Targeting the Y217C missense mutation at the mouse Trp53 locus.

(A) Targeting strategy. The Trp53 gene is within a 17 kb-long EcoRI (RI) fragment (black boxes are for coding sequences and white boxes for UTRs). The targeting construct contains: (1) a 1.5 kb-long 5’ homology region; (2) a Lox-Stop-Lox (LSL) cassette with a neomycin selection gene (Neo), four transcriptional stops (STOP) and an EcoRI site, flanked by LoxP sites (arrowheads); (3) p53 exons, including the Y217C (YC) missense mutation in exon 6 (asterisk) and an additional BanII site; (4) a 2.8 kb-long 3’ homology region; and (5) the diphteria α-toxin (DTA) gene for targeting enrichment. Proper recombinants with a p53LSL-Y217C allele were identified by a 2.4 kb-long band after PCR with primers a and b, and confirmed by bands of 635 and 224 bp after PCR with primers c and d and BanII digestion. They were also verified by Southern blot with the indicated probe as containing a 10.5 kb EcoRI band. Two recombinant ES clones were injected into blastocysts to generate chimeras, and germline transmission was verified by genotyping with primers c and d and BanII digestion. Excision of the LSL cassette was performed in vivo, by breeding p53+/LSL-Y217C male mice with females carrying the PGK-Cre transgene. (B-D) Screening of recombinant ES clones (+) by PCR with primers a and b (B); PCR with primers c and d then BanII digestion (C); Southern blot (D). (E) Genotyping of mouse embryonic fibroblasts (MEFs) from an intercross of p53+/Y217C mice, by PCR with primers c and d and BanII digestion. (F) p53Y217C sequence around codon 217. The introduced Y217C missense mutation and the mutation creating an additional BanII restriction site are highlighted (asterisks). (G) WT and p53Y217C/Y217C MEFs express similar p53 mRNA levels. Total RNA was extracted, then p53 mRNAs were quantified by real-time qPCR, normalized to control mRNAs and the amount in WT cells was assigned the value of 1. Primer sequences are listed in Table S6.

p53Y217C/Y217C cells exhibit alterations in p53 abundance and subcellular localization and defective responses to DNA damage.

(A) Increased p53 protein levels in p53Y217C/Y217C and p53+/Y217C MEFs. MEFs of the indicated genotypes were treated or not with 10 μM Nutlin 3a for 24 h, then protein extracts were immunoblotted with antibodies against Mdm2, p53, p21 and actin. (B) The transactivation of classical p53 target genes Cdkn1a/p21 and Mdm2 is impaired in p53Y217C/Y217C cells. WT, p53Y217C/Y217C and p53-/- MEFs were treated as in (A), then (top) mRNAs were quantified in 5-6 independent experiments using real-time PCR, with results normalized to control mRNAs and mean RNA amounts in unstressed p53+/+ cells assigned a value of 1; or (bottom) ChIP assays were performed at the Cdkn1a and Mdm2 promoters in 2-3 independent experiments with an antibody against p53 or rabbit IgG as a negative control. Immunoprecipitates were quantified using real-time PCR, normalized to data over an irrelevant region, and the amount in unstressed p53+/+ cells was assigned a value of 1. (C) Assessment of p53WT and p53Y217C subcellular localization by cellular fractionation. WT and p53Y217C/Y217C MEFs were treated or not with doxorubicin for 24 hr, submitted to cellular fractionation, then protein extracts were immunoblotted with antibodies against p53 or the fraction controls Tubulin for cytoplasm (Cp.), Nup98 for nucleoplasm (Np.) and histone H3 for chromatin (χin). (D) Assessment of p53WT and p53Y217C subcellular localization by immunofluorescence. WT, p53Y217C/Y217C and p53-/- MEFs were treated with 10 μM Nutlin 3a for 24 h, then stained with antibodies against p53 (red) or actin (green) and DNA was counterstained with DAPI (blue). (E) Absence of a cell cycle arrest response in p53Y217C/Y217C MEFs. Asynchronous cell populations of p53+/+, p53Y217C/Y217C and p53-/- MEFs were analyzed 24 hr after 0, 3 or 12 Gy ɣ-irradiation. Results from 3 independent experiments. (F) Absence of a p53-dependent apoptotic response in p53Y217C/Y217C thymocytes. Age-matched mice of the indicated genotypes were left untreated or submitted to 10 Gy whole-body ɣ-irradiation then sacrificed after 4 hr and their thymocytes were stained with Annexin V-FITC and analyzed by FACS, in 2 independent experiments. ***P<0.001, **P<0.01, *P<0.05, °P=0.09, ns: non-significant by Student’s t tests.

p53Y217C/Y217C mice exhibit female-specific perinatal lethality.

