Functional studies of human PAX9 paired domain mutations

Mutations in the paired-domain transcription factor PAX9 are associated with non-syndromic tooth agenesis which primarily affects posterior dentition. Of the 11 published mutations in the PAX9 gene, 6 are missense mutations which localize to the evolutionarily conserved DNA-binding domain, the paired-domain. Objectives: The aim of this study was to perform a functional and molecular characterization of the 6 missense mutations in the paired domain of the PAX9 gene. Methods: Using site-directed mutagenesis, we generated all six missense mutations in a PAX9 expression vector. Immunofluorescence and immunoblotting were used to localize the subcellular expression pattern of the mutant proteins. Electromobility shift assay was used to analyze the ability of the mutant proteins to bind a consensus binding site for the paired domain. Transcriptional assays using a Bmp4 promoter-reporter construct were used to determine the ability of the mutant proteins to regulate expression of a Pax9 effector gene, Bmp4. Results: All of the missense mutant PAX9 proteins were found to localize in the nucleus of transfected cells. Gel-shift assays using proteins from nuclear extracts showed that, with the exception of the G51 mutant protein, the remainder of the mutant proteins were unable to, or only partly able to interact with DNA. The luciferase reporter assay showed similar results. All of the mutant PAX9 proteins had at least a partial loss in transcriptional activity. Remarkably, G51 only showed a very slight decreasein the ability to activate transcription. Conclusion: Our demonstration of differential functional changes for the PAX9 mutant proteins is suggestive of the presence of a genotype-phenotype correlation for PAX9 mutations. The lack of observed defects for the G51 protein indicates an alternative mechanism which contributes to the hypodontia phenotype.

This study was supported by NIH U24 (DE16472), TAMHSC-VPR grant, NIH K08(DE16346).