title: A dominant negative mutant of microphthalmia transcription factor (MITF) lacking two transactivation domains suppresses transcription mediated by wild type MITF and a hyperactive MITF derivative.
abstract: Microphthalmia transcription factor (MITF) positively regulates transcription of differentiation-related genes in several cell lineages, including melanocytes. Recent data also indicate a new important role for MITF as a factor that appears to be required for survival of melanoma cells, suggesting a possibility that abrogation of MITF function in transformed melanocytes could lead to a decreased survival via attenuating anti-apoptotic signals. Therefore, to gain a better understanding of the role which MITF plays in melanoma cell survival, it is important to find efficient means of abolishing the transactivation of its target genes. Recently, a dominant negative MITF lacking the N-terminus has been shown to down-regulate tyrosinase and Trp1 expression in normal melanocytes and mouse B16 melanoma cells. Here, a dominant negative mutant of the melanocyte-specific isoform of MITF is described carrying deletions of both N- and C-terminal transactivation domains. Cotransfection of this mutant resulted in a complete inhibition of the wild type MITF function as tested on both the reporter-linked tyrosinase promoter and an endogenous, ectopic MITF-triggered tyrosinase gene in U2-OS cells. The dominant negative construct also strongly repressed the activity of a hyperactive MITF-Vp16 chimera. Importantly, deletion of both activation domains was necessary to eliminate the residual transcription activity observed when only the N-terminal domain was removed and to achieve the repressive effect in human melanoma cells. If the activity of MITF plays a role in the long-term survival of malignant melanocytes, overexpression of a strong dominant negative MITF mutant might be a useful strategy to suppress its transactivation function.
authors.affiliation: Laboratory of Molecular Biology, University Hospital, 3rd Medical Faculty, Charles University, Prague, Czech Republic. jivach@upn.anet.cz
authors.forename: Jiri
authors.initials: J
authors.lastname: Vachtenheim
authors.forename: Blanka
authors.initials: B
authors.lastname: Drdová
mesh_terms: D000818:Animals; D019677:Artificial Gene Fusion; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D005799:Genes, Dominant; D005814:Genes, Viral / Q000235:genetics; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D051739:Microphthalmia-Associated Transcription Factor; D014442:Monophenol Monooxygenase / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology*; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013489:Suppression, Genetic / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology; D015533:Transcriptional Activation / Q000502:physiology
chemical_list: D004268:DNA-Binding Proteins; D017405:Herpes Simplex Virus Protein Vmw65; C497234:MITF protein, human; D051739:Microphthalmia-Associated Transcription Factor; C497235:Mitf protein, mouse; D011994:Recombinant Proteins; D014157:Transcription Factors; D014442:Monophenol Monooxygenase