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1.
Figure 6

Figure 6. Model of p53 repression of Per2 expression and modulation of the circadian clock. From: p53 Regulates Period2 Expression and the Circadian Clock.

p53 levels are regulated by many factors such as circadian timing, cellular stress, and MDM2. Both physiological and stress induced p53 binds to the p53 response element of Per2 promoter which overlaps with the BMAL1 / CLOCK binding site (E-box) and suppresses BMAL1 / CLOCK mediated transcription of Per2 thereby modulating the circadian clock.

Takao Miki, et al. Nat Commun. ;4:2444-2444.
2.
Figure 4

Figure 4. p53 controls circadian behavior of mice. From: p53 Regulates Period2 Expression and the Circadian Clock.

(a, c) Wheel running activity of WT and p53−/− mice. (b) Summary of periods for WT (n=10) and p53−/− mice (n=13). (d) Average difference in observed period length after re-entrainment of individual WT (n=8) and p53−/− (n=7) mice. (e) A 15 min light pulse applied at CT14 to free-running mice. (f) Summary of (e): WT (n=3) and p53−/− (n=4). (g) A 15 min light pulse applied at CT22 to free-running mice. (h) Summary of (g): WT (n=3) and p53−/− (n=5). Error bars, mean ± SEM. T-test: *p< 0.05.

Takao Miki, et al. Nat Commun. ;4:2444-2444.
3.
Figure 5

Figure 5. Actograms of phase shift response induced by Nutlin-3. From: p53 Regulates Period2 Expression and the Circadian Clock.

(a) WT or p53−/− mice were kept in a DD cycle and either vehicle (DMSO) (n=3) or Nutlin-3 (n=6) was injected at CT9. (b) Summary of phase advance (CT9). (c) Mice were kept in a 12 h/12 h LD cycle for 2 weeks, vehicle (n=6) or Nutlin-3 (n=4) were injected, and then mice were released into a DD cycle. (d) p53−/− mice given vehicle (n=5) or Nutlin-3 (n=6), as in (c). (e) Summary of phase advance of vehicle (-) or Nutlin-3 (+) injected mice. Error bars, mean ± SEM. T-test: *p< 0.05. N.S., not significant.

Takao Miki, et al. Nat Commun. ;4:2444-2444.
4.
Figure 3

Figure 3. p53 oscillates and modulates Per2 expression in the SCN. From: p53 Regulates Period2 Expression and the Circadian Clock.

(a) Immunofluorescence analysis of p53 expression in WT mouse SCN. (b) Quantification of p53 expression in the SCN (n=3). ANOVA: p< 0.05. post hoc t-test: *p< 0.05. (c) In situ hybridization detection of Per2 mRNA in SCN of WT and p53−/− mice. (d) Quantification of Per2 expression in the WT (square) and p53−/− (circle) mouse SCN (n=3). ANOVA: p< 0.05. post hoc t-test: *p< 0.05. (e) Immunofluorescence analysis of PER2 expression in SCNs from WT or p53−/− mice (f) Correlation between p53 protein levels, shown by immunofluorescence, and Per2 expression, shown by in situ hybridization, in Nutlin-3 (200 mg/kg) or DMSO injected SCN. (g) Statistical analysis of (f) (n=2). Scale bars, 200 µm. Error bars: mean ± SEM. T-test:.*p< 0.05.

Takao Miki, et al. Nat Commun. ;4:2444-2444.
5.
Figure 1

Figure 1. p53 modulates Per2 expression. From: p53 Regulates Period2 Expression and the Circadian Clock.

(a) Per2 or (b) Bmal1 promoter luciferase assay in wildtype (WT) MEF cells transfected with the indicated expression vectors, and harvested 24 h after transfection. (c) Per2 promoter luciferase assays in the absence or presence of Nutlin-3 (10 µM) using WT or p53−/− MEF cells. Western blotting using the indicated antibodies; (d) WT or (e) p53−/− MEF cells were treated with 10 µM Nutlin-3 and harvested at the indicated time points; (f) WT or p53−/− (g) MEF cells were treated with the indicated concentration of Nutlin-3 for 16 h. (h) WT or (i) p53−/− MEF cells were treated with γ-irradiation (10Gy) and harvested at indicated time points (Full-length immunoblots images for 1d and 1h are shown in ). Error bars; mean ± SEM (n>3). T-test: *p< 0.05. N.S., not significant.

Takao Miki, et al. Nat Commun. ;4:2444-2444.
6.
Figure 2

Figure 2. p53 binds to a Per2 promoter consensus region. From: p53 Regulates Period2 Expression and the Circadian Clock.

(a) Per2 promoter deletion luciferase assay. (b) Comparison of Per2 promoter among species (upper). Images of −45 bp WT and mutant Per2 promoter luciferase construct (lower). Luciferase assays were performed using: (c) −105 bp WT and mutant Per2 or (d) −105 bp and −45 bp Per2 promoter luciferase. (e) p53 binding to the Per2 promoter consensus region examined by EMSA. (f) Competition assay for p53 binding by EMSA. (g) Relative p53 binding to the Per2 promoter (E2) was examined by ChIP-qPCR assay. (h) ChIP-qPCR assay for p53 in mouse SCN. ANOVA: p< 0.05, post hoc t-test: *p< 0.05. (i) ChIP-qPCR assay (E2) for p53 in mouse Thymus. (j) ChIP-qPCR assay (E2) for BMAL1 in wildtype (clear) or p53−/− (shaded) mouse Thymus obtained at ZT8 and ZT20. (k) BMAL1/FLAG-CLOCK (B/F-C) binding to the Per2 promoter consensus region examined by EMSA. Nuclear extracts from the indicated genotype of fibroblast cells were used. (l) EMSA of BMAL1/CLOCK (B/C) binding using nuclear extract obtained from thymus. (m) Competitive binding assay to the Per2 promoter by EMSA using recombinant p53 and BMAL1/CLOCK from nuclear extract of p53−/− MEF cells. Error bars: mean ± SEM (n>3). T-test: *p< 0.05. N.S., not significant.

Takao Miki, et al. Nat Commun. ;4:2444-2444.

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