OVERLAPPING DOMAINS ON THE P53 PROTEIN REGULATE ITS TRANSCRIPTIONAL ACTIVATION AND REPRESSION FUNCTIONS

Wild-type p53 has been shown to inhibit transcription from several vir al and cellular promoters without known p53-binding sites, while trans activating promoters with p53-binding sites. Using a series of N- and C-terminal p53 deletion mutants and wild-type p53, we have defined the domains on p53 responsible for its transcriptional functions. To test transcriptional activation by p53 we have used a promoter-chloramphen icol acetyltransferase (CAT) construct containing synthetic p53-bindin g sites. To check transcriptional inhibition by p53 we have used a hum an cytomegalovirus immediate-early promoter construct, CMV-CAT. Using transient transfection-transcription assays in Saos-2 cells, we determ ined that the p53 transcriptional activation and repression domains ov erlap at the N-terminus. This suggests the possibility that the same t ranscriptional machinery is involved in both functions. A C-terminal d eletion up to amino acid 327 (del 393-327) eliminated repression of CM V-CAT, while preserving the transactivation function to a large extent . Using gluteraldehyde cross-linking experiments, we observed that the mutant del 393-327, which is transactivation-competent, but repressio n-defective, could not oligomerize. Thus, oligomerization of p53 is no t required for transactivation, but may be essential for repression. I nterestingly, transactivation by the oligomerization-defective mutant could be inhibited by cotransfection with a plasmid expressing the tra nsforming mutant p53-175H.