Dicing with death: Mitf regulates DICER

All transcription factors are equal, but some are more equal than others. That is to say all transcription factors regulate gene expression, but some are critical to the identity of a cell, acting to integrate multiple signal pathways and coordinate a wide range of cellular processes. First among equals is Mitf, the microphthalmia-associated transcription factor first isolated by Heinz Arnheiter’s lab 17 years ago (Hodgkinson et al., 1993) as a gene critically required for melanoblast survival and the differentiation of the retinal pigment epithelium during development. Mitf null mice consequently are devoid of all pigment cells. Soon after, the role of Mitf in the melanocyte lineage was revealed to include coordinating the expression of genes implicated in the manufacture of melanosomes, the specialized organelle that controls the manufacture of the pigment melanin (Cheli et al., 2010). Given that differentiation is associated with cell cycle arrest, recent years have also seen Mitf designated as a key regulator of cell division, driving a differentiation-associated cell cycle arrest via up-regulation of p16 and p21. Mitf can also promote cell division by suppressing p27 expression and senescence. The elevated expression of Mitf associated with its differentiation function is achieved, at least in part, via activation of the melanocortin 1 receptor MC1R by is ligand, the melanocyte-stimulating hormone MSH that is released by keratinocyes in response to UV irradiation. Binding of MSH to MC1R triggers increased cAMP levels and consequent phosphorylation and activation of CREB, a transcription factor that recognizes the melanocyte-specific Mitf promoter (Bertolotto et al., 1998). This pathway is used to enhance skin pigmentation as a photoprotective tanning response to UV exposure. Thus in addition to its role in survival and differentiation, Mitf is also charged with suppressing senescence and coordinating cell cycle entry and exit depending on its levels and activity. That a single transcription factor coordinates so many aspects of melanocyte biology is in itself striking, and one would have thought it a large enough role. Yet in the paper from Levy et al. (2010) a further string to Mitf’s bow is uncovered: it regulates the expression of the gene encoding the microRNA-processing enzyme DICER which has been determined through targeted disruption in mice to play a crucial role in the survival and function of a wide range of lineages. The first clue to this previously unsuspected role for Mitf was revealed by the analysis of miRNA expression in response to the stimulation of primary human melanocytes by the cAMP elevating agent forskolin, thereby mimicking the effects of MSH. While some up-regulated miRNAs, such as miR-211 that is embedded in the Mitf target gene TRPM1, exhibited an increase in both the precursor as well as the mature forms, suggesting an increase driven by a transcriptional response, others such as miR-17-3p and miR-92 were characterized by an increase in the levels of the mature species, but the precursor forms did not appear to be amplified. Although several possible mechanisms might have accounted for the post-transcriptional regulation of the panel of miRNAs that exhibited a similar differential effect of cAMP on their mature vs precursor forms, the authors focussed on DICER. Both protein and mRNA levels of DICER increased in response to cAMP signaling and importantly this effect was restricted to melanocytes, since forskolin treatment of fibroblasts that do not express the melanocyte-specific isoform of Mitf did not give rise to a similar precursor-mature miRNA switch. No effect of cAMP was observed for DROSHA, the enzyme that generates pre-miRNA. Further experiments, including the use of siRNA depletion or dominant-negative of Mitf as well as chromatin immunoprecipitation firmly implicated Mitf as a direct regulator of DICER expression through two consensus CATGTG motifs in its promoter. Consistent with this, DICER was detected in differentiated Mitf-positive melanocytes in the hair follicle, but not in the Mitf-negative stem cell population in the bulge. Having established DICER as an Mitf target the authors then proceeded to examine the consequences of DICER-loss in the melanocyte lineage using mice in which CRE-recombinase is targeted via the tyrosinase promoter. The results revealed that inactivation of the DICER gene in the melanocyte lineage led to a loss of melanocytes and consequently a white coat colour. Excision of the DICER gene in melanocytes in culture also led to cell death. Thus DICER is an essential Mitf-regulated gene in the melanocyte lineage. Inactivation of DICER will lead to large-scale de-regulation of miRNAs and it is likely that many will contribute to the phenotype arising from DICER loss. However, the authors went on to show that up-regulation of miR-17 correlated with decreased expression of the pro-apoptotic factor BIM, and that siRNA–mediated knockdown of BIM in melanocytes lacking DICER partially rescued the apoptotic phenotype associated with DICER loss. This is significant since inactivation of BIM in mice counteracts the melanocyte–loss associated with inactivation of BCL2, another Mitf target gene (McGill et al., 2002). Although regulation of BIM by miR-17 in melanocytes represents a good candidate to account for the effects of DICER loss, no doubt expression of the miR-17-92 cluster will also function in melanocytes through other anti-apoptotic pathways, as suggested by its role in regulating the Akt pathway in oligodendrocytes (Budde et al., 2010). The results described in Levy et al., together with the ability of Mitf to regulate BCL2, may provide some explanation for the loss of melanocytes in Mitf null mice, though it is not yet clear how early in development DICER is required to prevent melanocye death. It is possible that the role of DICER in melanocyte survival is restricted to post-migratory melanoblasts or differentiating melanocytes, as DICER expression is known to be critical for survival of post-migratory neural crest cells (Zehir et al., 2010). While the role of DICER in melanocyte survival is evident, perhaps increasing resistance of melanocytes to UV-induced cell death in response to solar radiation, the preferential expression of DICER in differentiated melanocytes may indicate that miRNAs are also likely to play direct roles in regulating melanogenesis. Moreover, the fact that Mitf is either amplified or mutated in melanoma also implies a de-regulation of miRNA processing via DICER in this disease. This study, while revealing the cAMP-Mitf-DICER axis, serves to highlight the critical roles played by miRNAs in the melanocyte lineage and will stimulate the search for miRNA function in melanocyte differentiation and melanoma progression.

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