A 3' UTR modification of the mitochondrial rieske iron sulfur protein in mice produces a specific skin pigmentation phenotype.

TO THE EDITOR The role of mitochondrial proteins in melanocyte function and pigmentation has been brought into the spotlight by a recent article in the Journal of Investigative Dermatology (Ni-Komatsu and Orlow, 2007). Using a zebrafish screening, two oxidative phosphorylation-related factors, prohibitin and the complex V (F1/F0 ATPase), were identified as modulators of pigmentation. In addition, alterations in skin pigmentation have previously been reported in some patients with mitochondrial disorders (Bodemer et al., 1999; Kubota et al., 1999). During our studies on mitochondrial function, we developed a mouse with a knock-in Rieske iron sulfur protein (RISP). The production of mice with the RISP genetic modification was approved by the University of Miami Institutional Animal Care and Use Committee. The RISP is a subunit of the mitochondrial oxidative phosphorylation complex III. The knocked-in gene was flanked by loxP sites for tissue-specific ablation. The construct contained a full-length, intact gene, but included a neomycin/thymidine kinase selection cassette in the 3′-untranslated region. Therefore, the knock-in gene should be functional, and defective only when exposed to the Cre recombinase, which would delete exon 2 of the RISP. This can be accomplished by crossing the knock-in mice with Cre-expressing transgenic mice (Diaz et al., 2005). Not surprisingly, mice homozygous for the knock-in allele were healthy and lived a normal life, as modification of the knock-in gene did not affect the coding sequence or splicing of the RISP gene. However, mice heterozygous or homozygous for the knock-in allele showed change in coat color starting at about 4–7 months of age. In mice, the coat color depends on the ratio and distribution of two melanin pigments, the eumelanins (black to brown pigments) and the pheomelanins (yellow to red pigments), derived from a common precursor (dopaquinone), which are synthesized by follicular melanocytes. The heterozygous mice acquired dark patches in the dorsal brown coat. With age, the dark patches eventually filled almost all the dorsal coat, although it remained unchanged in the ventral region (Figure 1a). In homozygous mice, the dark patches eventually turned gray (Figure 1c and d). This phenotype segregated with the presence and dosage of the knock-in allele. Figure 1 Coat color changes of mice with an RISP-knock-in gene Analyses of RNAs by northern blots showed that, as expected, the knock-in allele was larger than the endogeneous RISP transcript (Figure 2a and b). Western blot analyses showed that in the skin, the levels of RISP were markedly decreased in homozygous knock-in mice (Figure 2c). The secondary antibody against mouse immunoglobulins detected non-specific bands in the region of RISP in skin homogenates (endogenous immunoglobulins), but we were able to distinguish those from the RISP by analyzing a heart mitochondrial sample in parallel (Figure 2c). The RISP levels were not significantly altered in muscle, brain, heart, or liver (Figure 2d), demonstrating that the knock-in transcripts were correctly translated into a functional protein in most tissues. Figure 2 Characterization of the RISP-knock-in gene expression We isolated fibroblasts from homozygous floxed mice, but detected neither a complex III defect (not shown) nor a significant decrease in RISP (Figure 2e). As an additional control, we deleted the RISP gene in fibroblast cultures using a plasmid encoding the Cre-recombinase. The knockout fibroblasts showed no RISP and a reduction in subunit core 2 of complex III (lane 2 in Figure 2e). Compared with floxed fibroblasts, isolated melanocytes showed proportionally higher decrease in RISP when normalized to a mitochondrial marker (VDAC1; Figure 2e), suggesting that melanocytes are more sensitive to the knock-in allele. Unfortunately, the yield and life span of the isolated primary melanocytes were very limited, precluding further experiments with them. From our observations, we speculate that melanocytes have a specific regulation (at the transcriptional or translational level) of RISP expression. Although it is unclear how a pigmentation phenotype developed in these mice, defects in complex III are commonly associated with an increase in reactive oxygen species (ROS) production in the mitochondria. ROS are not only known mediators of UV-induced hyperpigmentation and can eventually lead to melanocyte apoptosis and hypopigmentation (Costin and Hearing, 2007), but also participate in the metabolism of dopaquinone (Mastore et al., 2005). However, we did not find increased ROS production in RISP-deficient fibroblasts (not shown) and the knockdown reductions in the RISP protein have been associated with a decrease in ROS (Bell et al., 2007). Accordingly, treatment of homozygous or heterozygous RISP knock-in mice with N-acetyl cysteine for 60 days (starting at age 2 months) failed to prevent changes in coat color (not shown). Staining of skin sections for melanin (using the Fontana–Masson silver method) did not show major differences between wild-type and homozygous knock-in mice. The fact that darkening of the coat is not observed in the ventral part suggests that the agouti could be involved in this phenomenon. Agouti protein antagonizes the effects of α-melanocyte-stimulating hormone, switching the melanogenesis from eumelanin to phaeomelanin. Differences in agouti expression along the dorsal–ventral axis, have been found (Vrieling et al., 1994). Although we cannot rule out the participation of ROS in the coat color phenotype, mitochondrial function may have a broader role in the modulation of skin pigmentation, as the findings of Ni-Komatsu and Orlow suggest. In any case, the mice described here provide a unique model for the study of pigmentation abnormalities in mitochondrial disorders.