Abstract
RBL1 (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 RBL1 by upstream tumor suppressor pathways remains incompletely understood. Here, we investigate the p53-dependent transcriptional regulation of RBL1 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 RBL1 promoter. Overexpression of p53 showed that p53 represses RBL1 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 RBL1 promoter, while E2F4, p107, and p130 can support this effect to a lesser extent. Overall, our findings demonstrate that p53 controls RBL1 expression indirectly through the p21-RB-E2F pathway by utilizing two E2F binding sites within the RBL1 promoter.