The microphthalmia (mi) gene is essential to the development of mast cells based on the selective mast cell defects in mi mutant mice. Mast cells play an important role in the pathogenesis of allergic and inflammatory disorders. In addition, mast cell hyperplasia results in disorders such as mastocytosis. Thus, mast cells are not only an important disease-specific cell lineage, but also provide an attractive system to study the functions of the microphthalmia protein (Mi) essential in development. The mi gene encodes a novel basic helix-loop-helix leucine zipper transcription factor and is closely related to the oncoprotein, Myc. Mast cells derived from mi mutant mice are incapable of differentiating into mature mast cells, suggesting its role as a regulator of mast cell development. Intriguingly, mice with mutations in the receptor tyrosine kinase, c-kit, or its ligand, stem cell factor, (SCF), display a phenotypic constellation in several tissue types remarkably similar to mi mutant mice even in biochemical detail. This similarity suggests the possibility that Mi and c-Kit may reside in the same pathway within cells, a pathway which is central to the survival, proliferation, and/or differentiation of mast cells. The sponsor's laboratory has recently discovered that the Mi protein is phosphorylated in response to c-Kit activation. Given that SCF is critical for mast cells, this observation may implicate such a central role for Mi. The overall goal of this project will be to elucidate the critical function of the Mi transcription factor in mast cell development.
The specific aims are: 1) To characterize Mi's expression, dimerization partners and function during mast cell development, 2) To examine the regulation of Mi via c-Kit signaling, and 3) To analyze potential genes transcriptionally regulated by Mi in mast cells. Mi expression and dimerization partners will be analyzed by immunohistochemistry, immunoprecipitation, and Western analysis with specific Mi antibodies developed in the laboratory. Transfections of wild type and mutant Mi will be performed in hematopoietic and mast cell lines and potential functional outcomes will be assessed. Potential gene target promoters will be examined with reported gene experiments and a stringent differential display strategy will be used to identify novel targets. Mi's transcriptional activity and phosphorylation will be analyzed in response to SCF signaling. Dr. David Fisher will supervise the project and head and advisory board of experts in hematopoiesis and mast cell biology formed to provide additional guidance and aid in the candidate's transition to an independent investigator.
| Takemoto, Clifford M; Yoon, Yo-Jin; Fisher, David E (2002) The identification and functional characterization of a novel mast cell isoform of the microphthalmia-associated transcription factor. J Biol Chem 277:30244-52 |
| Takeda, K; Takemoto, C; Kobayashi, I et al. (2000) Ser298 of MITF, a mutation site in Waardenburg syndrome type 2, is a phosphorylation site with functional significance. Hum Mol Genet 9:125-32 |
