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A role for a p21-E2F interaction during senescence arrest of normal human fibroblasts
C A Afshari1, M A Nichols, Y Xiong
1Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Abstract:
The family of E2F transcription factors forms different multiprotein complexes with cell cycle regulatory proteins to control the expression of genes important in cell proliferation. In this study, we identified four distinct E2F complexes present in aged and senescent normal, human diploid fibroblasts. Two appeared to be identical to the previously described G1-specific p130 and Rb-E2F complexes present in young G0-arrested cells. The other two were novel E2F complexes that contained the cyclin-dependent kinase inhibitor p21 (cip1/WAF1/Sdi1/CAP20/PIC1) complexed with Rb/CDK2/cyclin E or with the Rb-related p107/CDK2/ cyclin D. These p21-E2F complexes, while present in young G1 cells at very low levels, were elevated in senescent cells. The p21 containing E2F complexes were not detected during the S-phase in young cells. The DNA-binding stability of the p21 complexes was approximately 10 times greater than the stability of any other E2F complex or uncomplexed E2F. Addition of purified p21 protein to the S-phase-specific cyclin A/ CDK2-p107-E2F complex from young cells dissociated cyclin A and CDK2 from p107/E2F, suggesting an additional novel function for p21. Finally, expression of p21 specifically inhibited transcription from an E2F-dependent promoter but had no effect on a mutant E2 promoter. In addition to its inhibition of CDK enzymes and proliferating cell nuclear antigen function in DNA replication, these studies reveal a novel mechanism by which p21 mediates growth arrest: direct interaction with E2F complexes and negative regulation of E2F transcription factor activity.
Insights
Researchers discovered novel p21-E2F complexes in senescent cells that enhance DNA-binding stability and inhibit transcription. This reveals a new mechanism for p21-mediated growth arrest via direct interaction with E2F transcription factors.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- E2F transcription factors regulate cell proliferation genes through multiprotein complexes.
- Aged and senescent cells exhibit altered cell cycle control mechanisms.
Purpose of the Study:
- To identify and characterize E2F complexes in aged and senescent human diploid fibroblasts.
- To elucidate the role of p21 in regulating E2F activity and mediating growth arrest.
Main Methods:
- Identification and characterization of E2F complexes using biochemical techniques.
- Analysis of E2F complex stability and DNA-binding properties.
- Assays to determine the effect of p21 on E2F-dependent transcription.
Main Results:
- Four distinct E2F complexes were identified, including two novel complexes containing p21 (cyclin-dependent kinase inhibitor).
- p21-E2F complexes were elevated in senescent cells and showed significantly higher DNA-binding stability.
- p21 directly inhibited E2F-dependent transcription, suggesting a novel growth arrest mechanism.
Conclusions:
- p21 plays a critical role in mediating growth arrest by directly interacting with E2F transcription factors.
- This interaction leads to the negative regulation of E2F transcriptional activity, independent of its kinase inhibitory functions.
- The findings reveal a novel mechanism of cellular senescence regulation involving p21 and E2F.