Although cellular proto-oncogenes are suspected of playing important roles in both normal and abnormal cell proliferation and differentiation, the functions of most proto-oncogenes remain hypothetical. In the proposed research, we will investigate the participation of two nuclear proto-oncogenes, c-fos and c-jun, in the transition from quiescence to renewed growth and also during induced differentiation. Recently, these two nuclear oncogenes have been shown to act as a transactivating factor by forming a heterodimer complex and binding to the specific DNA sequence (AP-1 binding site) of promoters of target genes. Both c-fos and c-jun gene products are known to be rapidly induced in mouse fibroblast 3T3 cells during transition from G0 to G1 and also in mouse embryonic teratocarcinoma F9 cells during the early stage of differentiation into endoderm cells. The c-fos/c-jun heterodimer complex seems to comprise a primordial signal transduction system utilized in many different cellular processes such as proliferation and differentiation. A detailed knowledge of the heterodimer activation mechanism and an identification of its target genes are essential to understand fully this important signalling system. By means of the strategy described below, we propose to identify, isolate, and characterize the target genes regulated positively and negatively by the c-fos/c-jun heterodimer complex in proliferating 3T3 cells and in differentiating F9 cells. We will isolate double transformed cell lines of 3T3 and F9 transfected with chimeric recombinant constructs carrying sheep metallothionine promoter and c-fos and c-jun sequence. cDNA libraries will be constructed with mRNAs extracted from those transformed cells stimulated by Zn2+. By screening with specific subtracted probes, a set of cDNA clones coding for target genes will be isolated from the library, and the structures of the genes will be determined. WE will then isolate genomic clones corresponding to cDNA clones and characterize their structures including the promoter region. Finally, promoters of isolated target genes will be tested in vivo for their inducibility by c-fos/c-jun complex by transient transfection experiments using CAT reporter gene constructs. The results of this proposed project should allow important insights into the physiological roles played by proto-oncogene products during the cell cycle differentiation.
