NF-Y and CCAAT/enhancer-binding protein α synergistically activate the mouse amelogenin gene

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 alpha(C/EBP alpha) is a potent transactivator of the mouse X-chromosomal amelogenin gene acting at the C/EBP alpha 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 alpha binding site. Similar to the C/EBP alpha 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 alpha 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 alpha. We identified protein-protein interactions between C/ EBP alpha and NF-Y by a co-immunoprecipitation analysis. These results suggest that C/EBP alpha and NF-Y synergistically activate the mouse amelogenin gene and can contribute to its physiological regulation during amelogenesis.