The Tumor Suppressor p53 Downregulates p107 (RBL1) Through p21–RB/E2F Signaling and Tandem E2F Sites
Abstract
1. Introduction
2. Results
2.1. p53 Represses RBL1 Expression in a p21-Dependent Manner
2.2. p53-Mediated Regulation of RBL1 Is Retained in a Short Promoter Fragment Containing Conserved E2F Sites
2.3. E2F Binding Sites Are Essential for Cell Cycle–Dependent Regulation of RBL1
2.4. p53-Dependent Repression of RBL1 Requires Both Tandem E2F Binding Sites
2.5. E2F1–4, RB, and p107 Bind to the RBL1 Promoter Through the Two E2F Binding Sites
2.6. p53-Dependent Repression of RBL1 Is Mediated by RB with Partial Support from DREAM
3. Discussion
4. Materials and Methods
4.1. Cell Lines and Knockouts
4.2. Cell Culture and Treatments
4.3. Flow Cytometry
4.4. RNA Isolation and Quantitative RT-PCR
4.5. Cloning and Mutagenesis
4.6. Luciferase Promoter Reporter Assays
4.7. Chromatin Immunoprecipitation (ChIP)
4.8. DNA Affinity Purification
4.9. SDS-PAGE and Western Blotting
4.10. Statistics
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
BMYB | B-Myb transcription factor |
β-Actin | Beta-actin |
CDK | Cyclin-dependent kinase |
CDKN1A | Cyclin-dependent kinase inhibitor 1A (p21) |
CDC25C | Cell division cycle 25C phosphatase |
ChIP | Chromatin immunoprecipitation |
DMSO | Dimethyl sulfoxide |
DREAM | Dimerization partner, RB-like, E2F, and MuvB complex |
E2F | E2 promoter-binding factor |
FACS | Fluorescence-activated cell sorting |
FCS | Fetal calf serum |
FOXM1 | Forkhead box M1 transcription factor |
GAPDH | Glyceraldehyde-3-phosphate dehydrogenase |
HRP | Horseradish peroxidase |
LIN37 | Lin-37 cell cycle regulatory protein |
LIN54 | Lin-54 cell cycle regulatory protein |
LIN9 | Lin-9 cell cycle regulatory protein |
MDM2 | Mouse double minute 2 homolog |
mRNA | Messenger ribonucleic acid |
MuvB | Multi-vulva class B core complex |
NFYA | Nuclear transcription factor Y subunit alpha |
PBS | Phosphate-buffered saline |
PCR | Polymerase chain reaction |
PVDF | Polyvinylidene difluoride |
RB | Retinoblastoma protein |
RBL1 | Retinoblastoma-like protein 1 (p107) |
RLU | Relative light unit |
RT-qPCR | Reverse transcription quantitative polymerase chain reaction |
SDS-PAGE | Sodium dodecyl sulfate polyacrylamide gel electrophoresis |
SEM | Standard error of the mean |
TBS-T | Tris-buffered saline with Tween 20 |
U6 | U6 small nuclear RNA |
WT | Wild-type |
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Promoter | −27 E2F Dist | E2F Prox −9 | TSS | |
---|---|---|---|---|
Human RBL1 WT | 5′-CAGCTGCAGA | TTTTCGCGCG | CTTTGGCGCA | GGTGGTTGTG-3′ |
Human RBL1 E2F Proximal | 5′-CAGCTGCAGA | TTTTCGCGCG | CAGCTCACCA | GGTGGTTGTG-3′ |
Human RBL1 E2F Distal | 5′-CAGCTGCAGA | TCACTCGACG | CTTTGGCGCA | GGTGGTTGTG-3′ |
Human RBL1 E2F Double | 5′-CAGCTGCAGA | TCACTCGACG | CAGCTCACCA | GGTGGTTGTG-3′ |
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Azzahrani, K.; Alqahtani, F. The Tumor Suppressor p53 Downregulates p107 (RBL1) Through p21–RB/E2F Signaling and Tandem E2F Sites. Int. J. Mol. Sci. 2025, 26, 9903. https://doi.org/10.3390/ijms26209903
Azzahrani K, Alqahtani F. The Tumor Suppressor p53 Downregulates p107 (RBL1) Through p21–RB/E2F Signaling and Tandem E2F Sites. International Journal of Molecular Sciences. 2025; 26(20):9903. https://doi.org/10.3390/ijms26209903
Chicago/Turabian StyleAzzahrani, Khaled, and Faleh Alqahtani. 2025. "The Tumor Suppressor p53 Downregulates p107 (RBL1) Through p21–RB/E2F Signaling and Tandem E2F Sites" International Journal of Molecular Sciences 26, no. 20: 9903. https://doi.org/10.3390/ijms26209903
APA StyleAzzahrani, K., & Alqahtani, F. (2025). The Tumor Suppressor p53 Downregulates p107 (RBL1) Through p21–RB/E2F Signaling and Tandem E2F Sites. International Journal of Molecular Sciences, 26(20), 9903. https://doi.org/10.3390/ijms26209903