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. 2016 May 16;107(6):734–745. doi: 10.1111/cas.12933

Figure 2.

Figure 2

FUCA1 is a direct target gene of p53. (a) HCT116 p53+/+ or p53−/− cells were treated with or without 5‐fluorouracil (5‐FU), and p53 ChIP sequencing analysis was carried out. Genomic locus of FUCA1 is shown together with the results obtained. ChIP sequencing analyses were carried out using antibodies against p53, H3K27ac, H3K4me1, H3K4me3, and phospho‐RNAP II. A p53 binding site (p53RE) was identified within intron 1 of the FUCA1 gene. (b) ChIP assay was carried out for FUCA1 intron 1, which contains the p53RE. A Saos2 cell line that stably expresses a temperature‐sensitive p53 (ts‐p53) was used to analyze p53 binding to the FUCA1 promoter. p53 binding to p53RE was analyzed at the non‐permissive (38°C) and permissive (32°C) temperatures. The positions amplified by PCR (131‐bp fragment was amplified) are shown. (c) The 131‐bp fragments within intron 1 containing wild‐type or mutant p53RE were cloned upstream of a firefly luciferase reporter gene with a minimal promoter, and a luciferase reporter assay was carried out. Constructs were tested for transactivation by WT p53 and p53‐V143A. The assay was undertaken 24 h post‐transfection. mt, mutant.