Transcription of the c-fos proto-oncogene is activated rapidly and transiently in many mammalian cell types treated with growth factors. In many cases, a specific sequence element in the c-fos promoter termed the Serum Response Element (SRE), is required. A nuclear factor, Serum Response Factor (SRF), which binds specifically to the SRE has been cloned. In order to understand how growth factors transmit their signals to the nucleus to rapidly activate new gene expression, SRFs mechanism of regulation will be studied in vivo and in vitro. While there is evidence that SRF is required for serum regulation of the fos gene, it is unknown how SRF is regulated. Biochemical approaches have not revealed a clear mechanism for SRF regulation. Therefore, SRF regulation will be studied using an in vivo assay we have developed to measure its function. A minimal regulatory domain of SRF required for regulation by growth factors has been identified and we will make further mutations to define critical elements of this region. This information will be critical for directing biochemical studies. An SRF-complexing protein, p62TCF, has been suggested to control SRF function. There is strong evidence, however, that it is not required and experiments are proposed to focus on the TCF- independent path way for SRF activation. Receptor tyrosine kinase mutants will also be used to distinguish cellular signalling pathways required for c-fos induction. Two putative inhibitory domains in SRF have been identified using GAL4-SRF constructs. These domains may be involved in regulation of SRF such that their functional significance will be further investigated. Methods are also proposed to identify SRF-complexing proteins that bind to regulatory domains defined above. Expression of c-fos is rapidly repressed after activation. The SRE has been shown to be sufficient for both activation and repression and fos protein production may cause the repression. The mechanism of repression will be investigated to determine whether SRF is involved and if so, which domains. Several methods are proposed to measure this activity and to separate SRF's activation and putative repression functions. The mechanism of transcriptional activation by SRF will be studied in vitro. This may help determine the critical functions of SRF regulated by serum. We have found that SRF interacts with TFIIF in vitro and propose that SRF activates transcription by recruiting TFIIF to the transcriptional initation complex. This SRF-TFIIF interaction will be further investigated to support this model.
Showing the most recent 10 out of 30 publications
