An important question in development concerns the mechanisms controlling cell numbers as they determine the availability of differentiated cells in the adult, of stem cells capable of regenerating damaged tissues, and of cells potentially becoming malignant. An ideal system to address the question of cell number determination is provided by neural crest-derived melanin-bearing pigment cells (melanocytes) which are not only important for coloration of the integument but also for seeing and, at least in mammals, for hearing. A crucial factor determining the numbers of melanocytes is the transcriptional regulator MITF which is well known for its multiple roles in melanocyte development, differentiation, maintenance, replenishment in hair follicles, and melanoma formation. MITF, however, is not a single protein but a family of isoforms generated by alternative promoter use, alternative splicing, and a variety of post-translational modifications. We have previously reported that in mice with a mutation in serine-73, whose phosphorylation influences MITF activity, alternative splicing leads to the selective exclusion of exon 2B, i.e. precisely the exon of which the serine-73 codon is part. This splice change is due to changes in the interaction with serine/arginine-rich splice factors. We also find that the absence of exon 2B is associated with changes in the levels of the cell cycle regulators p21 and diaphanous-1/p27 and, consequently, the rate of cell proliferation. In fact, as shown by others, elimination of MITF exon 2B is occasionally found in melanoma cells. To selectively test the role of serine-73 in vivo, we incorporate mutated codons at position 73 into the mature mRNA by also mutating in cis the alternative splice donor at the exon 2A/2B junction. We have prepared appropriate targeting constructs, tested corresponding minigenes for exon 2B incorporation in vitro, and are currently introducing these mutations into mice using ES cell technology. These experiments will help us to determine the relative importance of alternative splicing and post-translational modification of MITF for cell number control during normal development and in malignancies.

Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2009
Total Cost
$821,631
Indirect Cost
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Debbache, Julien; Zaidi, M Raza; Davis, Sean et al. (2012) In vivo role of alternative splicing and serine phosphorylation of the microphthalmia-associated transcription factor. Genetics 191:133-44
Arnheiter, Heinz (2012) Warp, weft, and the tapestry of nature. Pigment Cell Melanoma Res 25:403
Arnheiter, Heinz (2012) On the edge. Pigment Cell Melanoma Res 25:1
Zaidi, M Raza; Davis, Sean; Noonan, Frances P et al. (2011) Interferon-? links ultraviolet radiation to melanomagenesis in mice. Nature 469:548-53
Bharti, Kapil; Debbache, Julien; Wang, Xin et al. (2010) The basic-helix-loop-helix-leucine zipper gene Mitf: analysis of alternative promoter choice and splicing. Methods Mol Biol 647:237-50
Arnheiter, Heinz (2010) Sex-specific coloration for display and camouflage. Pigment Cell Melanoma Res 23:480-1
Arnheiter, Heinz (2010) The discovery of the microphthalmia locus and its gene, Mitf. Pigment Cell Melanoma Res 23:729-35
Arnheiter, Heinz (2010) Of patches, blotches, speckles and stripes. Pigment Cell Melanoma Res 23:479
Wen, Bin; Chen, Yu; Li, Huirong et al. (2010) Allele-specific genetic interactions between Mitf and Kit affect melanocyte development. Pigment Cell Melanoma Res 23:441-7
Bauer, Georg L; Praetorius, Christian; Bergsteinsdottir, Kristin et al. (2009) The role of MITF phosphorylation sites during coat color and eye development in mice analyzed by bacterial artificial chromosome transgene rescue. Genetics 183:581-94

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