Figure 1
From: IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis
The IER5 gene is a p53 target gene.
(A–C) IER5 expression was analyzed by Northern blotting. HCT116 p53 (+/+), HCT116 p53 (−/−) were subjected to γ-ray irradiation (20 Gy) (A). Normal human fibroblasts (MRC5 cells) transfected with control lentivirus or lentivirus expressing p53 shRNA were subjected to γ-ray irradiation (30 Gy) (B). Cell lines carrying wild-type p53 were treated with Adriamycin (1 μM) for 24 hrs (MCF7, U2OS and MRC5) or 19 hrs (A549) (C). (D) HCT116 p53 (+/+), HCT116 p53 (−/−) were treated with 5-FU (0.038 mM) for 24 hrs. Expression of IER5, p53 and p21 (representative p53 target gene) were analyzed by Western blotting. (E) Cell lines carrying wild-type p53 were treated with Adriamycin (1 μM) for 24 hrs (MRC5, MCF7, U2OS) or 19 hrs (A549). Expression of IER5 and p53 were analyzed by Western blotting. (F) Genomic locus of IER5 is shown together with the ChIP-seq and 4C-seq results. HCT116 p53 (+/+) cells treated with or without 5-FU were used for ChIP-seq analysis. ChIP-seq analyses were performed using antibodies against p53, H3K27ac, H3K4me1, H3K4me3 and phospho-RNAP II. Two p53 binding sites RE1 (11 kb downstream) and RE2 (46 kb downstream) were identified. Chromatin interaction was detected by 4C-seq analysis in HCT116 p53+/+ and HCT116 p53−/− cells with or without 5-FU. Primers were designed around RE2 and the genomic regions interacting with RE2 were determined. (G) The positions and nucleotide sequences of RE1 and RE2. (H) Double-stranded synthetic oligonucleotides containing RE1, RE2 or mutant RE2 (the sequences are shown in G) were inserted into the luciferase reporter plasmid containing a minimal promoter. The assay was performed 24 hrs post-transfection. The experiment was run in triplicate and data are represented as the mean-fold activation ±SD.
