Identification of CCAAT/enhancer-binding protein alpha as a transactivator of the mouse amelogenin gene.
Amelogenin expression is ameloblast-specific and developmentally regulated at the temporal and spatial levels. In a previous transgenic mouse analysis, the expression pattern of the endogenous amelogenin gene was recapitulated by a reporter gene driven by a 2. 2-kilobase mouse amelogenin proximal promoter. To understand the molecular mechanisms underlying the spatiotemporal expression of the amelogenin gene during odontogenesis, the mouse amelogenin promoter was systematically analyzed in mouse ameloblast-like LS8 cells. Deletion analysis identified a minimal promoter (-70/+52) containing a CCAAT/enhancer-binding protein (C/EBP)-binding site upstream of the TATA box. In transient transfection assays, C/EBPalpha up-regulated the promoter activity in a dose-dependent manner. The C/EBP-binding site was necessary for both C/EBPalpha-mediated transactivation and basal promoter activity. Electrophoresis mobility shift assays demonstrated that C/EBPalpha bound to its cognate site in the amelogenin promoter and that the binding was specific. Endogenous C/EBPalpha was detected in LS8 cells, and overexpression of exogenous C/EBPalpha in LS8 cells was able to increase the expression level of the endogenous amelogenin protein. The activity of the amelogenin promoter in rat parotid Pa-4 cells and Madin-Darby canine kidney cells was minimal, ranging from 20 to 30% of the activity in ameloblast-like cells. Transient transfection experiments showed that C/EBPalpha transactivated the mouse amelogenin reporter gene in Pa-4 cells, but not in Madin-Darby canine kidney cells. Taken together, these data indicate that C/EBPalpha is a bona fide transcriptional activator of the mouse amelogenin gene in a cell type-specific manner.
Zhou
Y L
YL
Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, California 90033, USA.
Snead
M L
ML
D053523:Amelogenin; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000502:physiology*; D003746:Dental Enamel Proteins / Q000235:genetics*; D004285:Dogs; D005786:Gene Expression Regulation; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000502:physiology*; D011401:Promoter Regions, Genetic; D051381:Rats; D015533:Transcriptional Activation; D014162:Transfection
D053523:Amelogenin; C507200:Amelx protein, mouse; C507201:Amelx protein, rat; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D003746:Dental Enamel Proteins; D009687:Nuclear Proteins