SP-C is a hydrophobic pulmonary surfactant protein which enhances the spreading and stability of surfactant phospholipids to prevent alveolar collapse. This application seeks to determine the mechanisms regulating the ontogenic and cell specific expression of the surfactant protein C (SP-C) gene. We have generated transgenic mice bearing regions of the human SP-C gene that directs CAT reporter gene expression. These mice express SP-C transgenes in epithelial cells of the distal region of the lung in a distribution consistent with localization of SP-C protein in human lung. Expression of the SP-C CAT transgene is developmentally and hormonally regulated. DNase hypersensitivity, gel shift analysis, and DNA footprinting will be used to identify the cis-active elements in the transgene. We will use transfection studies to analyze deletion constructs of the original SP-C CAT construct and functionally map cis- active regulating elements of the SP-C DNA in immortalized cells from lung tumors of SP-C-SV4O large T antigen transgenic mice. CAT assays will be conducted to detect promoter activation by the SP-C cis-active regulatory element(s). SP-C cis-active elements which function in vitro will be used to generate transgenic mice to demonstrate lung specific regulation in vivo. Regions of the SP-C gene implicated in cell specific expression will be used as probes to screen lung expression cDNA libraries to isolate clones encoding the SP-C gene binding proteins. Biochemical and functional analysis of the SP-C DNA binding clones will be used to characterize the protein-SP-C DNA binding. These studies seek to 1) identify the cis-acting elements which determine transcriptional control of SP-C in developing mouse lung, 2) test the function of SP-C elements to activate expression in immortalized lung cells and in vivo, and 3) to identify and characterize the transacting protein(s) which activate SP-C gene expression in an appropriate cell restricted manner.
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