(A) Distribution of weaned mice obtained from p53+/- or p53+/Y217C intercrosses. Obs: observed numbers of mice at weaning (P21); exp: expected numbers assuming a Mendelian distribution without sex distortion; f/m: observed female/male ratios. Consistent with previous reports the observed distribution of weaned mice from p53+/- intercrosses did not conform to values expected for a Mendelian distribution without sex distortion (υ=5; χ2= 16.3>15.09), indicating a significant deficit in female p53-/- mice (top). The distribution of weaned mice from p53+/Y217C intercrosses diverged even more from values for a Mendelian distribution without sex distortion (υ =5; χ2= 91.02>15.09), due to a striking deficit in female p53Y217C/Y217C mice (bottom). The female frequency is significantly lower for p53Y217C/Y217C (3/535) than for p53-/- (8/196) females (P=0.0018 in a Fischer’s test). (B) Exencephaly is frequently observed in p53Y217C/Y217C female embryos. Top: the distribution of E12.5-16.5 embryos from heterozygous (p53+/Y217C) intercrosses or heterozygous-homozygous (p53+/Y217C x p53Y217C/Y217C) crosses is shown. f or m exenc.: number of female or male embryos with exencephaly; o.a.: embryos with other abnormalities. Below, examples of female p53Y217C/Y217C embryos at E12.5, 13.5 and 16.5 exhibiting exencephaly (arrows) are each shown (center) together with a normal embryo from the same litter (bottom). (C) Distribution of mice at birth from the indicated crosses.

Oncogenic effects of the mutant protein in p53Y217C/Y217C male mice. (A-B)

In homozygous males, p53Y217C leads to accelerated tumor onset and death (A), and aggressive metastatic tumors (B); n=cohort size. (C) Hematoxylin and eosin (H&E) staining of sections from the lung (top) and spleen (bottom) of p53-/- and p53Y217C/Y217C male mice, showing metastases in p53Y217C/Y217C animals. Normal organ structures are shown, with “A” indicating pulmonary alveoli, and “WP” and “RP” standing for splenic white and red pulp, respectively. In the lung section of the p53Y217C/Y217C mouse, the rectangle indicates a lymphoma area. In the spleen section of the p53Y217C/Y217C mouse, the typical splenic structures are absent due to massive tissue homogenization of the spleen by lymphoma cells.

Increased expression of genes associated with white blood cell chemotaxis and inflammation in p53Y217C/Y217C male thymi. (A-B)

RNAseq analysis of thymi from p53Y217C/Y217C (n=3) and p53-/- (n=3) 8 weeks-old male mice. Volcano plot (A), with differentially expressed genes (DEGs), downregulated (blue) or upregulated (red) in p53Y217C/Y217C cells. Unsupervised clustering heat-map plot (B), with 192 DEGs ranked according to log2 fold changes. (C) Gene Ontology (GO) analysis of DEGs. Out of 192 DEGs, 141 are associated with at least one GO term, according to the Gene Ontology enRIchment anaLysis and visuaLizAtion tool (GOrilla). For each GO term, enrichment was calculated by comparing with the full list of 19759 genes associated with a GO term, and results are presented here with a color scale according to P-value of enrichment. This analysis mainly revealed an enrichment for genes associated with white blood cell chemotaxis/migration and inflammation, as shown here. Complete results of the analysis are presented in Figure S9. (D) RT-qPCR analysis of the indicated genes in thymi from 8 weeks-old p53+/+, p53Y217C/Y217C and p53-/- male mice. Means + s.e.m. (n=3 per genotype). ***P<0.001, **P<0.01, *P<0.05, °P<0.075 by Student’s t tests. (E) Gene set enrichment analysis (GSEA) of transcriptomes from p53Y217C/Y217C and p53-/- thymic cells. GSEA identified 13 gene sets enriched in p53Y217C/Y217C cells, with normalized enrichment scores (NES) > 2. Nominal p-values (NOM p-val) and false discovery rate q-values (FDR q-val) are indicated.

Protein sequence alignments showing homology between mouse p53 Tyrosine 217 and human p53 Tyrosine 220.

