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Regulation of the cyclin D3 promoter by E2F1
Yihong Ma1, Jing Yuan, Mei Huang
1Program in Molecular Oncology, H. Lee Moffitt Comprehensive Cancer Center, Tampa, Florida 33612, USA.
Abstract:
We have previously demonstrated that ectopic expression of E2F1 is sufficient to drive quiescent cells into S phase and that E2F1 expression can contribute to oncogenic transformation. Key target genes in this process include master regulators of the cell cycle, such as cyclin E, which regulates G(1) progression, and cyclin A, which is required for the initiation of DNA synthesis. In the present work, we present novel evidence that a second G(1) cyclin, cyclin D3, is also potently activated by E2F1. First, an estrogen receptor-E2F1 fusion protein (ER-E2F1) potently activates the endogenous cyclin D3 mRNA upon treatment with 4-hydroxytamoxifen, which induces nuclear accumulation of the otherwise cytosolic fusion protein. Furthermore, trans-activation of cyclin D3 by ER-E2F1 occurs even in the presence of the protein synthesis inhibitor cycloheximide and thus appears direct. Second, all of the growth-stimulatory members of the E2F family (E2F1, -2, and -3A) potently activate a cyclin D3 promoter reporter, whereas growth-restraining members of the family (E2F4, -5, and -6) have little effect. Third, recombinant E2F1 binds with high affinity to the cyclin D3 promoter in vitro. Fourth, chromatin immunoprecipitation assays demonstrate that endogenous E2F1 is associated with the cyclin D3 promoter in vivo. Finally, mapping experiments localize the essential E2F regulatory element of the cyclin D3 promoter to a noncanonical E2F site in the promoter between nucleotides -143 and -135 relative to the initiating methionine codon. We conclude that in addition to cyclins E and A, E2F family members can also activate one member of the D-type cyclins, further contributing to the ability of the stimulatory E2F family members to drive cellular proliferation.
Insights
The E2F1 transcription factor directly activates cyclin D3 gene expression, promoting cell cycle progression. This finding expands the known targets of E2F1, highlighting its role in driving cellular proliferation and oncogenesis.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Ectopic expression of E2F1 drives quiescent cells into S phase and contributes to oncogenic transformation.
- Key E2F1 targets include cyclin E and cyclin A, regulators of G1 progression and DNA synthesis initiation.
- The role of D-type cyclins in E2F1-mediated cell cycle control requires further elucidation.
Purpose of the Study:
- To investigate whether E2F1 activates cyclin D3, another G1 cyclin.
- To determine the mechanism and specificity of E2F1-mediated cyclin D3 activation.
- To understand the contribution of cyclin D3 to E2F1's proliferative effects.
Main Methods:
- Utilized an estrogen receptor-E2F1 fusion protein (ER-E2F1) to study endogenous cyclin D3 mRNA activation.
- Employed cycloheximide to assess the directness of ER-E2F1 trans-activation.
- Tested E2F family members' effects on a cyclin D3 promoter reporter.
- Performed in vitro binding assays and chromatin immunoprecipitation (ChIP) to confirm E2F1-promoter interaction.
- Localized the E2F binding site on the cyclin D3 promoter through mapping experiments.
Main Results:
- ER-E2F1 treatment potently activated endogenous cyclin D3 mRNA, even with protein synthesis inhibition, indicating direct activation.
- Growth-stimulatory E2F family members (E2F1, -2, -3A) activated the cyclin D3 promoter reporter, while growth-restraining members (E2F4, -5, -6) did not.
- Recombinant E2F1 bound the cyclin D3 promoter in vitro, and endogenous E2F1 was associated with the promoter in vivo.
- A critical E2F regulatory element was mapped to a noncanonical site (-143 to -135) on the cyclin D3 promoter.
Conclusions:
- E2F family members directly activate cyclin D3 gene expression.
- This activation contributes to the ability of stimulatory E2F members to drive cellular proliferation.
- Cyclin D3 is a novel target gene of E2F, alongside cyclins E and A, in regulating the cell cycle.