NF-Y and CCAAT/enhancer-binding protein alpha 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/EBPalpha) is a potent transactivator of the mouse X-chromosomal amelogenin gene acting at the C/EBPalpha 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/EBPalpha binding site. Similar to the C/EBPalpha 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/EBPalpha 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/EBPalpha. We identified protein-protein interactions between C/EBPalpha and NF-Y by a co-immunoprecipitation analysis. These results suggest that C/EBPalpha and NF-Y synergistically activate the mouse amelogenin gene and can contribute to its physiological regulation during amelogenesis.
Xu
Yucheng
Y
Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, California 90033, USA.
Zhou
Yan Larry
YL
Luo
Wen
W
Zhu
Qin-Shi
QS
Levy
Daniel
D
MacDougald
Ormond A
OA
Snead
Malcolm L
ML
D053523:Amelogenin; D000818:Animals; D001483:Base Sequence; D023081:CCAAT-Binding Factor / Q000502:physiology*; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000502:physiology*; D017931:DNA Primers; D003746:Dental Enamel Proteins / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression / Q000502:physiology*; D006801:Humans; D017403:In Situ Hybridization; D051379:Mice; D011401:Promoter Regions, Genetic; D012689:Sequence Homology, Nucleic Acid; D014960:X Chromosome
D053523:Amelogenin; C507200:Amelx protein, mouse; D023081:CCAAT-Binding Factor; D022763:CCAAT-Enhancer-Binding Protein-alpha; D017931:DNA Primers; D003746:Dental Enamel Proteins