The Nuclear Migration, DNA Binding, and Promoter Regulatory Potential of WT MITF in the Presence of Mutant MITF Isoforms

(A) Nuclear-cytoplasmic distribution of MITF in the presence of the mutants (scale bars represent 10 μm) was studied by confocal microscopy. The WT and mutant MITF were differentially tagged by GFP or RFP.

(B) Amounts of nuclear MITF were calculated by western blotting from three different experiments and are represented as a graph.

(C) EMSA gels representative of three experiments show DNA-binding ability of the WT-control-vector (CV), WT-p.Arg318del, WT-p.Lys307Asn, and p.Arg318del-p.Lys307Asn combinations (^∗supershifted band) to the M-box and E-box DNA sequences.

(D) The dominant effect of p.Arg318del on WT and p.Lys307Asn proteins was studied by a dual luciferase reporter assay with the TYRP promoter. In increasing concentrations (0, 50, 100, and 200 ng), p.Arg318del was co-transfected with a constant amount of WT or p.Lys307Asn (100 ng), along with 200 ng of a luciferase-promoter construct (TYRP or FGF19), 1:1,000 Renilla luciferase vector, and empty vector to equalize the amounts of transfected DNA. The p.Arg318del mutant in increasing amounts had a dominant-negative effect on WT MITF (solid bars) and p.Lys307Asn (open bars).

(E) Effect of mutant zebrafish mitfa overexpression on the average melanocyte numbers of 36 hpf zebrafish embryos. The WT ABTL strain of zebrafish was maintained under standard conditions of fish husbandry according to NIH Animal Use and Care Committee guidelines. WT mitfa and the isoforms harboring the two human mutations, cloned in pCS2+, were linearized and injected into the cells of freshly fertilized zebrafish eggs at the single-cell stage. Linearized pCS2+ vector (EV) was also injected in equal amounts as an experimental control. The volume of injection solution per embryo was ∼1 nL. The melanocytes on a specified dorsal-trunk region of 34–36 hpf zebrafish embryos were photographed and then counted for 30 different embryos pooled from three different trials. Representative images of 48 hpf embryos are shown (scale bars represent 0.1 mm).

(F) Nuclear localization of TFE3 in the presence of the two MITF mutants (scale bars represent 10 μm) was studied by confocal microscopy. All comparisons are in reference to the WT MITF or EV; error bars represent standard error p values were calculated with the two-sided Student’s t test: ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005.