p53 drives a transcriptional program that elicits a non-cell-autonomous response and alters cell state in vivo

Abstract

Cell stress and DNA damage activate the tumor suppressor p53, triggering transcriptional activation of a myriad of target genes. The molecular, morphological, and physiological consequences of this activation remain poorly understood in vivo. We activated a p53 transcriptional program in mice by deletion of Mdm2, a gene that encodes the major p53 inhibitor. By overlaying tissue-specific RNA-sequencing data from pancreas, small intestine, ovary, kidney, and heart with existing p53 chromatin immunoprecipitation (ChIP) sequencing, we identified a large repertoire of tissue-specific p53 genes and a common p53 transcriptional signature of seven genes, which included Mdm2 but not p21 Global p53 activation caused a metaplastic phenotype in the pancreas that was missing in mice with acinar-specific p53 activation, suggesting non-cell-autonomous effects. The p53 cellular response at single-cell resolution in the intestine altered transcriptional cell state, leading to a proximal enterocyte population enriched for genes within oxidative phosphorylation pathways. In addition, a population of active CD8+ T cells was recruited. Combined, this study provides a comprehensive profile of the p53 transcriptional response in vivo, revealing both tissue-specific transcriptomes and a unique signature, which were integrated to induce both cell-autonomous and non-cell-autonomous responses and transcriptional plasticity.

Keywords: Mdm2; p53; signature; single-cell sequencing; transcriptome.

Fig. 1.

Acute p53 activation in Mdm2^FM/− CAG-CreER^Tg mice. (A) Treatment regimen and time point of euthanasia (SAC). (B) Weight loss at euthanasia as a percent of weight on injection day in FM (Mdm2^FM/−), Tg (Mdm2^+/− CAG-CreER^Tg), and FMTg (Mdm2^FM/− CAG-CreER^Tg) mice with Student’s t test for statistical analysis. (C) Correlation between percent recombination of the Mdm2^FM allele and relative p21 mRNA levels in FMTg mice normalized to Tg mice. Graph is divided into arbitrary quadrants. (D) Western blot for p53 protein expression compared with vinculin (Vinc). Quantification of p53/Vinc signal, relative to Tg sample within a given tissue type, is indicated below the bands. H, heart; I, intestine; K, kidney; O, ovary; P, pancreas. (E and F) qRT-PCR of tissues from FMTg mice normalized to Tg mice. Expression normalized to Rplp0. (G) H&E staining. Yellow lines, crypts; light green arrows, dilated tubules; dark green arrows, protein cast; yellow arrows, infiltrating immune cells. (Scale bars, 100 µm.) (H) Quantification of crypt loss in intestines of mice 24 h after tamoxifen. Numbers are total crypts counted within ten 40× fields, and dots indicate biological replicates with Student’s t test for statistical analysis. (I) Number of protein casts per kidney; dots indicate biological replicates with Student’s t test for statistical analysis. (J) Representative images of CK19 immunohistochemistry and quantification; dots indicate biological replicates with Student’s t test for statistical analysis. (Scale bars, 100 µm.) (K) Quantification of immune cells. Number of immune cells within five 40× fields; dots indicate biological replicates. All data are presented as mean ± SD from individual mice.

Fig. 2.

RNA and ChIP-sequencing data reveal acute p53 transcriptional response. (A) Table depicting the total number of DEGs that resulted from DESeq2 analysis of bulk RNA-sequencing data of five tissues after p53 activation. DEGs have a P value of <0.05. (B) Bar graph depicting the percentage of total DEGs per tissue that have a p53 binding site (BS) anywhere within their promoter as per Kenzelmann Broz et al. (12) ChIP-sequencing data. These genes are henceforth referred to as differentially expressed p53 target genes. (C) Pie graphs depicting the percentage of up-regulated and down-regulated differentially expressed p53 target genes (TGs) for each tissue. (D) Top 10 enriched pathways from GSEA analysis of Hallmark and GO pathways for differentially expressed p53 target genes in FMTg. Red bars highlight the Hallmark p53 pathway in each dataset.

