In Vivo Role of Alternative Splicing and Serine Phosphorylation of the Microphthalmia-Associated Transcription Factor

Abstract

The microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix leucine zipper protein that plays major roles in the development and physiology of vertebrate melanocytes and melanoma cells. It is regulated by post-translational modifications, including phosphorylation at serine 73, which based on in vitro experiments imparts on MITF an increased transcriptional activity paired with a decreased stability. Serine 73 is encoded by the alternatively spliced exon 2B, which is preferentially skipped in mice carrying a targeted serine-73-to-alanine mutation. Here, we measured the relative abundance of exon 2B + and exon 2B 2 RNAs in freshly isolated and FACS-sorted wild-type melanoblasts and melanocytes and generated a series of knock-in mice allowing forced incorporation of either alanine, aspartate, or wild-type serine at position 73. None of these knock-in alleles, however, creates a striking pigmentation phenotype on its own, but differences between them can be revealed either by a general reduction of Mitf transcript levels or in heteroallelic combinations with extant Mitf mutations. In fact, compared with straight serine-73 knock-in mice with their relative reduction of 2B + Mitf, forced incorporation of alanine 73 leads to greater increases in MITF protein levels, melanoblast and melanocyte numbers, and extent of pigmentation in particular allelic combinations. These results underscore, in vivo, the importance of the link between alternative splicing and post-translational modifications and may bear on the recent observation that exon 2B skipping can be found in metastatic melanoma.A LTERNATIVE splicing and post-translational modifications are among the major mechanisms by which individual genes generate multiple protein products. In fact, modern sequencing technologies and proteomic analyses have shown that most if not all multiexon pre-mRNAs give rise to alternatively spliced mature mRNAs (Nilsen and Graveley 2010) and that post-translational modifications modulate the activities of most eukaryotic proteins (Mann and Jensen 2003). In a simple sense, then, the two mechanisms are linked as post-translational modifications depend on the presence of the particular exons encoding the modifiable residues or proteolytic cleavage sites. Here, we present a mouse model that allows us to probe this link in vivo by separately targeting a splice site and a codon for a biologically relevant phosphoacceptor site in the gene encoding the pigment cell transcription factor, microphthalmia-associated transcription factor (MITF). In fact, pigmentation is particularly suitable for such studies as it provides for an easily visible and highly sensitive readout of gene function.Mitf plays a crucial role in the development and function of melanin-bearing pigment cells in skin, eye, and inner organs (Hodgkinson et al. 1993;Arnheiter et al. 2006;Arnheiter 2010). Its protein product is a basic helix-loop-helix leucine zipper transcription factor that regulates target gene promoters by binding specific E box...

A Polymorphism in IRF4 Affects Human Pigmentation through a Tyrosinase-Dependent MITF/TFAP2A Pathway

“…This showed that the major IRF4 product in melanocytes is the full length transcript; the smaller alternative product was barely detectable (Figure S5b). Similarly, in mouse melanoblasts isolated from E15.5 and E17.5 embryos and melanocytes from P1 and P7 neonatal pups (Debbache et al, 2012), the full-length Irf4 transcript was the only transcript detected at all developmental stages (Figure S6). We therefore conclude that the regulatory element in intron 4 affects the production of the full-length IRF4 transcript.…”

“…We analyzed the expression of Mitf , Tfap2a , Irf4 and Tyr in melanoblasts isolated from E15.5, E17.5, P2 and P7 mouse embryos (Debbache et al, 2012). Mitf and Tfap2a are both expressed at the embryonic and postnatal stages whereas Irf4 is detected at a low level at the two postnatal stages (Figure 7a).…”

MITF—the first 25 years

“…The consequences for coat color phenotypes of the different alleles in mice are often intriguing. Some alleles or allele combinations, for instance, can lead to a black head spot on an otherwise completely white mouse, while others can lead to a white head spot on an otherwise completely black mouse; some allele combinations can even yield tricolored (white/tan/black) mice (Debbache et al 2012).…”

Tfe3 and Tfeb Transcriptionally Regulate Peroxisome Proliferator-Activated Receptor γ2 Expression in Adipocytes and Mediate Adiponectin and Glucose Levels in Mice

“…This particular phosphorylation, on three serine residues located on the C-terminal region, induces TFEB stabilization and increases transcriptional activity (66). Knock-in mutants of Mitf at serine 73 have been reported to exhibit melanocytic phenotypic effects in certain contexts (67). Further studies are needed to establish the role phosphorylation of Tfe3 plays in vivo, specifically in tissues involved in energy metabolism.…”