title: C/EBPalpha and C/EBPdelta activate the clara cell secretory protein gene through interaction with two adjacent C/EBP-binding sites.
abstract: 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)alpha is involved in controlling differentiation-dependent gene expression in the distal lung. In this study, immunofluorescence studies demonstrated high level expression of C/EBPdelta in the bronchiolar epithelium as well as lower levels of C/EBPalpha. Cotransfection studies in the lung epithelial cell line A549 showed that both C/EBPalpha and C/EBPdelta activate the murine CCSP gene and that a C/EBP-response element resides in the proximal CCSP promoter. C/EBPdelta exhibits an approximately 2-fold higher transactivation potential than does C/EBPalpha. 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/EBPalpha and C/EBPdelta, 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/EBPalpha and C/EBPdelta can bind to both C/EBP sites, whereas in DNase I footprint analyses, the interaction of C/EBPalpha with the proximal site was weak. Furthermore, electrophoretic mobility shift assays demonstrated that C/EBPalpha and C/EBPdelta preferentially form heterodimers at both binding sites. Cotransfections with C/EBPalpha and C/EBPdelta together resulted in a superinduction of the CCSP promoter, indicating a regulatory role for the C/EBPalpha-C/EBPdelta heterodimers. Our findings demonstrate that C/EBPalpha and C/EBPdelta regulate the CCSP gene through a compound response element and suggest that these factors are important for the differentiation-dependent expression of CCSP.
authors.affiliation: Department of Medical Nutrition, Karolinska Institutet, Huddinge, Sweden.
authors.forename: T N
authors.initials: TN
authors.lastname: Cassel
authors.forename: L
authors.initials: L
authors.lastname: Nordlund-Möller
authors.forename: O
authors.initials: O
authors.lastname: Andersson
authors.forename: J A
authors.initials: JA
authors.lastname: Gustafsson
authors.forename: M
authors.initials: M
authors.lastname: Nord
mesh_terms: D000230:Adenocarcinoma / Q000473:pathology; D000818:Animals; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D019556:COS Cells; D002454:Cell Differentiation; D002522:Chlorocebus aethiops; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D019281:Dimerization; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation / Q000502:physiology*; D008175:Lung Neoplasms / Q000473:pathology; D051379:Mice; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry / Q000502:physiology*; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D015533:Transcriptional Activation; D014162:Transfection; D014598:Uteroglobin
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C556390:Scgb1a1 protein, mouse; D014598:Uteroglobin