Using targeted transgenic reporter mice to study promoter-specific p53 transcriptional activity

Abstract

The p53 transcription factor modulates gene expression programs that induce cell cycle arrest, senescence, or apoptosis, thereby preventing tumorigenesis. However, the mechanisms by which these fates are selected are unclear. Our objective is to understand p53 target gene selection and, thus, enable its optimal manipulation for cancer therapy. We have generated targeted transgenic reporter mice in which EGFP expression is driven by p53 transcriptional activity at a response element from either the p21 or Puma promoter, which induces cell cycle arrest/senescence and apoptosis, respectively. We demonstrate that we could monitor p53 activity in vitro and in vivo and detect variations in p53 activity depending on the response element, tissue type, and stimulus, thereby validating our reporter system and illustrating its utility for preclinical drug studies. Our results also show that the sequence of the p53 response element itself is sufficient to strongly influence p53 target gene selection. Finally, we use our reporter system to provide evidence for p53 transcriptional activity during early embryogenesis, showing that p53 is active as early as embryonic day 3.5 and that p53 activity becomes restricted to embryonic tissue by embryonic day 6.5. The data from this study demonstrate that these reporter mice could serve as powerful tools to answer questions related to basic biology of the p53 pathway, as well as cancer therapy and drug discovery.

Fig. 1.

Generation of reporter mice that express EGFP under the control of p53. Schematic (Upper) and chromatogram (Lower) of the p21_p53RE-EGFP reporter (A) and Puma_p53RE-EGFP reporter (B) constructs used for targeted transgenesis.

Fig. 2.

EGFP expression is an indicator of p53 activity. A, RT-qPCR analysis of mRNA levels of the Egfp reporter or of endogenous p21 and Puma in p21_p53RE-EGFP (Left) and Puma_p53RE-EGFP (Right) MEFs treated with DMSO or increasing amounts of nutlin for 8 h. The increase in Egfp mRNA levels was dependent on nutlin concentration (P < 0.0001; two-way ANOVA) and correlated with the increase in p21 and Puma mRNA levels (P < 0.05; t test). Error bars indicate SD of three independent experiments. B, Western blots showing a concentration-dependent increase in EGFP and p53 protein levels after overnight treatment with nutlin. Actin served as a loading control.

Fig. 3.

Induction of EGFP expression is p53-dependent. A, RT-qPCR (Upper) and Western blot (Lower) analysis of p21_p53RE-EGFP and Puma_p53RE-EGFP MEFs after overnight treatment with 10 μM nutlin, showing EGFP induction only in p53 wild-type (p53^+/+) but not p53-null (p53^KO/KO) MEFs. Changes in mRNA levels were determined relative to those in the respective untreated p53^+/+ reporter MEFs. Error bars indicate SD of four independent experiments. *P < 0.02; **P < 0.05; ***P < 0.0005 (t test). B, Flow cytometric analysis of MEFs derived from p21_p53RE-EGFP (Left) and Puma_p53RE-EGFP (Right) mice that were either p53 wild-type or p53-null. The bold lines and shaded histograms correspond to untreated and nutlin-treated MEFs, respectively. Data shown are representative of three independent experiments. C, Fluorescence imaging of unfixed p21_p53RE-EGFP and Puma_p53RE-EGFP MEFs that were either p53 wild-type or p53-null, as well as of p53 wild-type MEFs lacking either reporter. The MEFs were treated with DMSO or 10 μM nutlin overnight. Bright-field images are included to illustrate the presence of nonfluorescent MEFs. D, RT-qPCR (Upper) and Western blot (Lower) analysis of p21_p53RE-EGFP MEFs after overnight treatment with 400 nM doxorubicin showing EGFP induction only in p53 wild-type (p53^+/+) but not p53-null (p53^KO/KO) MEFs. Changes in mRNA levels were determined relative to those in untreated p53^+/+ reporter MEFs. Error bars indicate SD of three independent experiments. *P < 0.01 (t test).

Fig. 4.

EGFP expression and p53 activity are dependent on the response element, tissue, and stimulus. A, Flow cytometric analysis of MEFs derived from p21_p53RE-EGFP (black histogram) and Puma_p53RE-EGFP (red histogram) mice or from mice lacking either reporter (shaded histogram). Graphs shown are representative of three independent experiments. B, RT-qPCR analysis of spleen and thymus tissue from untreated p21_p53RE-EGFP and Puma_p53RE-EGFP mice. Egfp mRNA levels were normalized to that in the untreated p21_p53RE-EGFP thymus. Within either tissue, Egfp expression was not significantly affected by the reporter construct [not significant (ns): P > 0.1; t test]. There was a significant tissue-specific difference in Egfp expression from the p21_p53RE-EGFP reporter (*P < 0.01; t test) but not the Puma_p53RE-EGFP reporter (ns: P > 0.1; t test). Error bars indicate SD of six independent experiments. C, RT-qPCR analysis of spleen and thymus tissue from age-matched p21_p53RE-EGFP and Puma_p53RE-EGFP mice, harvested 6 or 24 h after treatment with 8-Gy ionizing radiation. Changes in Egfp mRNA levels were determined relative to basal levels in the respective tissues from nonirradiated mice, which are represented by the red line (i.e., fold induction of 1). At 24 h, radiation-induced Egfp expression was dependent on both tissue type (P < 0.01; two-way ANOVA) and reporter construct (P < 0.01; two-way ANOVA). Error bars indicate SD of at least three independent experiments. *P < 0.01; **P < 0.05; ns (P > 0.5) (t test). D, RT-qPCR analysis of spleen and thymus tissue from age-matched p21_p53RE-EGFP and Puma_p53RE-EGFP mice, harvested 24 h after injection with 0.01 mg/g doxorubicin. Changes in Egfp mRNA levels were determined relative to basal levels in the respective tissues from control mice, which are represented by the red line (i.e., fold induction of 1). Doxorubicin-induced Egfp expression was dependent on both tissue type (P < 0.02; two-way ANOVA) and reporter construct (P < 0.02; two-way ANOVA). Error bars indicate SD of three independent experiments. *P < 0.05; ns (P > 0.1) (t test).

Fig. 5.

p53 is transcriptionally active in early embryonic development. Fluorescence microscopy of freshly harvested E3.5 blastocysts (A and B) and E6.5 embryos (C). ICM, inner cell mass; TE, trophectoderm.