The Tumor Suppressor p53 Downregulates p107 (RBL1) Through p21–RB/E2F Signaling and Tandem E2F Sites

<i>RBL1</i> (p107) is a member of the retinoblastoma (RB) family of pocket proteins involved in cell cycle regulation and E2F transcriptional repression. While its promoter contains conserved E2F motifs, the integrated regulation of <i>RBL1</i> by upstream tumor suppressor pathways remains incompletely understood. Here, we investigate the p53-dependent transcriptional regulation of <i>RBL1</i> and dissect the contribution of its tandem E2F binding sites to this mechanism. Luciferase assays in synchronized cells demonstrated that these two conserved E2F sites are required for cell cycle-dependent activation of the <i>RBL1</i> promoter. Overexpression of p53 showed that p53 represses <i>RBL1</i> promoter activity in an E2F site-dependent manner. Using HCT116 p21 knockout cells, we revealed that this p53-dependent repression is mediated by p21. Chromatin immunoprecipitation confirmed dynamic in vivo binding of E2F1-3 and E2F4, while DNA pull-down assays revealed specific in vitro recruitment of RB, p107, and E2F1-4 to the two E2F sites, along with weak binding of MuvB components. Additional experiments in RB^-/- and LIN37^-/- knockouts showed that RB/E2F repressing complex plays the main role in repressing the <i>RBL1</i> promoter, while E2F4, p107, and p130 can support this effect to a lesser extent. Overall, our findings demonstrate that p53 controls <i>RBL1</i> expression indirectly through the p21-RB-E2F pathway by utilizing two E2F binding sites within the <i>RBL1</i> promoter.