Related Experiment Videos
Regulation of mdm2 expression by p53: alternative promoters produce transcripts with nonidentical translation
Y Barak1, E Gottlieb, T Juven-Gershon
1Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Abstract:
The mdm2 proto-oncogene product binds to the p53 tumor suppressor protein and inhibits its ability to trans-activate target genes. One such target gene is mdm2 itself, which is therefore considered a component of a p53 negative feedback loop. Two tandem p53-binding motifs residing within the first intron of the murine mdm2 gene confer upon it p53-mediated activation. We now report that in murine cells p53 activates an internal mdm2 promoter (P2) located near the 3' end of intron 1, resulting in mRNA whose transcription starts within exon 2. P2 is activated by p53 within artificial constructs, as well as within the context of the chromosomal mdm2 gene. Activation follows either the introduction of overexpressed wild-type p53 into cells or the induction of endogenous wild-type p53 by ionizing radiation. The upstream, constitutive (P1) mdm2 promoter is only mildly affected by p53, if at all. The p53-derived mdm2 transcripts lack exon 1 and a few nucleotides from exon 2. As the first in-frame AUG of mdm2 is located within exon 3, the two types of mdm2 transcripts should possess similar coding potentials. Nevertheless, in vitro conditions, where each of these transcripts yields a distinct translation profile, reflect the differential usage of translation initiation codons. Initiation of translation at internal AUG codons, which occurs also in vivo, gives rise to MDM2 polypeptides incapable of binding to p53. In vitro translation profiles of the various mdm2 transcripts could be manipulated by changing the amounts of input RNA. Thus, p53 can modulate both the amount and the nature of MDM2 polypeptides through activation of the internal P2 promoter.
Insights
The p53 tumor suppressor protein activates a second mdm2 promoter (P2), generating distinct mdm2 transcripts. This regulation influences the amount and function of MDM2 proteins, impacting the p53 feedback loop.
Area of Science:
- Molecular Biology
- Oncology
- Gene Regulation
Background:
- The mdm2 proto-oncogene product inhibits the p53 tumor suppressor protein.
- mdm2 is a direct transcriptional target of p53, forming a negative feedback loop.
- p53-binding motifs in the mdm2 gene regulate its expression.
Purpose of the Study:
- To investigate the role of an internal mdm2 promoter (P2) in p53-mediated gene regulation.
- To determine how p53 influences mdm2 transcription and translation.
- To elucidate the impact of p53 on MDM2 protein isoforms.
Main Methods:
- Analysis of mdm2 promoter activity in murine cells.
- Introduction of overexpressed or induced wild-type p53.
- Assessment of mdm2 transcript variants and their translation.
- In vitro translation assays.
Main Results:
- p53 activates the internal mdm2 P2 promoter, leading to transcription initiation within exon 2.
- p53-induced mdm2 transcripts lack exon 1 and part of exon 2.
- Differential translation initiation occurs from distinct mdm2 transcripts, producing different MDM2 polypeptides.
- p53 can modulate MDM2 protein quantity and type via P2 promoter activation.
Conclusions:
- p53 regulates mdm2 expression through both the P1 and P2 promoters.
- Activation of the P2 promoter generates alternative mdm2 transcripts and non-functional MDM2 proteins.
- This provides a novel mechanism for p53 to control MDM2 levels and function.