Xenopus pitx3 target genes lhx1 and xnr5 are identified using a novel three-fluor flow cytometry-based analysis of promoter activation and repression.

Background: Pitx3 plays a well understood role in directing development of lens, muscle fiber, and dopaminergic neurons; however, in Xenopus laevis, it may also play a role in early gastrulation and somitogenesis. Potential downstream targets of pitx3 possess multiple binding motifs that would not be readily accessible by conventional promoter analysis. Results: We isolated and characterized pitx3 target genes lhx1 and xnr5 using a novel three-fluor flow cytometry tool that was designed to dissect promoters with multiple binding sites for the same transcription factor. This approach was calibrated using a known pitx3 target gene, Tyrosine hydroxylase. Conclusions: We demonstrate how flow cytometry can be used to detect gene regulatory changes with exquisite precision on a cell-by-cell basis, and establish that in HEK293 cells, pitx3 directly activates lhx1 and represses xnr5. (C) 2017 Wiley Periodicals, Inc.