p53 downregulates MEF expression in human cell lines. (A) Relative amount of MEF mRNA was examined in HCT116 p53^+/+ and p53^−/− cells. (B) HCT116 p53^−/− cells were transfected with control pcDNA3.1 empty vector or p53 plasmid. Forty-eight hours post-transfection, MEF mRNA level was analyzed. (C) HCT116 p53^+/+ and p53^−/− cells were untreated or treated with nutlin-3 for 24 h. Total RNA was isolated for analysis. (D) HepG2 cells were treated with 20 µM nutlin-3 for 48 h then total RNA was recovered. (E–G) A549, 16HBE14o- and HEK293 cells were transfected with si-GL2 (control) or si-p53 as described in ‘Materials and Methods’ section. Total RNA was isolated and analyzed for MEF mRNA. For (A–G) MEF mRNA level, assessed by quantitative RT-PCR, was normalized to GAPDH or 18S rRNA, which served as internal controls. Values are mean ± SD of triplicate measurements. *P < 0.05; **P < 0.01 versus control cells, assessed by Student’s t-test or ANOVA with Tukey–Kramer test (for (C)). (H) Endogenous MEF and p53 protein expressions in nuclear extracts of HCT116 p53^+/+ and p53^−/− cells were examined by western blotting. (I) HCT116 p53^+/+ and p53^−/− cells were transfected with control pcDNA3.1 empty vector, or p53 plasmid for p53^−/− cells. Forty-eight hours post-transfection, nuclear extracts were isolated. (J) HCT116 p53^+/+ and p53^−/− cells were untreated or treated with Nutlin-3 (5 µM) for 24 h, then the nuclear extracts were isolated. (K and L) A549 and 16HBE cells were transfected with si-GL2 (control) or si-p53. Forty-eight hours after transfection, nuclear extracts were isolated. For (H–L), protein levels of MEF and p53 were analyzed by western blotting. γ-tubulin was used as internal control in these experiments.

p53 suppresses MEF promoter activity. (A) HCT116 p53^−/− cells were transiently transfected with MEF (–849/+181 bp) promoter construct or pGL3b vector (0.2 µg) and the indicated amount of p53 plasmid or pcDNA3.1 empty vector. Luciferase activity was determined 48 h after transfection of plasmids and is expressed as fold activation over the pGL3b vector. Values are the mean ± SE of triplicate platings. ***P < 0.001 versus pcDNA3.1, determined by ANOVA with Tukey–Kramer test. n.s, not significant. (B) HCT116 p53^−/− cells were transiently transfected with the indicated MEF promoter constructs (0.2 µg) and p53 plasmid (0.1 µg) or pcDNA3.1 empty vector (as control). Luciferase activity was determined 48 h after transfection of plasmids and is expressed as fold activation over the pcDNA3.1 vector (con) in each promoter construct. Values are mean ± SE of triplicate platings. **P < 0.01; ***P < 0.001 versus control, assessed by Student’s t-test. (C) The –849 bp 5′-flanking region of MEF. Nucleotide sequence of 5′-flanking region of human MEF gene is shown. The site indicated (+1) denotes the start site of the first exon. The predicted binding sites for E2F1 are marked on the sequence.

E2F1 increases MEF promoter activity by binding to E2F site. (A) HCT116 p53^−/− cells were transiently transfected with MEF (–849/+181 bp) promoter construct or pGL3b vector (0.2 µg) and the indicated amounts of E2F1 plasmid or pcDNA3.1 empty vector. Luciferase activity was determined 48 h after transfection and is expressed as fold activation over the pGL3b vector. Values are the mean ± SE of triplicate platings. ^†††P < 0.001 versus pGL3b; ***P < 0.001 versus pcDNA3.1, determined by ANOVA with Tukey–Kramer. (B) HCT116 p53^−/− cells were transiently transfected with the indicated MEF promoter constructs (0.2 µg) and E2F1 plasmid or pcDNA3.1 vector (0.1 µg). Luciferase activity was determined 48 h after transfection and is expressed as fold activation over the pcDNA3.1 vector in each promoter construct. Values are mean ± SE of triplicate platings. ***P < 0.001 versus control, assessed by Student’s t-test. (C) HCT116 p53^−/− cells were transfected with 0.2 µg pGL3b vector or MEF (–200/+181 bp) promoter wild-type (WT), MT1, MT2 or MT1&2. (Left) MT1, MT2 represent MEF (–200/+181 bp) promoter containing mutated E2F site. Luciferase activity was determined 48 h after transfection and is expressed as fold activation over pGL3b vector. Values are mean ± SE of triplicate platings. ***P < 0.001 versus WT, assessed by ANOVA with Tukey–Kramer test. (D) E2F1 binding on MEF promoter was determined by ChIP assay using nuclear extracts of HCT116 p53^−/− cells transiently transfected with E2F1 or pcDNA3.1 vector (1.0 µg). CDC6 promoter was used as positive control and GAPDH promoter was used as negative control for E2F1 binding. Upper panel illustrates the MEF promoter region (–204 bp/+181 bp) in which binding was assessed. (E) Nuclear extract from HEK293 cells was used to assess the endogenous binding of E2F1 on MEF promoter. CDC6 and GAPDH promoters were used as positive and negative controls, respectively.

