Molecular Basis of PITX2 Regulation of FoxJ1 Expression During Odontogenesis

Objective:

Axenfeld-Rieger syndrome (ARS) patients with PITX2 point mutations exhibit a wide range of dental anomalies and mild craniofacial dysmorphisms. To understand the molecular basis of these anomalies, identifying new targets of PITX2 and studying its biochemical interactions are important. FoxJ1 was identified as a PITX2 target, indicating a new role for FoxJ1 in craniofacial/tooth morphogenesis.

Methods:

Bioinformatics, Immunohistochemistry, Western Blot, Chromatin Immunoprecipitation assays, Cell culture, Transient transfection, Co-Immunoprecipitation assays and GST Pull-down assays.

Results:

Immunohistochemistry detected FoxJ1 expression in the inner enamel epithelium of developing tooth germ, oral epithelium, tongue epithelium, sub-mandibular salivary gland and hair follicles during embryonic day 18.5 and postnatal day 1 mice tissues. Chromatin immunoprecipitation assays demonstrate PITX2 binding to the FoxJ1 promoter, indicating that PITX2 regulates FoxJ1 expression. PITX2 activates the FoxJ1 promoter in transfected cell culture system. Also, Lef-1 and ß-catenin interact with PITX2 to synergistically regulate the FoxJ1 promoter. FoxJ1 and PITX2 physically interact and synergistically regulate FoxJ1 expression. Furthermore, a combinatorial interaction between FoxJ1, PITX2, Lef-1, and ß-catenin dramatically increase the synergistic activation of the FoxJ1 promoter. A specific PITX2 ARS mutant is unable to activate FoxJ1 and provides a molecular basis for craniofacial/ tooth anomalies observed in these patients.

Conclusion:

Our data shows a first time evidence of FoxJ1 as a new gene to be expressed during craniofacial/tooth morphogenesis and also a direct downstream target of PITX2. Its expression is regulated by PITX2, Lef-1, and ß-catenin. Also, FoxJ1 interacts with the PITX2, Lef-1, and ß-catenin complex to regulate FoxJ1 promoter in a positive feed back fashion. Also our data demonstrate how transcription factors act in concert in regulating FoxJ1 expression which emphasizes the significance of studying protein-protein interactions in explaining the complex nature of genetic defects.

Support for this research was provided from grant DE 13941 from the NIDCR