Identification of CCAAT/enhancer-binding protein alpha as a transactivatorof 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 recapitulate d by a reporter gene driven by a 2.2-kilobase mouse amelogenin proximal pro moter. To understand the molecular mechanisms underlying the spatiotemporal expression of the amelogenin gene during odontogenesis, the mouse amelogen in promoter was systematically analyzed in mouse ameloblast-like LS8 cells. Deletion analysis identified a minimal promoter (-70/+52) containing a CCA AT/enhancer-binding protein (C/EBP)-binding site upstream of the TATA box. In transient transfection assays, C/EBP alpha up-regulated the promoter act ivity in a dose-dependent manner. The C/EBP-binding site was necessary for both C/EBP alpha-mediated transactivation and basal promoter activity. Elec trophoresis mobility shift assays demonstrated that C/EBP alpha bound to it s cognate site in the amelogenin promoter and that the binding was specific . Endogenous C/EBPa was detected in LS8 cells, and overexpression of exogen ous C/EBPa in LS8 cells was able to increase the expression level of the en dogenous amelogenin protein. The activity of the amelogenin promoter in rat parotid Pa-4 cells and Madin-Darby canine kidney cells was minimal, rangin g from 20 to 30% of the activity in ameloblast-like cells. Transient transf ection experiments showed that C/EBPa transactivated the mouse amelogenin r eporter gene in Pa-4 cells, but not in Madin-Darby canine kidney cells. Tak en together, these data indicate that C/EBP alpha is a bona fide transcript ional activator of the mouse amelogenin gene in a cell type-specific manner .