Fig. 3.

Activation of p53 up-regulates a p53 signature. (A) Venn diagram, generated using InteractiVenn, overlaying the differentially expressed p53 target genes for each tissue (31). (B) Log₂ fold change values for the seven overlapping p53 target genes in A. (C) ChIP-qPCR for FMTg (Mdm2^FM/− CAG-CreER^Tg) pancreas (n = 4) of seven overlapping p53 target genes presented as p53 ChIP/IgG ChIP. Chrm5 encodes an acetylcholine receptor that serves as a negative control for p53 binding. ns, not significant. Student’s t test was used for statistical analysis.

Fig. 4.

Exogenous activation of p53 through IR. (A) qRT-PCR of irradiated (IR) wild-type (WT; n = 3) and p53-null (n = 5) pancreas, heart, kidney, ovary, and intestine compared with unirradiated WT (n = 2) for seven overlapping p53 target genes. Expression normalized to Rplp0. Statistical analysis was conducted with ANOVA utilizing Dunnett’s test for multiple comparisons against irradiated WT mice. All data are presented as mean from individual mice. (B) Western blot for p53 protein expression compared with vinculin (Vcl). Quantification of p53/Vcl signal, relative to WT sample within a given tissue type, is indicated below the bands. H, heart; I, intestine; K, kidney; O, ovary; P, pancreas. *Nonspecific band. (C) Western blot for p53 and phosphorylated p53 at serine 15 (p-p53 ser15) and Vcl. Quantification of p53 or p-p53/Vcl signal, relative to first Tg sample, is indicated below the bands and graphed to the right with Student’s t test for statistical analysis. ns, not significant. Data are presented as mean ± SD from individual mice.

Fig. 5.

The pancreatic response to p53 activation in FMTg mice is non-cell-autonomous. (A) Treatment regimen and time point of euthanasia (SAC) for FMMist (Mdm2^FM/− Mist1^CreERT2) and Mist (Mdm2^+/− Mist1^CreERT2) mice. (B) Percent recombination of the conditional Mdm2^FM allele as determined via qPCR assay in Mist, Tg (Mdm2^+/− CAG-CreER^Tg), FMMist, and FMTg (Mdm2^FM/− CAG-CreER^Tg) mice. Statistical analysis was conducted with ANOVA utilizing Dunnett’s test for multiple comparisons against Tg mice. (C) qRT-PCR for relative Eda2r and Gtse1 mRNA levels within the pancreas of Mist, Tg, FMMist, and FMTg mice. Expression normalized to Rplp0. Statistical analysis was conducted with ANOVA utilizing Dunnett’s test for multiple comparisons against Tg mice. ns, not significant. (D) H&E staining on pancreas samples. (Scale bars, 100 µm.) (E) Quantification of immune cells. Number of immune cells within five 40× fields; dots indicate biological replicates with a Student’s t test for statistical analysis. ns, not significant. All data are presented as mean ± SD from individual mice.

Fig. 6.

scRNA-seq in FMTg intestines provides insight into cell-states driven by p53 activation. (A, i) Total number of cells analyzed after quality control metrics ran with Seurat V3 for one FMTg (Mdm2^FM/− CAG-CreER^Tg) intestine sample and one Tg (Mdm2^+/− CAG-CreER^Tg) intestine sample. (A, ii) Uniform Manifold Approximation and Projection (UMAP) of labeled cell populations. (A, iii) Portion of specific cell populations in FMTg and Tg samples. (B) Total number of goblet and paneth, tuft, distal enterocytes, and memory CD4+ T cells in FMTg and Tg intestines. (C) Total number of all enterocytes and T cells per group. (D) Number of cells that encompass proximal enterocyte population 2 and CD8+ T cell population 2 in each intestine sample. (E) GSEA analysis on defining features for proximal enterocyte population 2. (F) GSEA analysis on defining features for CD8+ T cell group 2.