Fig 2: Combined in-silico and chromatin immunoprecipitation (ChIP) identifies p53 down-stream DNA-binding sites (p53BS) in the DPYD gene. (A) Schematic representation of the approach for in silico screening for putative p53BS within the DPYD gene and 20 kb upstream and downstream of the gene. (B) Fold-enrichment over untreated control of the p53BS (R-0.84) and other binding sites predicted by the in-silico analysis in mouse liver DNA as detected by ChIP analysis following IV 5-FU (150 mg/kg) administration for 6 hr (N = 3). (NS = Not significant) (C) Validation of p53DBS enrichment when treated as in (B), binding to p21 promoter is used as positive control. (N = 3) P-values were determined by multiple t-test.

Fig 3: Model depicting the repression of DPYD by p53 following thymidylate synthase (TS) inhibition, dTTP imbalance and DNA damage. P53 transcriptionally represses DPYD expression and negatively impacts the catabolism of pyrimidines following inhibition of TS. In extension, this results in an imbalance in the dTTP/dUTP ratio and a need to salvage cellular levels of dTTP to maintain uninterrupted DNA replication and repair. However, in the presence of 5-FU this might cause increased cell death due to reduced catabolism and increased incorporation of the 5-FU metabolites FdUTP and FUTP into DNA and RNA respectively.

Fig 4: p53-dependent repression of DPYD expression in intact liver and impact of human p53 polymorphic variants on liver expression of DPYD. (A and B) Fold-change in expression of DPYD mRNA and protein in livers of p53+/+ (wild-type,) and p53−/− mice. P-values are determined Multiple t test.and one way Anova [NT vs 5-FU in p53+/+ mice for DPYD mRNA is p = 0.08; DPYD protein p = 0.0021and NT vs 5-FU in p53−/− mice for DPYD mRNA is p = 0.040; DPYD protein p = 0.09] (C) HUPKI Codon R72P MEF-R/R or MEF-P/P were treated with 5-FU (384 μM) up to 24 hr and DPYD protein expression in MEF-P/P or MEF-R/R allele was evaluated by western blot. (D) DPYD protein expression in Normal human fibroblast cell line harboring Codon R72P polymorphism, i.e., NHF P/P and NHF R/R is evaluated by western blot after treatment with 5-FU for the indicated times.

Fig 5: The tumor suppressor p53 represses dihydropyrimidine dehydrogenase (DPYD) expression. (A) mRNA and protein expression of DPYD in HCT-116 p53 +/+ and HCT-116 p53 −/− cell lines at indicated times after 5-FU (384 μM) treatment. (B) Fold-expression of mRNA in A549 and H460 cell lines at 24 hr after 5-FU (384 μM) treatment with and without siRNA knockdown of p53 (P = 0.0011 N = 3). (C) H3K9 Acetylation at DPYD promoter following 5-FU treatment for indicated time points. Values are normalized in the sequence [input > IgG > total H3 > No Treatment (NT)] (N = 3). (D,E,F) Protein expression of DPYD in A549, U87MG, HT-1080 and H460 is shown after western blotting at the indicated time points with and without siRNA knockdown of p53. (G) H3K4me3 and H3K27me3 at DPYD promoter at 24hrs after 5-FU treatment. Values normalized in the sequence [input > IgG > total H3] (N = 3).