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Cell cycle analysis of E2F in primary human T cells reveals novel E2F complexes and biochemically distinct forms of
T Chittenden1, D M Livingston, J A DeCaprio
1Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
Abstract:
The transcription factor E2F activates the expression of multiple genes involved in cell proliferation, such as c-myc and the dihydrofolate reductase gene. Regulation of E2F involves its interactions with other cellular proteins, including the retinoblastoma protein (Rb), the Rb-related protein p107, cyclin A, and cdk2. We undertook a detailed analysis of E2F DNA-binding activities and their cell cycle behavior in primary human T cells. Three E2F DNA-binding activities were identified in resting (G0) T cells with mobilities in gel shift assays distinct from those of previously defined E2F complexes. One of these activities was found to be a novel, less abundant, Rb-E2F complex. The most prominent E2F activity in resting T cells (termed complex X) was abundant in both G0 and G1 but disappeared as cells entered S phase, suggesting a possible role in negatively regulating E2F function. Complex X could be dissociated by adenovirus E1A with a requirement for an intact E1A conserved region 2. However, X failed to react with a variety of antibodies against Rb or p107, implicating the involvement of an E1A-binding protein other than Rb or p107. In addition to these novel E2F complexes, three distinct forms of unbound (free) E2F were resolved in gel shift experiments. These species showed different cell cycle kinetics. UV cross-linking experiments suggested that a distinct E2F DNA-binding protein is uniquely associated with the S-phase p107 complex and is not associated with Rb. Together, these results suggest that E2F consists of multiple, biochemically distinct DNA-binding proteins which function at different points in the cell cycle.
Insights
This study reveals multiple forms of the E2F transcription factor in human T cells, each with distinct DNA-binding activities and cell cycle roles. These findings clarify E2F regulation during cell proliferation.
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
- Transcription Factor Function
Background:
- The transcription factor E2F regulates genes essential for cell proliferation.
- E2F activity is modulated by interactions with proteins like retinoblastoma protein (Rb) and p107.
- Understanding E2F's dynamic behavior is crucial for comprehending cell cycle control.
Purpose of the Study:
- To analyze E2F DNA-binding activities and cell cycle-dependent behavior in primary human T cells.
- To identify novel E2F complexes and their interactions with regulatory proteins.
- To elucidate the distinct roles of various E2F forms throughout the cell cycle.
Main Methods:
- Gel shift assays to detect and characterize E2F DNA-binding activities.
- Analysis of E2F complexes in different cell cycle phases (G0, G1, S).
- UV cross-linking experiments to identify protein associations.
Main Results:
- Three novel E2F DNA-binding activities were identified in resting T cells, distinct from known complexes.
- A prominent complex (complex X) abundant in G0/G1 phases disappeared in S phase, suggesting negative regulation.
- Distinct unbound E2F species with varying cell cycle kinetics were resolved, and an S-phase specific E2F-p107 association was observed.
Conclusions:
- E2F comprises multiple, biochemically distinct DNA-binding proteins.
- These different E2F forms exhibit unique cell cycle kinetics and regulatory functions.
- The findings provide a more nuanced understanding of E2F's role in cell cycle progression.