p53 suppresses MEF expression during cellular senescence. (A and B) HCT116 p53^+/+ and p53^−/− cells were treated with doxorubicin (Dox) for 24 h and incubated for 5 days to induce cellular senescence. Cells were fixed and stained with X-gal to detect SA-β-gal activity. Stained cells were photographed at phase contrast with 20-fold magnification (Scale bar, 100 µm) (A). The nuclear extracts from Dox-treated or untreated cells were subjected to immunoblotting using the indicated antibodies. γ-tubulin was used as internal control (B). (C) HCT116 p53^+/+ cells were untreated or treated with Dox for 24 h and incubated for 3 days. Total RNA was recovered and MEF mRNA expression was examined by Q-PCR. Amount of MEF mRNA was normalized to GAPDH and expressed as relative amount compared to control cells. Values are mean ± SD of triplicate platings. **P < 0.01, determined by Student’s t-test. (D) Cellular senescence was induced in HCT116 p53^+/+ cells by doxorubicin treatment similar to (A). p53 binding to E2F1 was examined by immunoprecipitation of nuclear extracts. (E) E2F1 binding on MEF promoter was examined by ChIP assay using nuclear extracts of control or Dox-induced senescent HCT116 p53^+/+ cells. (F) Negative regulatory mechanism between p53 and MEF. (Left) DNA damage stimulates p53 that enhances its binding to E2F1. This leads to reduced recruitment of E2F1 to the MEF promoter and the suppression of MEF transcription. (Right) MEF upregulates MDM2 (described in ref. 16), which inhibits p53.

MEF mRNA level is upregulated in p53^−/− mice tissues. (A–F) Total RNA isolated from the indicated tissues of p53^+/+ and p53^−/− mice was analyzed for the expression of MEF by real-time quantitative RT–PCR. MEF mRNA level was normalized to mouse 18S rRNA (internal control). Results represent mean ± SD (n = 3). *P < 0.05, **P < 0.01 against p53 wild-type mice assessed by Student’s t-test.

E2F1 upregulates MEF expression in human cell lines. (A–E) Cells were transiently transfected with pcDNA3.1 vector (con) or the indicated amount of E2F1 (A) or 1.0 µg E2F1 (B–E). Forty-eight hours after transfection, total RNA was extracted and analyzed for MEF mRNA expression. (F–H) si-GL2 or si-E2F1 (50 nM) was transfected into the indicated cell lines and MEF mRNA expression was assessed 48 h post-transfection. For (A–H) MEF mRNA level, determined by quantitative RT–PCR, was normalized to GAPDH or 18S rRNA (internal control) and expressed as relative amount of mRNA. Values are mean ± SD of triplicate measurements. *P < 0.05; **P < 0.01; ***P < 0.001 versus control, assessed by ANOVA with Tukey–Kramer (A) or Student’s t-test (B–H). (I–J) MEF and E2F1 protein expressions were examined by western blotting in nuclear extracts of HCT116 p53^−/− (I) or 16HBE14o- cells (J) transiently transfected with E2F1 or pcDNA3.1 empty vector. γ-tubulin was used as internal control.

p53 inhibits E2F1 binding to MEF promoter and reduces E2F1-induced MEF promoter activation. (A and B) HCT116 p53^−/− and HEK293 cells were transiently transfected with MEF (–204/+181 bp) promoter construct or pGL3b vector (0.2 µg), pcDNA3.1 or E2F1 (0.1 µg) and the indicated amounts of p53 plasmid. Luciferase activity was determined 48 h after transfection and is expressed as fold activation over the pGL3b vector. Values are the mean ± SE of triplicate platings. ^†††P < 0.001 versus pGL3b; ^###P < 0.001 versus pcDNA3.1; *P < 0.05; ***P < 0.001 versus E2F1, determined by ANOVA with Tukey–Kramer. (C) HCT116 p53^−/− cells were transiently transfected with p53 and E2F1 plasmids. Nuclear extracts were analyzed for p53-E2F1 association by IP using antibody specific to p53, E2F1 or control mouse IgG. Immunoprecipitates were loaded onto SDS–PAGE gel, blotted and probed with E2F1 or p53 antibodies. (D) HCT116 p53^−/− cells were transiently transfected with E2F1 and p53 plasmid or pcDNA3.1 empty vector. Nuclear extracts were isolated and used for ChIP assay to determine E2F1 binding on MEF promoter and CDC6 promoter in the presence or absence of p53. (E) Endogenous protein level of E2F1 was examined in nuclear extracts of HCT116 p53^+/+ and p53^−/− cells. γ-Tubulin was used as internal control.