C/EBPα and C/EBPδ activate the Clara cell secretory protein gene through interaction with two adjacent C/EBP-binding sites

The Clara cell secretory protein (CCSP) gene is a cell-specific differentiation marker for the bronchiolar Clara cell. Previous studies suggest that CCAAT/enhancer binding protein (C/EBP)α is involved in controlling differentiation-dependent gene expression in the distal lung. In this study, immunofluorescence studies demonstrated high level expression of C/EBPδ in the bronchiolar epithelium as well as lower levels of C/EBPα. Cotransfection studies in the lung epithelial cell line A549 showed that both C/EBPα and C/EBPδ activate the murine CCSP gene and that a C/EBP-response element resides in the proximal CCSP promoter. C/EBPδ exhibits an approximately 2-fold higher transactivation potential than does C/EBPα. DNase I footprint analyses revealed a footprint region located at -100 to -62 bp, corresponding to two C/EBP-binding sites. Mutation of either site resulted in abolished or strikingly reduced transactivation of the CCSP promoter by C/EBPα and C/EBPδ, as well as impaired binding of both factors, indicating that the two C/EBP-binding sites form a compound response element. In electrophoretic mobility shift assays, it was shown that C/EBPα and C/EBPδ can bind to both C/EBP sites, whereas in DNase I footprint analyses, the interaction of C/EBPα with the proximal site was weak. Furthermore, electrophoretic mobility shift assays demonstrated that C/EBPα and C/EBPδ preferentially form heterodimers at both binding sites. Cotransfections with C/FBPα and C/EBPδ together resulted in a superinduction of the CCSP promoter, indicating a regulatory role for the C/EBPα-C/EBPδ heterodimers. Our findings demonstrate that C/EBPα and C/EBPδ regulate the CCSP gene through a compound response element and suggest that these factors are important for the differentiation-dependent expression of CCSP.