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J. Biol. Chem., Vol. 282, Issue 41, 29882-29889, October 12, 2007

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CCAAT/Enhancer-binding Protein (C/EBP) Maintains Amelogenin Expression in the Absence of C/EBP in Vivo^*

Yucheng Xu, Yan Larry Zhou, Frank J. Gonzalez, and Malcolm L. Snead¹

From the The Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, California 90033 and Laboratory of Metabolism, NCI, National Institutes of Health, Bethesda, Maryland 20892

C/EBP is implicated to regulate mouse amelogenin gene expression during tooth enamel formation in vitro. Because enamel formation occurs during postnatal development and C/EBP-deficient mice die at birth, we used the Cre/loxP recombination system to characterize amelogenin expression in C/EBP conditional knock-out mice. Mice carrying the Cre transgene under the control of the human keratin-14 promoter show robust Cre expression in the ameloblast cell lineage. Mating between mice bearing the floxed C/EBP allele with keratin-14-Cre mice generate C/EBP conditional knock-out mice. Real-time PCR analysis shows that removal of one C/EBP allele from the molar enamel epithelial organ of 3-day postnatal mice results in dramatic decrease in endogenous C/EBP mRNA levels and coordinately altered amelogenin mRNA abundance. Conditional deletion of both C/EBP alleles further diminishes C/EBP mRNA levels; however, rather than ablating amelogenin expression, we observe wild-type amelogenin mRNA abundance levels. We examined C/EBP and nuclear factor YA expression, two transcription factors that had previously been shown to modestly participate in amelogenin expression, in vitro but found no significant changes in either of their mRNA abundance levels comparing conditional knock-out mice with wild-type counterparts. Although the abundance of C/EBP is also unchanged in C/EBP conditional knock-out mice, in vitro we find that C/EBP activates the mouse amelogenin promoter and synergistically cooperates with nuclear factor Y, suggesting that C/EBP can functionally substitute for C/EBP to produce an enamel matrix competent to direct biomineralization.

Received for publication, March 12, 2007 , and in revised form, August 16, 2007.

^* This work was supported by National Institute for Dental and Craniofacial Research, National Institutes of Health Grant DE-06988. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1 and Figs. S1 and S2.

¹ To whom correspondence should be addressed: The Center for Craniofacial Molecular Biology, University of Southern California, CSA 142, 2250 Alcazar St., Los Angeles, CA 90033. Tel.: 323-442-3178; Fax: 323-442-2981; E-mail: mlsnead{at}usc.edu.

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