Portions of the DNA binding domains from the mouse (residues 208-228) and human (residues 211-231) p53 proteins are shown, with identical residues in bold, and mouse Tyrosine 217 and human Tyrosine 220 in red.

Uncropped images of the western blot presented in Figure 2A.

All images are from the same blot, cut in two below the 35KDa marker before incubation with the indicated antibodies.

Uncropped images of the fractionation assays presented in Figure 2C.

Chemiluminescence and colorimetric scans obtained with a ChemiDoc imaging system (Bio- Rad) were superposed to show the entire membranes, ladder lanes (λ), and chemiluminescence signals from subcellular fractions.

Cell cycle arrest responses of p53+/+, p53Y217C/Y217C and p53-/- mouse embryonic fibroblasts.

Asynchronous MEFs were exposed to 0-12 Gy ɣ-irradiation, then after 24 hr cells were labelled with BrdU for 1 hr and analyzed by FACS. A typical experiment for cells of each genotype and condition is shown, with % cells in G1, S or G2/M mentioned in each panel.

Apoptotic responses of p53+/+, p53-/- and p53Y217C/Y217C thymocytes.

Mice were ɣ-irradiated (or not) and their thymocytes were recovered and analyzed by FACS after annexin V-FITC staining. A typical experiment for cells of each genotype and condition is shown. Numbers indicate % cells either alive (live), early apoptotic (early) or mid to late apoptotic (mid+late).

Evidence of aberrant chromosome X inactivation in p53Y217C/Y217C and p53-/- female embryos.

The expression of Xist and 3 X-linked genes (Maob, Usp9x and Pls3) was quantified in neurospheres from 2 p53+/+, 5 p53Y217C/Y217C and 3 p53-/- female embryos by using real-time PCR, with results normalized to control mRNAs and mean RNA amounts in WT neurospheres assigned a value of 1. *P<0.05, °P=0.09, ns: non-significant by Student’s t tests.

p53+/Y217C and p53+/- mice exhibit similar tumor onset and spectra.

No significant difference was observed between heterozygous p53+/- and p53+/Y217C mice, neither in spontaneous tumor onset and survival (A), nor in tumor spectra (B); n=cohort size. Mice of both sexes were included in this study.

RNA-seq analysis from the thymi of 8 weeks-old p53+/+, p53Y217C/Y217C and p53-/- male mice.

(A) Heat-Map plot, with 717 differentially expressed genes suggestive of a loss of function (LOF), a separation of function (SOF) or a gain of function (GOF) for the p53Y217C mutant, ranked according to log2 fold change (n=number of genes). (B) Evidence of LOF in p53Y217C/Y217C cells for genes encoding Puma, p21 and Zmat3. Data from 3 mice per genotype. ***P<0.001, **P<0.01, *P<0.05, °P=0.07, ns: non-significant by Student’s t tests.

Gene ontology analysis of differentially expressed genes.

The 192 differentially expressed genes (DEGs) between p53-/- and p53Y217C/Y217C thymocytes were analyzed with the Gene Ontology enRIchment anaLysis and visuaLizAtion tool (GOrilla). Out of 192 genes, 141 were associated with a GO term. For each GO term, enrichment was calculated by comparing with the full list of 19759 genes associated with a GO term. For example, genes with GO term #0071621 (granulocyte chemotaxis) represent 79/19759 genes associated with a GO term, but 8/141 of the identified DEGs, which represents a 14.19-fold enrichment.

Gene set enrichment analysis (GSEA) of transcriptomes from p53Y217C/Y217C and p53-/- thymic cells: examples of GSEA enrichment plots.

Examples of GSEA enrichment plots indicating differences in immune responses (A), mitochondrial function (B) and chromatin plasticity (C) are shown.

Viability of Mdm2 or Mdm4 loss in a p53Y217C/Y217C background.

Mouse distributions from the indicated crosses were determined at weaning.

Evidence of dystocia in pregnant p53Y217C/Y217C females.

Results of the mating of two (a and b) p53Y217C/Y217C females (F) with a p53+/Y217C male (M) are shown. Both females were rapidly pregnant after encountering a male, but had to be sacrificed due to extended labor and pain during their first (female a) or third (female b) delivery.

Ccl17, Ccl9, Ccr3, Cxcl10, S100a8 and S100a9 are associated with GO terms of white blood cell migration chemotaxis/and inflammation.

Primers used in this study.