Functional Antagonism between Msx2 and CCAAT/Enhancer-binding Protein α in Regulating the Mouse Amelogenin Gene Expression Is Mediated by Protein-Protein Interaction

Yan Larry Zhou,Yaping Lei,Malcolm L Snead

doi:10.1074/jbc.m002031200

Yan Larry Zhou, Yaping Lei + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m002031200

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Abstract

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.

Highlights

Enamel is the only epithelially derived calcified tissue in vertebrates
Electrophoresis mobility shift assays demonstrate that Msx2 interferes with the binding of CCAAT/enhancer-binding protein (C/EBP)␣ to its cognate site in the mouse amelogenin minimal promoter, Msx2 itself does not bind to the same promoter fragment
Msx2 Was a Transcriptional Repressor of Mouse Amelogenin Promoter—To determine whether Msx2 could regulate the promoter activity of the mouse amelogenin gene, a Msx2 expression plasmid was cotransfected into ameloblast-like LS8 cells with a series of 5Ј-deletion amelogenin-promoter reporter constructs

Summary

Introduction

Enamel is the only epithelially derived calcified tissue in vertebrates. Amelogenin, the major organic component of enamel matrix, is essential to the proper regulation of enamel mineralization. Extensive homologies (70% identity) in the 300-nucleotide region upstream of the transcription initiation site exist between the murine, bovine, and human X-chromosomal amelogenin gene, suggesting that this region is likely involved in the transcriptional regulation of tissue-specific amelogenin gene expression. The minimal promoter of the mouse amelogenin gene (Ϫ70/ϩ52) was identified, which contains a CCAAT/enhancer-binding protein (C/EBP) consensus binding site, and C/EBP␣ activated amelogenin transcription in a celltype specific manner through binding to its cognate site [14]. The C/EBPs consist of a family of related basic region leucine zipper transcription factors that are critical regulators of cellular differentiation and function in multiple tissues. The expression of C/EBPs is tissue- and stage-specific during development. Msx and C/EBP␣ Antagonism on the Amelogenin Promoter tion with either components of basal transcription machinery or other transcription factors [31,32,33,34,35]

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