CCAAT/Enhancer-binding protein δ(C/EBP δ) maintains amelogenin expression in the absence of C/EBPα in vivo

C/ EBP alpha is implicated to regulate mouse amelogenin gene expression during tooth enamel formation in vitro. Because enamel formation occurs during postnatal development and C/ EBP alpha- deficient mice die at birth, we used the Cre/ loxP recombination system to characterize amelogenin expression in C/ EBP alpha 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 alpha allele with keratin14- Cre mice generate C/ EBP alpha conditional knock- out mice. Real- time PCR analysis shows that removal of one C/EBP alpha allele from the molar enamel epithelial organ of 3- day postnatal mice results in dramatic decrease in endogenous C/EBP alpha mRNAlevels and coordinately altered amelogeninm RNA abundance. Conditional deletion of both C/ EBP alpha alleles further diminishes C/ EBP alpha mRNAlevels; however, rather than ablating amelogenin expression, we observe wild- type amelogenin mRNA abundance levels. We examined C/ EBP beta 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 delta is also unchanged in C/ EBP alpha\ conditional knock- out mice, in vitro we find that C/ EBP delta activates the mouse amelogenin promoter and synergistically cooperates with nuclear factor Y, suggesting that C/ EBP delta can functionally substitute for C/ EBP alpha to produce an enamel matrix competent to direct biomineralization.