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. 2011 Aug;20(4):899-912.
doi: 10.1007/s11248-010-9468-4. Epub 2010 Dec 3.

Mice defective in p53 nuclear localization signal 1 exhibit exencephaly

Anouk Regeling  1 , Heather L Armata, Judy Gallant, Stephen N Jones, Hayla K Sluss
Affiliations

Mice defective in p53 nuclear localization signal 1 exhibit exencephaly

Anouk Regeling et al. Transgenic Res. 2011 Aug.

Abstract

p53 is a major suppressor of human malignancy. The protein levels and activity are tightly regulated in cells. Early experiments identified nuclear localization signal 1 (NLS1) as a regulator of p53 localization. We have generated mice bearing a mutation in p53 ( NLS1 ), designated p53 ( NLS1 ). Our experiments confirm a role for NLS1 in regulating p53 function. Murine embryonic fibroblasts generated from homozygous p53 ( NLS1 ) animals are partially defective in cell cycle arrest and do not respond to inhibitory signals from oncogenic Ras. In addition, p53-dependent apoptosis is abrogated in thymocytes. Contrary to predicted results, fibroblasts from homozygous p53 ( NLS1 ) animals have a greater rate of proliferation than p53-null cells. In addition, p53 ( NLS1 ) cells are more resistant to UV-induced death. Surprisingly, the homozygous p53 ( NLS1 ) animals exhibit embryonic and peri-natal lethality, with a significant portion of the animals developing exencephaly. Thus, p53 ( NLS1/NLS1 ) embryos exhibit a reduced viability relative to p53-null mice. These studies indicate that the NLS1 is a major regulator of p53 activity in vivo.

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Conflict of interest statement

Conflict of interest The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
Mutation of Lysines 316–318 (NLS1) in the p53 locus. a Organization of the p53 locus. Filled boxes represent exons. Restriction enzymes sites are indicated. b The targeting vector encodes a triple Lysine to Alanine missense mutation in exon 9. A floxed NeoR gene was introduced in intron 9 to permit positive selection of ES cells. c Homologous recombination of the targeting vector in ES cells is illustrated. Probes used for Southern blot analysis are shown (hatched boxes). d The NeoR gene is excised in the chimera due to expression of a protamine-Cre transgene. e Southern blot analysis of targeted ES cell DNA using a 5′ external probe. EcoR1 digest of ES cell DNA indicates the presence of the mutant allele (unexcised NeoR gene, 11 kb) and the wildtype allele (14 kb)
Fig. 2
Fig. 2
Growth characteristics of p53NLS1 cells. a Effects of p53 NLS1 mutation on cell growth. Cells were plated in triplicate and cell number was determined at the indicated times. b Loss of p53 NLS1 leads to defects in cell cycle arrest. Cell cycle arrest of heterozygous and homozygous p53NLS1 MEFs in response to gamma radiation, compared to wild type and p53−/− MEFs. Cells were harvested 18 h post-treatment. The decrease in S-phase in p53NLS1/NLS1 MEFs is significant (*, P<0.05) c Expression of p53NLS1 protein. Western analysis of wildtype (left panel) and p53NLS1/NLS1 cells (right panel) in response to various genotoxic stress [UV (50 J/m2), gamma irradiation (IR 20 Gy) and doxorubicin (Dox 50 μg/ml)] and harvested at 8 h post-treatment. α-tubulin is the loading control
Fig. 3
Fig. 3
p53NLS1/NLS1 cells escape Ras induced cellular senescence. a Ras infection does not reduce viability of p53NLS1/NLS1 cells. Cells were infected with pBabe (vector) or pBabe-HaRas were plated and growth recorded at the indicated time points. b p53NLS1/NLS1 cells do not senescence in response to Ras. Phase-contrast micrograph of cells infected with oncogenic HaRas stained with senescence marker β-galactosidase. c Number of cells staining positive for β-galactosidase in cells in response to oncogenic Ras
Fig. 4
Fig. 4
Analysis of p53NLS1 localization. Representative staining in cells with or without UV treatment. Staining of untreated p53NLS/NLS1 cells (top panels), wildtype cells treated with UV (middle panels), and UV-treated p53NLS1/NLS1 cells (bottom panels). Cells were stained 18 h post-treatment with the anti-p53 antibody Ab-7 and DAPI. Nuclear accumulation in response to UV is defective in p53NLS1 cells
Fig. 5
Fig. 5
Loss of p53 Lys316–318 (NLS1) abrogates p53-dependent death. a Protection of CD4+/CD8+ thymocytes in p53NLS1/NLS1 animals. Mice were treated in vivo with IR (8 Gy) or untreated and the thymocytes harvested 24 h post treatment. Cells were stained for CD4 and CD8. b Loss of p53 NLS1 leads to increased survival in thymocytes. Thymocytes were removed from untreated and treated (8 Gy) animals 16 h post-treatment. The percent of viable cells is presented (cells negative for annexin-V and 7AAD). c Loss of p53 NLS1 leads to increased survival in E1A-MEFs. MEFs were exposed to UV radiation (5 J/m2) and survival was measured at 18 and 24 h post-treatment. All numbers are normalized to the number of cells in untreated samples. Increased survival in p53NLS1/NLS1 MEFs compared to p53−/− MEFs is significant (*, P < 0.05). d Defective NOXA induction in response to UV in p53NLS1 cells. The expression of NOXA mRNA was measured by quantitative real time PCR analysis of UV-treated MEFs (6 h post-treatment). The amount of Gapdh mRNA in each sample was used to calculate relative mRNA expression (mean ± -S.E.M.; n = 3). The induction of NOXA was presented as the ratio of induced in wildtype/induced in p53NLS1 MEFs
Fig. 6
Fig. 6
p53NLS1/NLS1 animals develop exencephaly. a Embryonic Day 15.5 embryos from a heterozygous p53NLS1 cross. Wildtype animal (on left), with complete closure of hindbrain. p53NLS1/NLS1 animals in the middle (female, NLS1-1) and right (male, NLS1-2), showing hindbrain exencephaly. b p53NLS1/NLS1 exencephalic embryo showing the eversion and exposure of neural tissue (arrows) pictured in frontal section (left panel) and back sagittal section (right panel)
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