Publications (6)13.07 Total impact
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Article: In vivo role of alternative splicing and serine phosphorylation of the microphthalmia-associated transcription factor.
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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.Genetics 02/2012; 191(1):133-44. · 4.01 Impact Factor -
Article: Allele-specific genetic interactions between Mitf and Kit affect melanocyte development.
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ABSTRACT: The tyrosine kinase receptor KIT and the transcription factor MITF, each required for melanocyte development, have been shown to interact functionally both in vitro and in vivo. In vitro, KIT signaling leads to MITF phosphorylation, affecting MITF activity and stability. In vivo, the presence of the Mitf (Mi-wh) allele exacerbates the spotting phenotype associated with heterozygosity for Kit mutations. Here, we show that among a series of other Mitf alleles, only the recessive Mitf (mi-bws) mimics the effect of Mitf (Mi-wh) on Kit. Intriguingly, Mitf (mi-bws) is characterized by a splice defect that leads to a reduction of RNAs containing MITF exon 2B which encodes serine-73, a serine phosphorylated upon KIT signaling. Nevertheless, other Mitf alleles that generally affect Mitf RNA levels, or carry a serine-73-to-alanine mutation that specifically reduces exon 2B-containing RNAs, do not show similar interactions with Kit in vivo. We conclude that the recessive Mitf (mi-bws) is a complex allele that can display a semi-dominant effect when present in a Kit-sensitized background. We suggest that human disease variability may equally be due to complex, allele-specific interactions between different genes.Pigment Cell & Melanoma Research 03/2010; 23(3):441-7. · 5.06 Impact Factor -
Article: The basic-helix-loop-helix-leucine zipper gene Mitf: analysis of alternative promoter choice and splicing.
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ABSTRACT: The activity of transcription factors is often regulated by Post-translational modifications. A precondition for such modifications is the presence, in the corresponding mRNAs, of the exons that either directly encode the modifiable residues in question, or encode protein domains that influence their modification indirectly. The inclusion or exclusion of coding exons is regulated predominantly by alternative splicing but can also depend on promoter choice and polyadenylation site selection. Information about exon inclusion and exclusion, both qualitatively and quantitatively, is particularly important for experiments designed to mutate endogenous codons because such mutations can alter splicing patterns. Therefore, we here describe methods employed to quantitate exon inclusion and exclusion, using as example a mouse transcription factor gene, Mitf.Methods in molecular biology (Clifton, N.J.) 01/2010; 647:237-50. -
Article: The role of MITF phosphorylation sites during coat color and eye development in mice analyzed by BAC transgene rescue
08/2009; 109. -
Article: The role of MITF phosphorylation sites during coat color and eye development in mice analyzed by bacterial artificial chromosome transgene rescue.
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ABSTRACT: The microphthalmia-associated transcription factor (Mitf) has emerged as an important model for gene regulation in eukaryotic organisms. In vertebrates, it regulates the development of several cell types including melanocytes and has also been shown to play an important role in melanoma. In vitro, the activity of MITF is regulated by multiple signaling pathways, including the KITL/KIT/B-Raf pathway, which results in phosphorylation of MITF on serine residues 73 and 409. However, the precise role of signaling to MITF in vivo remains largely unknown. Here, we use a BAC transgene rescue approach to introduce specific mutations in MITF to study the importance of specific phospho-acceptor sites and protein domains. We show that mice that carry a BAC transgene where single-amino-acid substitutions have been made in the Mitf gene rescue the phenotype of the loss-of-function mutations in Mitf. This may indicate that signaling from KIT to MITF affects other phospho-acceptor sites in MITF or that alternative sites can be phosphorylated when Ser73 and Ser409 have been mutated. Our results have implications for understanding signaling to transcription factors. Furthermore, as MITF and signaling mechanisms have been shown to play an important role in melanomas, our findings may lead to novel insights into this resilient disease.Genetics 08/2009; 183(2):581-94. · 4.01 Impact Factor -
Article: ERK-regulated differential expression of the Mitf 6a/b splicing isoforms in melanoma.
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ABSTRACT: The master regulator of the melanocyte lineage Mitf is intimately involved in development as well as melanoma, controlling cell survival, differentiation, proliferation and metastasis/migration. Consistent with its central role, Mitf expression and Mitf post-translational modifications are tightly regulated. An additional potential level of regulation is afforded by differential splicing of Mitf exon-6 leading to the generation of two isoforms that differ by the presence of six amino-acids in the Mitf (+) isoform and which have differential effects on cell cycle progression. However, whether the ratio of the two isoforms is regulated and whether their expression correlates with melanoma progression is not known. Here, we show that the differential expression of the Mitf 6a/b isoforms is dependent on the MAPKinase signalling, being linked to the activation of MEK1-ERK2, but not to N-RAS/B-RAF mutation status. In addition, quantification of Mitf 6a/b splicing forms in 86 melanoma samples revealed substantially increased levels of the Mitf (-) form in a subset of metastatic melanomas. The results suggest that differential expression of the Mitf 6a/b isoforms may represent an additional mechanism for regulating Mitf function and melanoma biology.Pigment Cell Melanoma Res.
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2012
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National Institutes of Health
- Section on Gamete Development
Bethesda, MD, USA
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