Loss of DNA-binding Activities of 219InsG-PAX9 is Associated with Molar Oligodontia

The mammalian PAX9 gene encodes a protein that plays pivotal roles in organogenesis, specifically in embryonic patterning and morphogenesis. Previously, we reported that a frameshift mutation (219InsG) in the paired DNA-binding domain of PAX9 is associated with molar Oligodontia. This paired domain mutation likely inactivates the DNA-binding activities of 219InsG-PAX9 protein (J. Dent. Res. 81(SI):166,2002). Objectives: The objectives of this study are to assess the functional consequences of 219InsG mutation in PAX9 and to provide an understanding of the transcriptional mechanisms that control PAX9-mediated pathway in tooth development. Method: The experimental method involved the use of gel mobility shift assay to examine the differences in the DNA-binding activities of 219InsG-PAX9 and wild-type PAX9. We chose the Drosophila even skipped promoter (e5) as our putative Pax9 binding site and have constructed a PAX9 luciferase reporter containing six tandem copies of e5 upstream of the TK promoter (6e5TKLuc). Result: Our results show that 219InsG-PAX9 does not bind to the paired domain recognition motif (GTTC) present in e5. Tansient co-transfection assays with the 6e5TKLuc reporter in COS-7 and NIH-3T3 is currently being used to examine the differences in transcriptional activation between the two proteins. Conclusion: Loss of DNA-binding activities of 219InsG-PAX9 abrogates PAX9-mediated transcriptional regulation of target genes during early tooth development and contributes to the observed tooth phenotype. Ultimately, our study will define a novel mechanism for PAX9-mediated regulation of normal tooth development. Supported by THECB-ATP 004949 0143 1999 (RDS).