Functional antagonism between Msx2 and CCAAT/enhancer-binding protein alpha in regulating the mouse amelogenin gene expression is mediated by protein-protein interaction

Ameloblast-specific amelogenin gene expression is spatiotemporally regulate d during tooth development. In a previous study, the CCAAT/enhancer-binding protein alpha (C/EBP alpha) was identified as a transcriptional activator of the mouse amelogenin gene in a cell type-specific manner. Here, Msx2 is shown to repress the promoter activity of amelogenin-promoter reporter cons tructs independent of its intrinsic DNA binding activity. In transient cotr ansfection assays, Msx2 and C/EBP alpha antagonize each other in regulating the expression of the mouse amelogenin gene. Electrophoresis mobility shif t assays demonstrate that Msx2 interferes with the binding of C/EBP alpha t o its cognate site in the mouse amelogenin minimal promoter, although Msx2 itself does not bind to the same promoter fragment. Protein-protein interac tion between Msx2 and C/EBP alpha is identified with co-immunoprecipitation analyses. Functional antagonism between Msx2 and C/EBP alpha is also obser ved on the stably transfected a,a-kilobase mouse amelogenin promoter in ame loblast-like LS8 cells, Furthermore, the carboxyl-terminal residues 183-267 of Msx2 are required for protein-protein interaction, whereas the amino-te rminal residues 2-97 of Msx2 play a less critical role. Among three family members tested (C/EBP alpha, -beta, and -gamma), Msx2 preferentially intera cts with C/EBP alpha. Taken together, these data indicate that protein-prot ein interaction rather than competition for overlapping binding sites resul ts in the functional antagonism between Msx2 and C/EBP alpha in regulating the mouse amelogenin gene expression.