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J. Biol. Chem., Vol. 281, Issue 23, 16090-16098, June 9, 2006

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NF-Y and CCAAT/Enhancer-binding Protein Synergistically Activate the Mouse Amelogenin Gene^*

Yucheng Xu, Yan Larry Zhou, Wen Luo, Qin-Shi Zhu, Daniel Levy, Ormond A. MacDougald^¶, and Malcolm L. Snead¹

From the Center for Craniofacial Molecular Biology and Department of Biochemistry and Molecular Biology, University of Southern California, Los Angeles, California 90033 and ^¶Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0622

Amelogenin is the major protein component of the forming enamel matrix. In situ hybridization revealed a periodicity for amelogenin mRNA hybridization signals ranging from low to high transcript abundance on serial sections of developing mouse teeth. This in vivo observation led us to examine the amelogenin promoter for the activity of transcription factor(s) that account for this expression aspect of the regulation for the amelogenin gene. We have previously shown that CCAAT/enhancer-binding protein (C/EBP) is a potent transactivator of the mouse X-chromosomal amelogenin gene acting at the C/EBP cis-element located in the –70/+52 minimal promoter. The minimal promoter contains a reversed CCAAT box (–58/–54) that is four base pairs downstream from the C/EBP binding site. Similar to the C/EBP binding site, the integrity of the reversed CCAAT box is also required for maintaining the activity of the basal promoter. We therefore focused on transcription factors that interact with the reversed CCAAT box. Using electrophoretic mobility shift assays we demonstrated that NF-Y was directly bound to this reversed CCAAT site. Co-transfection of C/EBP and NF-Y synergistically increased the promoter activity. In contrast, increased expression of NF-Y alone had only marginal effects on the promoter. A dominant-negative DNA binding-deficient NF-Y mutant (NF-YAm29) dramatically decreased the promoter activity both in the absence or presence of exogenous expression of C/EBP. We identified protein-protein interactions between C/EBP and NF-Y by a co-immunoprecipitation analysis. These results suggest that C/EBP and NF-Y synergistically activate the mouse amelogenin gene and can contribute to its physiological regulation during amelogenesis.

Received for publication, September 26, 2005 , and in revised form, February 10, 2006.

^* This work was supported by NIDCR, 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 Fig. S1.

¹ To whom correspondence should be addressed: CSA 142, CCMB, University of Southern California, 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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