Related Experiment Videos
Overlapping domains on the p53 protein regulate its transcriptional activation and repression functions
M A Subler1, D W Martin, S Deb
1Department of Microbiology, University of Texas Health Science Center at San Antonio 78284.
Abstract:
Wild-type p53 has been shown to inhibit transcription from several viral and cellular promoters without known p53-binding sites, while transactivating 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-chloramphenicol acetyltransferase (CAT) construct containing synthetic p53-binding sites. To check transcriptional inhibition by p53 we have used a human cytomegalovirus immediate-early promoter construct, CMV-CAT. Using transient transfection-transcription assays in Saos-2 cells, we determined that the p53 transcriptional activation and repression domains overlap at the N-terminus. This suggests the possibility that the same transcriptional machinery is involved in both functions. A C-terminal deletion up to amino acid 327 (del 393-327) eliminated repression of CMV-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 repression-defective, could not oligomerize. Thus, oligomerization of p53 is not required for transactivation, but may be essential for repression. Interestingly, transactivation by the oligomerization-defective mutant could be inhibited by cotransfection with a plasmid expressing the transforming mutant p53-175H.
Insights
Wild-type p53 protein regulates gene transcription. Researchers found its activation and repression functions overlap at the N-terminus, with oligomerization essential for repression but not activation.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- The p53 protein is a crucial tumor suppressor involved in regulating cellular responses to stress.
- Wild-type p53 exhibits dual transcriptional roles: activating genes with specific binding sites and inhibiting others.
- Understanding the specific domains and mechanisms governing these functions is key to p53 research.
Purpose of the Study:
- To delineate the specific domains of the p53 protein responsible for its transcriptional activation and repression functions.
- To investigate the role of p53 oligomerization in its transcriptional activities.
- To explore the interplay between different p53 mutants and their transcriptional effects.
Main Methods:
- Utilized a series of N- and C-terminal p53 deletion mutants alongside wild-type p53.
- Employed transient transfection-transcription assays with chloramphenicol acetyltransferase (CAT) reporter constructs.
- Conducted gluteraldehyde cross-linking experiments to assess protein oligomerization.
Main Results:
- Identified overlapping N-terminal domains for both p53 transcriptional activation and repression.
- Demonstrated that a C-terminal deletion mutant (del 393-327) retained transactivation but lost repression capabilities.
- Observed that this repression-defective mutant could not oligomerize, suggesting oligomerization is crucial for repression, not activation.
- Showed that transactivation by the oligomerization-defective mutant was inhibited by a transforming p53 mutant (p53-175H).
Conclusions:
- The N-terminus of p53 harbors domains critical for both transcriptional activation and repression, implying shared regulatory machinery.
- p53 oligomerization is essential for its transcriptional repression activity but not for transactivation.
- The findings provide insights into the complex regulatory mechanisms of p53 transcriptional function and its potential role in cancer.