Related Experiment Videos
Transcription of the E2F-1 gene is rendered cell cycle dependent by E2F DNA-binding sites within its promoter
E Neuman1, E K Flemington, W R Sellers
1Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
Abstract:
The cell cycle-regulatory transcription factor E2F-1 is regulated by interactions with proteins such as the retinoblastoma gene product and by cell cycle-dependent alterations in E2F-1 mRNA abundance. To better understand this latter phenomenon, we have isolated the human E2F-1 promoter. The human E2F-1 promoter, fused to a luciferase cDNA, gave rise to cell cycle-dependent luciferase activity upon transfection into mammalian cells in a manner which paralleled previously reported changes in E2F-1 mRNA abundance. The E2F-1 promoter contains four potential E2F-binding sites organized as two imperfect palindromes. Gel shift and transactivation studies suggested that these sites can bind to E2F in vitro and in vivo. Mutation of the two E2F palindromes abolished the cell cycle dependence of the E2F-1 promoter. Thus, E2F-1 appears to be regulated at the level of transcription, and this regulation is due, at least in part, to binding of one or more E2F family members to the E2F-1 promoter.
Insights
The human E2F-1 promoter drives cell cycle-dependent gene expression. E2F transcription factor binding to the E2F-1 promoter is crucial for this regulation, influencing E2F-1 mRNA levels.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- The transcription factor E2F-1 plays a critical role in cell cycle regulation.
- E2F-1 levels are controlled by protein interactions and mRNA abundance, which varies with the cell cycle.
Purpose of the Study:
- To investigate the transcriptional regulation of the human E2F-1 gene.
- To identify the regulatory elements within the E2F-1 promoter responsible for cell cycle-dependent expression.
Main Methods:
- Isolation and cloning of the human E2F-1 promoter fused to a luciferase reporter gene.
- Transfection into mammalian cells to assess luciferase activity.
- Gel shift and transactivation assays to study E2F binding.
- Site-directed mutagenesis of putative E2F-binding sites.
Main Results:
- The isolated human E2F-1 promoter exhibited cell cycle-dependent luciferase activity, mirroring endogenous E2F-1 mRNA patterns.
- The promoter contains four potential E2F-binding sites organized as two imperfect palindromes.
- Gel shift and transactivation studies confirmed E2F binding to these sites in vitro and in vivo.
- Mutation of the E2F-binding palindromes abolished the promoter's cell cycle-dependent activity.
Conclusions:
- Human E2F-1 gene expression is regulated at the transcriptional level.
- Binding of E2F family members to the E2F-1 promoter is a key mechanism for this transcriptional regulation.
- This finding provides insight into the control of cell cycle progression by E2F-1.