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 regulated during tooth development. In a previous study, the CCAAT/enhancer-binding protein alpha (C/EBPalpha) 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/EBPalpha 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/EBPalpha 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/EBPalpha is identified with co-immunoprecipitation analyses. Functional antagonism between Msx2 and C/EBPalpha is also observed on the stably transfected 2.2-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/EBPalpha, -beta, and -gamma), Msx2 preferentially interacts with C/EBPalpha. 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/EBPalpha in regulating the mouse amelogenin gene expression.
Zhou
Y L
YL
The Center for Craniofacial Molecular Biology, The University of Southern California, Los Angeles, California 90033, USA.
Lei
Y
Y
Snead
M L
ML
D053523:Amelogenin; D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology*; D003746:Dental Enamel Proteins / Q000235:genetics*; D005786:Gene Expression Regulation; D018398:Homeodomain Proteins; D051379:Mice; D009687:Nuclear Proteins / Q000502:physiology*; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000502:physiology*
D053523:Amelogenin; C507200:Amelx protein, mouse; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D003746:Dental Enamel Proteins; D018398:Homeodomain Proteins; C084119:MSX2 protein; D009687:Nuclear Proteins; D012097:Repressor Proteins; D004247:DNA