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. 2012 May;191(1):133-44.
doi: 10.1534/genetics.111.135996. Epub 2012 Feb 23.

In vivo role of alternative splicing and serine phosphorylation of the microphthalmia-associated transcription factor

Julien Debbache  1 , M Raza Zaidi, Sean Davis, Theresa Guo, Keren Bismuth, Xin Wang, Susan Skuntz, Dragan Maric, James Pickel, Paul Meltzer, Glenn Merlino, Heinz Arnheiter
Affiliations

In vivo role of alternative splicing and serine phosphorylation of the microphthalmia-associated transcription factor

Julien Debbache et al. Genetics. 2012 May.

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- 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.

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Figures

Figure 1
Figure 1
Detection of Mitf isoforms in melanocytes in vivo. (A) GFP+ melanoblasts from bitransgenic (iDct-GFP) embryos and postnatal pups of the indicated ages were isolated and subjected to sorting by FACS. Corresponding RNA-seq data were analyzed as described in Materials and Methods. The ratios of the reads across exon 2A/exon 3 and exon 2B/exon 3 [2B−/(2B− + 2B+)] as well as the total number of reads analyzed are shown. (B) Relative abundance of Mitf 2B+ and Mitf 2B− RNA prepared from in vivo isolated P1 and P3 melanocytes, expressing GFP and FACS-sorted as for A, was determined by qRT–PCR using the appropriate primer pairs (Table S1). In addition, RNA from melanocyte cell lines derived from either Cdkn2a−/−; Mitf+/Mitf+ and Cdkn2a−/−; Mitfmi-S73A/Mitfmi-S73A P1 dorsal skins and cultured for 10 passages served as controls. The calculated percentages correspond to the ratio of 2B−/(2B− + 2B+).
Figure 2
Figure 2
Generation of four knock-in Mitf alleles. (A) Schematic representation of the Mitf genomic structure and the mutations generated in Mitfmi-S73Aneo [previously described (Bismuth et al. 2008) and re_targeted for the purpose of this study] and in the three novel splice alleles. Sequence modifications at the exon 2A–2B junction as well as at the S73 codon are highlighted in red, and the position of the neomycin resistance cassette in intron 2 is shown. (B and C) Differential restriction profile of the Mitf locus before and after _targeting and Southern blot analysis of wild-type and one line each of homozygous _targeted mice. DNA was digested as indicated and probed with the respective 5′ and 3′ probes shown in B.
Figure 3
Figure 3
Mitf exon 2B alternative splicing profile at the RNA and protein level in melanocyte lines. (A) RNA extracts were prepared from passage 10 Cdkn2a−/− melanocyte lines from wild-type B6, Mitfmi-S73AΔNeo (S73A), and Mitfmi-S-S73AΔNeo (S-S73A) mice and subjected to RT–PCR. (B) Schematic diagram of select post-translational modifications of MITF seen in melanocytes and other cell types. Indicated are phosphorylation, sumoylation, and caspase cleavage sites, while sites for acetylation, ubiquitination, and other modifications are not shown. (C) Western blots of protein extracts of the cell lines used in A, probed with 6A5 anti-MITF antibodies. The double band seen in the wild-type sample corresponds to phosphorylated and nonphosphorylated exon 2B+ MITF. Mitfmi-S73A cells show both a minor band corresponding to 2B− MITF and a major band corresponding to 2B+ MITF, in addition to a band of higher molecular weight and one of lower molecular weight, likely representing another post-translationally modified form and a degradation product (arrows). Mitfmi-S-S73A cells show a predominant 2B+ MITF band and no 2B− MITF band. Also, there is a band of higher molecular weight and one of lower molecular weight (arrows). MITF quantification relative to the endogenous β-tubulin is indicated below the gel.
Figure 4
Figure 4
Phenotypes associated with the four knock-in alleles alone and in combination with extant Mitf alleles. (A–J) Controls and Neo-cassette–containing lines. (A–D) White belly spots found in homozygotes of the indicated genotypes. Note that Mitfmi-S-S73A homozygotes show no belly spots, in contrast to the other lines. (E) Mitfmi-vga9 heterozygotes show no pigmentary phenotype while Mitfmi-vga9 homozygotes are completely white and microphthalmic (H). (F, G, I, and J) Heteroallelic combinations with Mitfmi-vga9. Note different degrees of white spotting with the different _targeted alleles. (K–R) Lines lacking the Neo cassette (Δneo). (K–N) Regardless of the genotype, all homozygotes are normally pigmented and indistinguishable by visual inspection from wild-type B6 mice, except that Mitfmi-S73AΔneo/Mitfmi-S73AΔneo mice have a tail with generally lighter pigmentation compared to the other genotypes. (O–R) Heteroallelic combinations with MitfMi-wh. Note the darker pigmentation seen in combinations with either Mitfmi-S73AΔneo or Mitfmi-S-S73AΔneo compared to combinations with Mitfmi-S-S73DΔneo, which yield mice with a coat that was only slightly darker, and combinations with Mitfmi-S-S73SΔneo, which yield mice that are indistinguishable from MitfMi-wh/Mitf+ heterozygotes. Each photograph in O–R represents littermates.
Figure 5
Figure 5
Melanocyte numbers in developing embryos. Embryos homozygous for either Mitfmi-S73AΔNeo or Mitfmi-S-S73AΔNeo and heterozygous for Kittm1Alf were X-gal–labeled and blue cells counted in selected regions at E12.5 and E15.5 and in P1 skin and P1 pigmented hair follicles. Numbers from 7 to 10 fields from at least three embryos are shown relative to those observed in Kittm1Alf/Kit+ control samples. Statistical significance (two-tailed unpaired t test) is indicated (*P < 0.1; **P < 0.01; ***P < 0.001).
Figure 6
Figure 6
Effects of the S73A mutation and exon 2B deletion on protein levels and DNA content in stable cell lines inducibly expressing MITF. ARPE-19 cells expressing either pBABE-ER (ER Ø), MITF wild-type S73 (ER WT), MITF S73A (ER S73A), or MITF 2B− (ER 2B−) proteins were treated with 4-OH-tamoxifen (TM) for 48 hr before collection, and total extracts were probed with 6A5 anti-MITF antibodies. (A) TM-treated, wild-type MITF expressing cells show the characteristic double band of S73-phosphorylated and nonphosphorylated MITF protein, S73A-MITF expressing cells only one band corresponding to nonphosphorylated MITF, and 2B− MITF-expressing cells a single band corresponding to the 2B− isoform. (B and C) ER MITF and ER only (ER Ø) expressing ARPE-19 cell lines were incubated with or without TM, harvested after 48 hr, and labeled for MITF and DNA content, using DAPI. (B) Flow cytometric analysis displays the percentage of cells expressing relatively low (L) or high (H) MITF intensity and (C) their relative cell cycle stages based on DNA content. Gating was determined empirically and applied equally for all samples.
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