Molecular Characteriziation of Human PAX9 Paired Domain Missense Mutations

Mutations in human PAX9, a paired domain containing transcription factor, are associated with posterior tooth agenesis. To date, six missense paired domain mutations have been described. However, the molecular mechanisms underlying their pathogenesis remain unclear. Objective: The goals of this study are to perform a comparative structural and functional analysis of the wild-type PAX9 protein and the gene product of the substitution mutations. Methods: The tertiary structure of the paired domains was analyzed using the crystal structure of the PAX6 paired domain bound with DNA as a template. To understand the effect of the mutations on DNA-binding activities, electromobility shift assays (EMSA) were performed using two high affinity paired-domain recognition sequence, CD19-2(A-ins) and e5. To ascertain the subcellular localization of the Ile87Phe protein, immunolocalization studies were utilized. Co-immunoprecipitation analysis was used to determine if the mutant proteins are able to form a heterodimeric complex with the Msx1 homoeoprotein. Results: Sequence alignment analysis revealed that each of the affected amino acid residues is remarkably conserved in the Pax gene family. We show that either the bonding between the DNA and protein is directly affected or the structure of the paired domain itself is distorted. Gel shift assays indicate that G51S, I87F, and K91E proteins are able to form complexes with CD19-2/A and e5. Immunocytochemistry data demonstrates that both wild type and mutant proteins are synthesized in mammalian cells and that nuclear localization is unaffected by all of the mutations. Protein-protein interactions with Msx1 are unaffected by the mutations. Conclusion: Our results suggest defects in DNA-binding are likely responsible for tooth agenesis in three of the six mutations studied. Additional functional studies are underway to determine the pathogenic mechanisms for the other mutations. This research was supported by NIH grant R01 DE13668 to RDS, NIH K08-DE14237 to HK.