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Originally published In Press as doi:10.1074/jbc.M002031200 on June 19, 2000

J. Biol. Chem., Vol. 275, Issue 37, 29066-29075, September 15, 2000
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Functional Antagonism between Msx2 and CCAAT/Enhancer-binding Protein alpha  in Regulating the Mouse Amelogenin Gene Expression Is Mediated by Protein-Protein Interaction*

Yan Larry Zhou, Yaping Lei, and Malcolm L. SneadDagger

From the The Center for Craniofacial Molecular Biology, The University of Southern California, Los Angeles, California 90033

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.

* This work was supported by Grant DE06988 from NIDCR, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: CSA142, CCMB, The University of Southern California, 2250 Alcazar St., Los Angeles, CA 90033. Tel.: 323-442-3178; Fax: 323-442-2981; E-mail: mlsnead@ hsc.usc.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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