C/EBP alpha and C/EBP delta activate the Clara cell secretory protein genethrough interaction with two adjacent C/EBP-binding sites

The Clara cell secretory protein (CCSP) gene is a cell-specific differentia tion marker for the bronchiolar Clara cell. Previous studies suggest that C CAAT/enhancer binding protein (C/EBP)alpha is involved in controlling diffe rentiation-dependent gene expression in the distal lung. In this study, imm unofluorescence studies demonstrated high level expression of C/EBP delta i n the bronchiolar epithelium as well as lower levels of C/EBP alpha. Cotran sfection studies in the lung epithelial cell line A549 showed that both C/E BP alpha and C/EBP delta activate the murine CCSP gene and that a C/EBP-res ponse element resides in the proximal CCSP promoter. C/EBP delta exhibits a n approximately 2-fold higher transactivation potential than does C/EBP alp ha. DNase I footprint analyses revealed a footprint region located at -100 to -62 bp, corresponding to two C/EBP-binding sites. Mutation of either sit e resulted in abolished or strikingly reduced transactivation of the CCSP p romoter by C/EBP alpha and C/EBP delta, as well as impaired binding of both factors, indicating that the two C/EBP-binding sites form a compound respo nse element. In electrophoretic mobility shift assays, it was shown that C/ EBP alpha and C/EBP delta can bind to both C/EBP sites, whereas in DNase 1 footprint analyses, the interaction of C/EBP alpha with the proximal site w as weak. Furthermore, electrophoretic mobility shift assays demonstrated th at C/EBP alpha and C/EBP delta preferentially form heterodimers at both bin ding sites. Cotransfections with C/EBP alpha and C/EBP delta together resul ted in a superinduction of the CCSP promoter, indicating a regulatory role for the C/EBP alpha-C/EBP delta heterodimers, Our findings demonstrate that C/EBP alpha and C/EBP delta regulate the CCSP gene through a compound resp onse element and suggest that these factors are important for the different iation-dependent expression of CCSP.