Transcriptional Mechanisms between Islet-1 and PITX2 regulating Craniofacial Morphogenesis

Islet-1 (ISL-1) was identified as a potential downstream target of the PITX2 protein using a bioinformatics screen for DNA elements, shared between known PITX targets. Isl-1 and Pitx2 have overlapping expression patterns in oral epithelium, and in molar and incisor developing tooth stages. Objectives: we aim to explore the molecular interactions between Isl-1 and PITX2 during craniofacial development. Methods: we use bioinformatic motif screen, transient transfections, real-time PCR, mutant mice, chromatin immunoprecipitation (ChIP) assays, electrophoretic mobility shift assays, GST pull-down assays, immunohistochemical (IHC) staining and biochemical assays. Results: IHC staining detected Isl-1 expression in differentiating odontoblasts and late stage pre-ameloblasts. ChIP assays demonstrated Pitx2 binding to the distal Isl-1 promoter and Isl-1 binding to the distal Pitx2 promoter. PITX2 activates the full-length Isl-1 promoter in the oral epithelium LS-8 cell line, but the activation was attenuated by co-transfection of Isl-1, however, Isl-1 minimally activates the PITX2 promoter and represses PITX2 activation of the PITX2 promoter in LS-8 cell line. Immunoprecipitation showed PITX2 directly interacts with ISL-1. GST pull-down assays mapped down the interacting domains of PITX2 and Isl-1, Isl-1 functionally interacts with the homeodomain and C-terminal tail residues of PITX2, while PITX2 interacts with the LIM domain of Isl-1. Conclusions: ISL-1 is a downstream target for PITX2, Isl-1 negatively feeds back to repress the activation of the Isl-1 promoter by PITX2. We demonstrate new Isl-1 expression patterns and new mechanisms for the regulated control of gene expression during tooth development. Support for this research was provided from DE 13941 from the National Institute of Dental and Craniofacial Research.