title: Nuclear import and export signals are essential for proper cellular trafficking and function of ZIC3.
abstract: Missense, frameshift and nonsense mutations in the zinc finger transcription factor ZIC3 cause heterotaxy as well as isolated congenital heart disease. Previously, we developed transactivation and subcellular localization assays to test the function of ZIC3 point mutations. Aberrant cytoplasmic localization suggested that the pathogenesis of ZIC3 mutations results, at least in part, from failure of appropriate cellular trafficking. To further investigate this hypothesis, the nucleocytoplasmic shuttling properties of ZIC3 have been examined. Subcellular localization assays designed to span the entire open-reading frame of wild-type and mutant ZIC3 proteins identified the presence of nucleocytoplasmic transport signals. ZIC3 domain mapping indicates that a relatively large region containing the zinc finger binding sites and a known GLI interacting domain is required for transport to the nucleus. Site-directed mutagenesis of critical residues within two putative nuclear localization signals (NLSs) leads to loss of nuclear localization. No further decrease was observed when both NLS sites were mutated, suggesting that mutation of either NLS site is sufficient for loss of importin-mediated nuclear localization. Additionally, we identify a cryptic CRM-1-dependent nuclear export signal (NES) within ZIC3, and identify a mutation within this region in a patient with heterotaxy. These results provide the first evidence that control of cellular trafficking of ZIC3 is critical for function and suggest a possible mechanism for transcriptional control during left-right patterning. Identification of mutations in mapped NLS or NES domains in heterotaxy patients demonstrates the functional importance of these domains in cardiac morphogenesis and allows for integration of structural analysis with developmental function.
authors.affiliation: Department of Pediatrics, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA.
authors.forename: James E J
authors.initials: JE
authors.lastname: Bedard
authors.forename: Jennifer D
authors.initials: JD
authors.lastname: Purnell
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Ware
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D019295:Computational Biology; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D008156:Luciferases; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D049790:Nuclear Export Signals / Q000235:genetics; D019913:Nuclear Localization Signals / Q000235:genetics; D009841:Oligonucleotides; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000235:genetics*; D013045:Species Specificity; D014157:Transcription Factors / Q000235:genetics*
chemical_list: D018398:Homeodomain Proteins; D049790:Nuclear Export Signals; D019913:Nuclear Localization Signals; D009841:Oligonucleotides; D014157:Transcription Factors; C491595:ZIC3 protein, human; D008156:Luciferases