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

Ameloblast-specific amelogenin gene expression is spatiotemporally regulated 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 constructs independent of its intrinsic DNA binding activity. In transient cotransfection assays, Msx2 and C/EBP alpha antagonize each other in regulating the expression of the mouse amelogenin gene. Electrophoresis mobility shift assays demonstrate that Msx2 interferes with the binding of C/EBP alpha to its cognate site in the mouse amelogenin minimal promoter, although Msx2 itself does not bind to the same promoter fragment. Protein-protein interaction between Msx2 and C/EBP alpha is identified with co-immunoprecipitation analyses. Functional antagonism between Msx2 and C/EBP alpha is also observed on the stably transfected a,a-kilobase mouse amelogenin promoter in ameloblast-like LS8 cells, Furthermore, the carboxyl-terminal residues 183-267 of Msx2 are required for protein-protein interaction, whereas the amino-terminal residues 2-97 of Msx2 play a less critical role. Among three family members tested (C/EBP alpha, -beta, and -gamma), Msx2 preferentially interacts with C/EBP alpha. Taken together, these data indicate that protein-protein interaction rather than competition for overlapping binding sites results in the functional antagonism between Msx2 and C/EBP alpha in regulating the mouse amelogenin gene expression.