ALUNG May 22/5

Glasser, Stephan W., Michael S. Burhans, Susan K. Eszterhas, Michael D. Bruno, and Thomas R. Korfhagen. Human SP-C gene sequences that confer lung epithelium-specific expression in transgenic mice. Am J Physiol Lung Cell Mol Physiol 278: L933–L945, 2000.—We used transgenic mice to identify cis-active regions of the human pulmonary surfactant protein C (SP-C) gene that impart tissueand cell-specific expression in vivo in the lung. Approximately 3.7 kb of genomic SP-C DNAupstream 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 2215 bp of the SP-C promoter. Analysis of SP-C promoter deletions demonstrate that sequences between 23.7 kb and 21.9 kb contain enhancer sequences that stimulate SP-C transgene expression. In situ hybridization studies demonstrate that deletion of the 21,910to 2215-bp region abolishes the ectopic bronchiolar expression seen with the original 3.7-kb SP-C promoter construct. Comparison of sequences from 2215 to 11 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 21,910to 2215-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.