the parental 3.7-kb human SP-C sequence in transgenic mice. Tissue CAT assays of mice generated with truncations or overlapping internal deletions of the 3.7-kb construct functionally map alveolar cell-specific regulatory elements to within −215 bp of the SP-C promoter. Analysis of SP-C promoter deletions demonstrate that sequences between −3.7 kb and −1.9 kb contain enhancer sequences that stimulate SP-C transgene expression. In situ hybridization studies demonstrate that deletion of the −1,910- to −215-bp region abolishes the ectopic bronchiolar expression seen with the original 3.7-kb SP-C promoter construct. Comparison of sequences from −215 to +1 bp identified consensus binding sites for the homeodomain transcription factor thyroid transcription factor-1 (TTF-1). Cotransfection assays of the human 3.7-kb SP-C or −1,910- to −215-bp SP-C deletion construct with a TTF-1 expression plasmid demonstrates that TTF-1 transactivates the human SP-C gene. These results suggest that the TTF-1 cis-active sites are important in directing cell-specific expression of the SP-C gene in vivo.