The p53 protein is a potent tumor and growth suppressor. Addition of wild-type p53 to transformation assays dramatically decreases the number of foci formed. Hundreds of alterations of the p53 gene have been found in tumors as diverse as colorectal carcinomas, lung and esophageal cancer, breast cancer, osteosarcomas, hepatocellular carcinomas, and lymphomas and leukemias. We have characterized the biochemical activity of p53 as that of a transcription factor. Fusion proteins were made using p53 and the DNA-binding domain of the yeast transcription factor GAL4. Wild-type p53/GAL4 fusion proteins specifically transactivated the reporter plasmid that contained GAL4 binding sites, while most p53 mutants did not. One mutant analyzed in this assay is especially interesting since it encodes a temperature-sensitive p53. Expression of p53val 135 which contains an ala to val substitution at amino acid 135 has no affect on growth of cells at 37 degrees C. However, upon shift to 32 degrees C, cells expressing p53val 135 stop growing. Analysis of p53val135 fused to the GAL4 DNA-binding domain in transactivation assays showed that it was inactive at 37 degrees C and regained transactivation function at 32 degrees C. In addition, wild-type but not mutant p53 has been shown to activate various DNA sequences fused to heterologous promoters. These experiments suggest a direct correlation between p53 function as a tumor suppressor and transactivator. In an attempt to look for targets of p53 transactivation, we asked whether p53 could activate its own promoter. In transient transfection assays, wild-type but not mutant p53 activated the p53 promoter. Deletion analysis of the p53 promoter delineated the sequences critical for autoregulation. The main focus of this proposal is to study the function of p53 as a transcription factor with the long term goal of dissecting p53's role in tumor suppression. Because those genes activated by p53 are probably critical components in the pathway of tumor suppression, we plan to identify downstream targets of p53 transactivation. Subtractive hybridization of mRNA from cells with a temperature-sensitive p53 mutant grown at permissive and nonpermissive temperatures will be used to identify downstream targets of p53 transactivation. The mechanism of autoregulation will be analyzed in detail, specifically identification of the factor(s) binding the p53 responsive element. The establishment of an in vitro system to analyze regulation of the p53 promoter and other target promoters will be extremely useful in analyzing interactions of p53 with the general transcription machinery.