The long-term objective of this project is to understand at a biochemical level how transcription of the c-fos proto-oncogene is induced by extracellular signals. Transcription of the c-fos gene is rapidly induced in cells exposed to mitogenic and differentation-promoting factors. These factors generally act by binding to specific, high-affinity cell surface receptors. How this interaction transduces a signal into the cell and how this signal eventually travels to the nucleus are poorly understood. Induction of c-fos transcription is a primary response to such signals, requiring no new protein synthesis. Moreover, it is the earliest known nuclear response. Therefore, a biochemical analysis of the mechanism of induction of c-fos by extracellular signals may elucidate the general mechanisms through which cell growth is regulated by these signals and how these signalling mechanisms are perturbed in cancer cells. Previous work has identified a number of potential intracellular intermediates in the signaling pathways through which c-fos transcription is induced. The first specific aim of this project is to purify and characterize one such intermediate, the protein that binds to the short DNA sequence required for induction of c-fos transcription by serum growth factors. Antibodies will be raised to the purified protein and used to study its response in vivo to extracellular signals. The second specific aim is to develop an in vitro assay for the proposed transcriptional stimulatory activity of this protein. The strategy is to prepare transcription templates carrying polymerized protein binding sites substituted for the identified upstream elements in the c-fos promoter, with the goal of observing in vitro transcription dependent on these sequences and on exogenously added protein. The third specific aim is to use gene transfer experiments to identify additional cis-acting regulatory sequences in the c-fos gene required for response to experimentally distinct intracellular signalling pathways. If such sequences are identified, the proteins that bind to these sites will also be purified and used to develop in vitro transcription assays.
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