The c-fos proto-oncogene and actin genes are transiently activated over 100-fold within minutes after quiescent fibroblasts are stimulated with platelet-derived growth factor and when the rat pheochromocytoma cell line PC12 is induced to differentiate with nerve growth factor (NGF). The activation of these genes represents a highly regulated program of gene expression that occurs in diverse cell types in response to multiple environmental stimuli. c-fos encodes a nuclear protein whose disruption leads to deregulated cell growth and differentiation suggesting that the normal function of this protein may be to control growth and differentiation events. This proposal focuses on NGF regulation of c-fos expression in PC12 cells. Two important questions are addressed. First, what is the biochemical pathway by which NGF activates and represses c-fos transcription? Second, what is the function of rapid c-fos activation during PC12 cell differentiation? The first step towards defining the mechanism of NGF regulation of c-fos expression will be to use transfection protocols to identify sequences within the c-fos gene that control transcriptional activation and repression. The c-fos regulatory sequences will then be employed in DNA binding experiments to identify and characterize c-fos transcriptional factors. Similar binding experiments will be used to test the possibility that the c-fos protein itself functions as a transcriptional factor. To identify other genes that may be critical for NGF induced differentiation, NGF non-responsive mutant PC12 cells will be generated using the technique of retrovirus insertion mutagenesis. This method allows the isolation of the genes that have been disrupted in the mutant cell line. The experiments should yield a significant amount of new information relevant to the process of tumorigenesis. Characterization of the nuclear factors that regulate c-fos transcription has the potential to define a new class of recessive oncogenes. Elucidating the mechanisms by which NGF controls neuronal differentiation could uncover the molecular basis of a variety of neurological diseases.
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