The molecular mechanisms governing normal mammalian lens development are not fully understood. As Myc cellular oncoproteins function as sequence specific transcription factors that regulate genes involved in key developmental processes, the PI has explored the role of these proteins in lens growth and differentiation. He has demonstrated previously that enforced c-Myc expression results in deregulated cell cycle control of lens fiber cells, while enforced L-Myc leads to their aberrant differentiation. Experiments proposed here continue to utilize the lens as a model developmental genetic system, in combination with biochemical and cell culture-based assays, to elucidate the basis for these differential phenotypic consequences on the molecular level. Regions of the c-Myc protein that are integral to its specific ability to drive cells through the cell cycle will be identified through replacement of L-Myc domains encoding transactivation function and/or DNA binding capacity with those of c-Myc and analysis of resultant phenotypes after targeted overexpression in the lens. To gain insight into the mechanisms by which activities of the cMyc transactivation domain are effected, interactive proteins will be searched for through a two-hybrid screening strategy. Finally, to ascertain the precise phase in the cell cycle wherein c-Myc executes its growth promoting functions, a dominant negative-Myc neutralization approach coupled with a novel lens complementation strategy will be implemented using lenses deficient for Rb, a protein that serves as gatekeeper of the GI/S transition. Together, the findings of these studies will further understanding of the role of Myc family proteins as key regulators through their association with cell cycle proteins and their transactivation of target genes controlling lens proliferation and differentiation.
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