Figure 4. ACER2 is a direct transcriptional target of p53.

(A) Overexpression of wild-type p53 enhances ACER2 promoter activity. p53-deficient H1299 cells were transiently co-transfected with the indicated luciferase reporter constructs and pRL-TK together with the expression plasmid for wild-type p53. Forty-eight hours after transfection, their luciferase activities were examined as in Fig. 2. (B) Mutant p53 has an undetectable effect on ACER2 promoter activity. H1299 cells were co-transfected with ACER2-P397 reporter construct and pRL-TK along with mutant p53 (R175H) expression plasmid. Forty-eight hours after transfection, luciferase activities were examined as in (A). (C) Schematic diagram of the luciferase reporter plasmids containing ACER2 promoter region with the indicated point mutations at the potential p53-binding sites (p53CBS1 and p53CBS2). Exons of ACER2 are indicated as filled boxes (upper) and the potential p53-binding sites are shown as open boxes (lower). (D) p53CBS1 is required for p53-dependent activation ACER2 promoter. The indicated luciferase reporter constructs were introduced into H1299 cells together with empty plasmid or with wild-type p53 expression plasmid. Forty-eight hours after transfection, their luciferase activities were determined as in (A). (E) Direct recruitment of p53 onto ACER2 promoter region in cells. H1299 cells were transfected with p53 expression plasmid or with the empty plasmid. Forty-eight hours after transfection, chromatin fragments were prepared and immunoprecipitated with p53 antibody or with control IgG. The co-precipitated DNA fragments were purified and subjected to qPCR analysis. p53-target p21 promoter was amplified as a positive control. (F) p53 activates the endogenous ACER2 transcription. H1299 cells were transiently transfected with the empty plasmid or with p53 expression plasmid. Forty-eight hours after transfection, total RNA was extracted and subjected to qRT-PCR with the indicated primer sets. (G) Adriamycin-dependent induction of the endogenous ACER2. p53-deficient H1299 cells and p53-proficient A549 cells were treated with or without 1 μM of adriamycin (ADR). Twenty-four hours after treatment, qRT-PCR was conducted. (H) p53 is required for DNA damage-induced ACER2 up-regulation. A549 cells were transfected with control siRNA or with p53-specific siRNA. Twenty-four hours after transfection, cells were treated with 1 μM of ADR. Twenty-four hours after treatment, qRT-PCR was conducted.