Fig. 3: GOF mutant p53 binds to the promoter and transactivates expression of the CTNNB1 gene. | Nature Communications

Fig. 3: GOF mutant p53 binds to the promoter and transactivates expression of the CTNNB1 gene.

From: Gain-of-function mutant p53 together with ERG proto-oncogene drive prostate cancer by beta-catenin activation and pyrimidine synthesis

Fig. 3

a Venn diagram showing the 1116 mutant p53-bound peaks shared by two replicates (Rep.1 and Rep. 2) of p53 R248 ChIP-seq in VCaP cells. b Gene Ontology (GO) analysis of 615 p53-occupied target genes identified by ChIP-seq in VCaP cells. c UCSC Genome Browser screenshots showing the occupancy of mutant p53 R248W in the CTNNB1 gene promoter in VCaP cells. d ChIP-qPCR analysis of mutant p53 R248W binding at the CTNNB1 promoter in VCaP cells. n.s., not significant. e Scheme showing the locations of p53 ChIP-qPCR amplicons and EMSA DNA probes as well as the DNA sequence of MP53BS in the CTNNB1 gene promoter. f ChIP-qPCR analysis of mutant p53 R248W binding at the CTNNB1 promoter in VCaP cells using three sequential pairs of primers shown in (e). g EMSA assay using biotin-labeled double-stranded (ds) DNA probes from the CTNNB1 promoter as indicated in (e) and nuclear extract from VCaP cells. DPC, DNA-protein complex. h EMSA assay using biotin-labeled and unlabeled ds DNA probe 1 as shown in (e) and nuclear extract from VCaP cells. i EMSA assay using biotin-labeled ds DNA probe 1 and nuclear extract from VCaP cells incubated with non-specific IgG (negative control) or anti-p53 antibody (DO-1). j Top, scheme showing the p53 missense mutants examined. Middle and Bottom, results of EMSA assay using biotin-labeled dsDNA probe 1 as shown in (e) or unlabeled probe and untagged p53 WT or indicated mutants purified from bacteria after GST cleavage. k,l Western blot (k) and RT-qPCR (l) analyses of indicated proteins and mRNAs in different clones of DU145 cells expressing MP53BS-targeting sgRNAs. m,n Western blot (m) and RT-qPCR (n) analyses of indicated proteins and mRNAs in control or the MP53BS KO #7 clone of DU145 cells expressing MP53BS-targeting sgRNAs. n.s. not significant. Data in (d, f, l) and (n) represented mean ± s.d. from three independent experiments. Both of the EMSA assays in (g, h, i) and (j), and the western blot assays in (k) and (m) were repeated two times independently with similar results. Data was performed by two-sided Fisher’s exact test from DAVID Bioinformatics Resources (https://david.ncifcrf.gov/) in (b). Two-tailed Student’s t test was performed in (d, f, l) and (n).

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