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.