title: [Analysis of nuclear localization and signal function of MITF protein predisposing to Warrdenburg syndrome].
abstract: OBJECTIVE
  To study the role of dysfunction of nuclear localization signals (NLS) of MITF protein in the pathogenesis of Waardenburg syndrome.
  
  
  METHODS
  Eukaryotic expression plasmid pCMV-MITF-Flag was used as a template to generate mutant plasmid pCMV-MITF△NLS-Flag by molecular cloning technique in order to design the mutagenic primers. The UACC903 cells were transfected transiently with MITF and MITF△NLS plasmids, and the luciferase activity assays were performed to determine their impact on the transcriptional activities of target gene tyrosinase (TYR). The oligonucleotide 5'-GAACGAAGAAGAAGATTT-3' was subcloned into pEGFP-N1 to generate recombinant eukaryotic expression plasmid pEGFP-N1-MITF-NLS. The NIH3T3 cells were transfected separately with MITF, MITF△NLS, pEGFP-N1 and pEGFP-N1-NLS plasmids, and their subcellular distribution was observed by immunoflorescence assays.
  
  
  RESULTS
  Expression plasmids for the mutant MITF△NLS with loss of core NLS sequence and pEGFP-N1-NLS coupled with MITF△NLS were successfully generated. Compared with the wild-type MITF, MITF△NLS was not able to transactivate the transcriptional activities of promoter TYR and did not affect the normal function of MITF. MITF△NLS was only localized in the cytoplasm and pEGFP-N1 was found in both the cytoplasm and nucleus, whereas pEGFP-N1-NLS was mainly located in the nucleus.
  
  
  CONCLUSIONS
  This study has confirmed the localization function of NLS sequence 213ERRRRF218 within the MITF protein. Mutant MITF with loss of NLS has failed to transactivate the transcriptional activities of target gene TYR, which can result in melanocyte defects and cause WS.
authors.affiliation: Department of Otorhinolaryngology, First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang 830011, P.R. China. fyong@xysm.net.
authors.forename: Hua
authors.initials: H
authors.lastname: Zhang
authors.forename: Juan
authors.initials: J
authors.lastname: Feng
authors.forename: Hongsheng
authors.initials: H
authors.lastname: Chen
authors.forename: Jiada
authors.initials: J
authors.lastname: Li
authors.forename: Hunjin
authors.initials: H
authors.lastname: Luo
authors.forename: Yong
authors.initials: Y
authors.lastname: Feng
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D020022:Genetic Predisposition to Disease / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D051739:Microphthalmia-Associated Transcription Factor / Q000235:genetics* / Q000378:metabolism; D018613:Microscopy, Confocal; D014442:Monophenol Monooxygenase / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D041681:NIH 3T3 Cells; D019913:Nuclear Localization Signals / Q000235:genetics*; D015533:Transcriptional Activation; D014162:Transfection; D014849:Waardenburg Syndrome / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism
chemical_list: D051739:Microphthalmia-Associated Transcription Factor; D019913:Nuclear Localization Signals; D049452:Green Fluorescent Proteins; D008156:Luciferases; D014442:Monophenol Monooxygenase