Fig 2: RFX7 up-regulates its target genes in response to stress. (A) The p53-dependent regulation of direct RFX7 target genes (Table 1) across 20 datasets from a meta-analysis (23). Genes were identified as significantly up-regulated (green; +1), down-regulated (red; −1), or not significantly differentially regulated (white; 0). The p53 Expression Score represents the summary across all 20 datasets. No meta-analysis data was available for NRSN2-AS1 and HNRNPUL2. (B) RT-qPCR data from U2OS cells depleted for RFX7 or p53 and treated with DMSO control, Nutlin-3a (N3A), Actinomycin D (AD) and Doxorubicin (Dox). RT-qPCR data normalized to ACTR10 and siControl DMSO levels. Mean and standard deviation is displayed. Statistical significance of RT-qPCR data obtained through a two-sided unpaired t-test, n = 6 replicates (two biological with three technical each).

Fig 3: RFX7 target gene expression correlates significantly positive with good prognosis in 11 cancer types. Kaplan-Meier plots of patients from TCGA cohorts. Patients were grouped into low, medium, and high based on the rank expression of 19 direct RFX7 target genes that display a p53 Expression Score > 5 (23). 11 out of 33 cancer types are displayed that show a significantly (Cox likelihood ratio test variable <0.05 and Cox likelihood ratio test group low versus high < 0.1) better prognosis when the expression of the RFX7 targets is higher. Only one cancer type (TCGA-COAD) showed a significantly poorer prognosis. Statistical significance obtained through the Cox proportional hazards (PH) model (Cox test variable). To correct for major confounding factors, gender and age were included into the multivariate regression analysis. Statistical significance of the rates of occurrence of events over time between the groups was obtained using the fitted Cox PH model (Cox test groups).

Fig 4: Analyses of RFX7 involvement in transcriptional regulation of genes upregulated upon MDMX depletion. (A,B) MEL202/CR-Ctrl and/CR-p53, i-shCtrl and i-shF1F3, containing either i-shCtrl or i-shMDMX shRNAs, were treated for 72 h with 10 ng/mL doxycycline (D) or Vehicle (V) after which RNA and protein was harvested and analyzed for the mRNA expression of RFX7 and the reported RFX7-target genes RFX5, PNRC1 and PDCD4 (A) and to evaluate the effect of MDMX depletion on RFX7 protein migration. Significant alterations (p < 0.05) in gene expression are indicated with *. These are the same samples as analyzed in Figure 3. (C) 92.1/i-shCtrl and 92.1/i-shF1F3, i-shCtrl and i-shMDMX cell lines were treated for 72 h with 10 ng/mL doxycycline, after which protein lysates were harvested and analyzed for RFX7 expression. These are the same samples as analyzed in Supplementary Figure S6. Original blots see Supplementary File S1.

Fig 5: p53 inhibits AKT and mTOR through DDIT4 and RFX7.A–C Western blot analysis of U2OS and RPE-1 cells transfected with indicated siRNAs and treated with 10 µM Nutlin-3a or DMSO control. Actin served as loading control. Densitometric quantification relative to siControl DMSO samples and actin levels. D Critical nodes in p53-dependend AKT and mTORC1 control. Blue nodes are direct p53 target genes in human. Black edges indicate activation or inhibition. Green and orange nodes are established activators and inhibitors of mTORC1, respectively, irrespective of p53. Saturated blue and orange nodes (RFX7 and DDIT4) were assessed for their p53-dependent mTORC1 control in this study. p53 appears to inhibit mTORC2-AKT signaling through RFX7-DDIT4. RFX7 is required for p53-mediated AKT and mTORC1 inhibition. RFX7 employs yet unknown targets to inhibit mTORC1 (indicated by a question mark).