New Dlx2 Transcriptional Regulatory Functions during Tooth Development

Dlx2 is required for the inductive formation of epithelial-derived organs including teeth. Objectives: because Dlx2 is required for tooth development and other tissues, we are working to define the molecular mechanisms required for Dlx2 expression and activity during tooth development. Dlx2, Lef-1, Pitx2, and β-catenin are expressed early in the dental epithelium and have overlapping expression patterns in the tooth bud and cap stages. We have identified interactions between Dlx2, PITX2 and Lef-1 and these interactions specifically modulate genes expressed during tooth development. Methods: we use transient transfections, real-time PCR, mutant mice, chromatin immunoprecipitation (ChIP) assays, electrophoretic mobility shift assays, immunochemical and biochemical assays. Results: ChIP assays have identified endogenous Dlx2 binding to the Msx2 and Dlx2 promoters. Dlx2 positively auto-regulates its promoter and physically interacts with Lef-1. Dlx2 and Lef-1 interact to synergistically activate the Dlx2 promoter. Dlx2 and PITX2 physically interact, which represses Dlx2 activation of the Msx2 promoter. Beta-catenin functionally interacts with Dlx2 and Lef-1 to further regulate the Dlx2 and Msx2 promoters. Lef-1 deletion mutants demonstrate a specific differential interaction with Dlx2 and beta-catenin. The Lef-1 interaction with Dlx2 is beta-catenin independent, as a Lef-1 isoform without the beta-catenin binding domain interacts and synergistically activates the Dlx2 promoter in concert with Dlx2. Conclusions: these data demonstrate that Dlx2 can regulate its own expression and interact with Lef-1 to synergistically regulate its promoter. Furthermore, Dlx2 activation of Msx2 expression is attenuated through its physical interaction with PITX2. Dlx2 interaction with Lef-1 is independent of beta-catenin, however, beta-catenin can increase the transcriptional activity of Dlx2. These are new transcriptional mechanisms for Dlx2 and support a role for dental patterning through control spatial and temporal expression. Support for this research was provided from grant DE13941 from the National Institute of Dental and Craniofacial Research.