Transforming growth factor alpha is a 50 amino acid mitogenic peptide that shares structural and functional homology with EGF. It is produced by many transformed cells, as well as developing and normal mammalian tissues, indicating a broad role in nature. Very little is known, however, about the regulatory mechanisms involved in TGF-alpha mRNA expression. The existence of cloned neoplastic cell lines derived by chemical transformation of a rat liver epithelial cell have provided a model in which to explore these mechanisms. Using certain cell lines from this lineage, it is the long range goal of this proposal to elucidate the mechanisms regulating the TGF-alpha gene and to identify how TGF-alpha mRNA expression is altered in its various contexts. Accordingly, we will: (1) perform nuclear run-on assays, and methylation and DNase I sensitivity assays to ascertain if regulatory control occurs at the level of transcription and is influenced by structural changes in the gene; (2) further define those sequences representing positive and negative regulatory elements of the TGF-alpha promoter by CAT assay using wild-type and mutagenized constructs containing the putative regulatory sequences; (3) recognize activities from cell extracts which bind to identified regulatory sequences by performing gel shift and DNA footprinting assays; (4) examine the role of 3' untranslated sequences in TGF-alpha mRNA stability, and hence TGF-alpha mRNA accumulation. This will be accomplished by preparing chimeric constructs which contain varying amounts of 3' untranslated sequences from the TGF-alpha gene and an easily identified reporter gene. These will be used in a transfection experiments and the stability of the resultant transcripts evaluated by S1 nuclease digestion analysis subsequent to treatment of the transfectants with actinomycin D.
