Genotoxic stresses such as DNA damage oxidative stress and

Genotoxic stresses, such as DNA damage, oxidative stress, and hypoxia, induce phosphorylation and stabilization of the p53 protein (Vogelstein et al., 2000). Activated p53 functions as a transcription factor and suppresses cell growth via transactivation of its target genes involved in apoptosis, hippo pathway arrest, and/or senescence (Nakamura, 2004; Vogelstein et al., 2000). Additionally, p53 regulates various biological processes, such as metabolic pathways, aging, development, reprogramming, and reproduction (Hu et al., 2007; Kang et al., 2009; Kruiswijk et al., 2015). Thus, p53 exhibits various physiological functions in a context-dependent and tissue-specific manner. Previous studies have identified a number of p53 target molecules using transcriptome analysis of specific cell lines, tissues, or mouse embryonic fibroblasts (MEFs) (Kenzelmann Broz et al., 2013; Masuda et al., 2006) (Table S1). However, no systematic analysis of p53 target genes in various tissues has been conducted thus far. To elucidate the role of p53 at individual tissue levels, we performed whole-body transcriptome analysis after X-ray-irradiation on p53+/+ and p53−/− mice. In our study, 280 samples from 24 tissues were evaluated by RNA sequence analysis. Our study is the comprehensive analysis of the p53 signalling pathway in vivo.
Materials and Methods
Results
Discussion The regulation of gene expression by p53 has been extensively analyzed (Idogawa et al., 2014; Kenzelmann Broz et al., 2013), however most of the previous studies analyzed one or two human/mouse cells or tissues as shown in Table S1. Since p53 mutations were observed in cancers from multiple tissues, the identification of p53-regulated genes in various cell types is essential to fully understand the p53 signalling network. Here, we conducted an RNA sequence-based whole-body transcriptome analysis using p53+/+ and p53−/− mice. In our study, 280 samples from 24 tissues were analyzed, andB transcriptome analysis of p53 regulated genes. Our results also indicated that the great majority of X-ray-responsive genes were regulated by p53 and further supported the crucial roles of p53 in the DNA damage response. In the previous analysis, cells or mice were treated with various dose of radiation (1–10Gy) and were analyzed at various time points (1–24h) (Table S1). p53 was induced at 3–6h after irradiation in thymus, spleen and small intestine, however sensitivity to irradiation varies between tissues (Fei et al., 2002; Komarova et al., 2000). p53 deficient mice exhibited apoptosis resistance and prolonged survival after 10Gy of radiation (Komarova et al., 2004), suggesting that p53 targets related with apoptotic pathway were induced in this condition. Genes related with apoptotic pathway were shown to be induced relatively late time point in response to severe DNA damage (Oda et al., 2000). Thus, we selected 10Gy and 24h in our study to identify p53 targets even in radiation resistant tissues. We also found remarkable differences in the DNA damage response across different tissues. >2000 genes were regulated in the thymus and spleen in p53+/+ mice. These two tissues are known to be sensitive to radiation-induced apoptosis (Gottlieb et al., 1997; Midgley et al., 1995) and commonly develop cancer in p53-defective mice (Donehower et al., 1995). Expression of p53, Atm, and Chek2 was strongly correlated with the number of p53-regulated genes as well as fold induction of Cdkn1a. Taken together, we revealed that Atm-Chek2-p53 axis was associated with DNA damage response and cancer susceptibility. However, p53 protein was increased in only nine tissues among 22 tissues analyzed. Interesting, both X-ray sensitive (thymus and spleen) and X-ray resistant tissues (testis and heart) exhibited high basal p53 expression with no or marginal p53 induction by X-ray. These results indicate that p53 protein expression cannot fully explain difference in DNA damage response among tissues. Further analyses of genes or pathways that are associated with the number of p53-resposive genes as well as p53 modification are necessary to understand the molecular mechanism of p53-mediated transcriptional regulation.