CELLULAR TRANSFORMATION BY AP-1 TRANSCRIPTION FACTORS

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Oncogenes that encode DNA-binding, transcription factors are likely to contribute to the cancerous state by directly altering gene regulation. The Jun and Fos oncoproteins, which belong to the family of transcription factors that bind AP-1 sites (TGACTCA), have been extensively characterized. Mutational analyses have defined regions of Jun and Fos that are important for oncogenesis, but the functional complexities of AP- 1 factors make it difficult to discriminate among potential molecular mechanisms. Moreover, although AP-1 sites are found in numerous promoters and can affect transcription, oncogenically relevant target genes have not yet been identified. We have developed a novel approach to studying oncogenesis mediated by AP-1 factors that involves yeast GCN4, an autonomous and heterologous AP-1 transcription factor whose function is not subject to the regulatory complexities of the Jun-Fos family. In rat embryo fibroblasts, GCN4 induces transcriptional activation through AP-1 sites, but unlike Jun and Fos1 it is unable to mediate cellular transformation in cooperation with Ha-ras. Analysis of chimeric proteins containing the GCN4 DNA-binding domain defined oncogenic functions on Jun and Fos that are distinct from generic transcriptional activation domains. The overall goal of this proposal is to use these and other AP-1 derivatives as probes for identifying oncogenically relevant target genes and for determining how Fos and Jun transform cells. To accomplish this, we propose the following experiments. First, oncogenically-relevant target genes will be identified by virtue of being differentially affected by oncogenic and non-oncogenic derivatives of GCN4 that are equally efficient at transcriptional activation through AP-1 sites. Rat embryo fibroblasts will be transfected in parallel with DNAs expressing Ha-ras, a cell-surface marker, and the GGN4 derivative of interest, and transfected cells will be isolated by panning (using antibodies to the cell surface marker). Differentially expressed genes will be identified by direct analysis of candidate genes likely to be important in cell growth, by PCR- based RNA display technology, and by subtractive cDNA libraries. Second, for a selected number of target genes, we will isolate the promoter regions and determine whether they can recapitulate the expression pattern. Further promoter analysis will be focused on identifying AP-1 and other DNA sequences involved in the discrimination between the GCN4 derivatives. The long-term goal is to determine the transcriptional mechanisms that are specifically correlated to cellular transformation. Third, using the yeast 2-hybrid system, we will search for proteins that interact with the N-terminal loop residues of Fos, a region that transforms cells when fused to the GCN4 bZIP domain. Oncogenic relevance of the identified proteins will be tested using our existing set of transforming and non-transforming Fos derivatives that differ by l or 2 amino acids. Genes passing the oncogenic relevance test will be sequenced, and the encoded proteins will be tested for their transcriptional properties on promoters identified and characterized in aims l and 2. Overall, these studies represent a novel approach for identifying and characterizing oncogenically-relevant targets of AP-1 transcription factors. The results should have broad significance because targets of other oncogenes and tumor suppressor genes are essentially unknown.