Figure 3.

The effect of P72 SNP on the transcriptional regulation of canonical p53 target genes by TP53 missense mutants. A) Group design for the pairwise analysis of RNA transcripts from cells transduced with p53 mutants. SKOV3 cells were freshly transduced with p53 mutants R248Q, R248W, and R273H with the P72 and R72 SNP. Three mutant pairs are considered as experimental replicates, and 3 technical replicates for each mutant are included. After transduction and 5-8 days of puromycin selection, RNA was collected, and sequencing libraries were generated. DESeq2 and pair-wise analysis were used to analyze reads between the P72 and R72 groups (n = 18) and within each mutant pair. B) Differentially expressed transcripts in p53 mutants with the P72 SNP vs the R72 SNP. 3' Tag RNA-Seq was conducted on 9 p53 mutants with the P72 SNP and 9 p53 mutants with the R72 SNP; 20 982 transcripts show no statistically significant changes between the mutants with the P72 SNP and mutants with the R72 SNP, 3528 transcripts were upregulated in cells transduced with the mutants with the P72 SNP compared with those transduced with the mutants with the R72 SNP (false discovery rate [FDR] < 0.01 and fold change > 1.5), and 2701 transcripts upregulated in the mutants with the R72 SNP relative to the P72 SNP (FDR < 0.01 and fold change > 1.5). C) Metascape analysis of pathways associated with upregulated genes. D) Heatmap showing changes in the expression of select p53-target genes. E) MA plot of p53 mutant R248W indicates altered gene levels in select p53 targets. F) The number of gene sets predicted to be affected by p53 mutants with either the P72 or the R72 SNP. G) Select hallmark gene sets from the gene set enrichment analysis (GSEA) differentially enriched in mutants with either the P72 or R72 SNP. Representative enrichment plots of E2F targets and apoptosis gene sets differentially enriched in mutants with the R72 SNP or with the P72 SNP, respectively, are shown.