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Xenopus pitx3 target genes lhx1 and xnr5 are identified using a novel three‐fluor flow cytometry–based analysis of promoter activation and repression
Author(s) -
Hooker Lara N.,
Smoczer Cristine,
Abbott Samuel,
Fakhereddin Mohamad,
Hudson John W.,
Crawford Michael J.
Publication year - 2017
Publication title -
developmental dynamics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.634
H-Index - 141
eISSN - 1097-0177
pISSN - 1058-8388
DOI - 10.1002/dvdy.24532
Subject(s) - biology , flow cytometry , transcription factor , somitogenesis , promoter , xenopus , microbiology and biotechnology , gene , genetics , embryonic stem cell , gene expression , somite
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 . Developmental Dynamics 246:657–669, 2017 . © 2017 Wiley Periodicals, Inc.