Figure 3 : ERAP1 is transcriptionally regulated by p53 via a bona fide p53RE.

From: p53 increases MHC class I expression by upregulating the endoplasmic reticulum aminopeptidase ERAP1

Figure 3

(a) Real-time qPCR validation of fold change in ERAP1 mRNA expression in HCT116 (p53−/−) cells transfected with wild-type p53 (p53WT) or one of 6 p53 mutants, compared with cells transfected with the pcDNA3.1 control. (b) Real-time qPCR analysis of relative basal ERAP1 mRNA expression levels in p53+/+ and p53−/− HCT116 cells. **P<0.01 (two-tailed Student’s t-test). (c) Western blot of ERAP1, ERAP2, p21, p53 and β-actin (loading control) in HCT116 (p53+/+) and HCT116 (p53−/−) cells. (d) Dual-luciferase assay results of p53WT or the p53 dominant-negative mutant R175H co-transfected with different ERAP1 RE constructs (pGL3-pro-ERAP1-RE1 and RE2, sequences listed in the table at the bottom panel) into HCT116 (p53−/−) cells. Data are presented as percentage luciferase activity relative to the pcDNA3.1 control vector-transfected cells. Luciferase assay using p21 promoter construct (pGL3-pro-p21) serves as the positive control. (e) Binding affinity of wild-type p53 (p53WT) or the six p53 mutants to the identified ERAP1 p53RE sequences (wild-type or mutant) was determined by ProLabel Protein–DNA binding assay. (f) Schematic representation of identified ERAP1 p53RE (RE2) in relation to the ChIP-seq peaks and genomic localization of ERAP1 gene. TSS: transcription start site. Data are representative of three independent experiments and error bars represent s.d. of technical replicates (mean±s.d.), n=3, in a,b,d,e.