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Transcription factor E2F-1 is upregulated in response to DNA damage in a manner analogous to that of p53
1Cancer Research Campaign Cell Transformation Group, Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee DD1 4HN, United Kingdom.
Abstract:
The transcription factor E2F-1 directs the expression of genes that induce or regulate cell division, and a role for E2F-1 in driving cells into apoptosis is the subject of intense discussion. Recently it has been shown that E2F-1 binds and coprecipitates with the mouse double-minute chromosome 2 protein (Mdm2). A domain of E2F-1 (amino acids 390 to 406) shows striking similarity to the Mdm2 binding domain of the tumor suppressor protein p53. It is known that interaction of Mdm2 with p53 through this domain is required for Mdm2-dependent degradation of p53. We show here that E2F-1 protein is upregulated in response to DNA damage. The kinetics of induction are dependent upon the source of DNA damage, i.e., fast and transient after irradiation with X rays and delayed and stable after irradiation with UVC, and thus match the kinetics of p53 induction in response to DNA damage. We show further that E2F-1 is also upregulated by treatment with the transcription inhibitor actinomycin D and with the kinase inhibitor DRB, as well as by high concentrations of the kinase inhibitor H7, all conditions which also upregulate p53. In our experiments we were not able to see an increase in E2F-1 RNA production but did find an increase in protein stability in UVC-irradiated cells. Upregulation of E2F-1 in response to DNA damage seems to require the presence of wild-type p53, since we did not observe an increase in the level of E2F-1 protein in several cell lines which possess mutated p53. Previous experiments showed that p53 is upregulated after microinjection of an antibody which binds to a domain of Mdm2 that is required for the interaction of Mdm2 with p53. Microinjection of the same antibody also increases the expression of E2F-1 protein, while microinjection of a control antibody does not. Furthermore, microinjection of Mdm2 antisense oligonucleotides upregulates E2F-1 protein, while microinjection of an unrelated oligonucleotide does not. These data suggest that E2F-1 is upregulated in a similar way to p53 in response to DNA damage and that Mdm2 appears to play a major role in this pathway.
Insights
The transcription factor E2F-1 is upregulated following DNA damage, similar to p53. Mouse double-minute chromosome 2 protein (Mdm2) appears crucial in this pathway, influencing E2F-1 protein stability and expression.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- The transcription factor E2F-1 regulates cell division and is implicated in apoptosis.
- E2F-1 interacts with mouse double-minute chromosome 2 protein (Mdm2), a known regulator of the tumor suppressor p53.
- The Mdm2-binding domain of E2F-1 resembles that of p53, suggesting a potential functional link.
Purpose of the Study:
- To investigate the regulation of E2F-1 protein levels in response to DNA damage.
- To explore the role of wild-type p53 and Mdm2 in the DNA damage-induced upregulation of E2F-1.
- To elucidate the mechanisms underlying E2F-1 stabilization and expression.
Main Methods:
- Upregulation of E2F-1 protein in response to various DNA damaging agents (X-rays, UVC) and inhibitors (actinomycin D, DRB, H7).
- Analysis of E2F-1 RNA and protein stability in response to DNA damage.
- Assessment of E2F-1 upregulation in cell lines with wild-type versus mutated p53.
- Microinjection of antibodies against Mdm2 and Mdm2 antisense oligonucleotides to study Mdm2's role.
Main Results:
- E2F-1 protein is upregulated by DNA damage, with kinetics mirroring p53 induction.
- UVC irradiation leads to increased E2F-1 protein stability, not RNA production.
- E2F-1 upregulation requires wild-type p53; it is not observed in cells with mutated p53.
- Interference with Mdm2 function (via antibody or antisense oligonucleotides) leads to E2F-1 upregulation.
Conclusions:
- E2F-1 is upregulated in response to DNA damage through a mechanism dependent on wild-type p53.
- Mdm2 plays a significant role in the DNA damage-induced upregulation of E2F-1, likely by stabilizing the protein.
- These findings suggest a parallel regulatory pathway for E2F-1 and p53 in response to cellular stress.