Volume 46, Issue 1, Pages 30-42 (April 2012) Distinct Regulatory Mechanisms and Functions for p53-Activated and p53-Repressed DNA Damage Response Genes in Embryonic Stem Cells  Mangmang Li, Yunlong He, Wendy Dubois, Xiaolin Wu, Jianxin Shi, Jing Huang  Molecular Cell  Volume 46, Issue 1, Pages 30-42 (April 2012) DOI: 10.1016/j.molcel.2012.01.020 Copyright © 2012 Elsevier Inc. Terms and Conditions

Molecular Cell 2012 46, 30-42DOI: (10.1016/j.molcel.2012.01.020) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Genomic Profiling of p53 and S18P (A) Venn diagrams of p53 and S18P peaks in mES cells untreated (Ctr) or treated with adriamycin (Adr) for 8 hr. (B) Genomic views of p53 and S18P peaks at the Mdm2 (left panel) and Cdkn1a (right panel) gene loci. (C) Histograms indicate that Adr has a more profound effect on S18P than on p53. (D) Upper panel: Venn diagram showing the overlap of p53 and S18P peaks. Lower panel, average peak intensity (tags/10 million) for each category of peaks. See also Figure S1 and Table S1. Molecular Cell 2012 46, 30-42DOI: (10.1016/j.molcel.2012.01.020) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 p53 Represses Many Core Transcription Factors in mES Cells (A) Identification of p53 direct target genes. Genes are rank-ordered according to their fold induction. (B) Real-time PCR validation of p53-dependent repression of nine core transcription factors of mES cells. (C) Western blot analysis of the repression of Oct4, Nanog, Sox2, and cMyc. (D) Real-time PCR analysis of dosage effect of adriamycin (Adr) treatment on the repression of p53-repressed core transcription factors. Numbers shown are concentrations of Adr (nM). (E) Western blot analysis of the dosage effect of Adr treatment on the repression of Oct4, Nanog, Sox2, and the activation of Mdm2 (positive control). For all panels, all error bars are SEM, n ≥ 3; ∗p < 0.05; ∗∗p < 0.01; see also Figure S2 and Table S2. Molecular Cell 2012 46, 30-42DOI: (10.1016/j.molcel.2012.01.020) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Promoter Binding of p53 Correlates with Activation (A) Heatmaps showing p53 and S18P binding in each region of each p53 target. Genes are ordered according to expression change. Each horizontal line represents one gene. Red (Adr) or green (Ctr, untreated), binding; white, no binding. (B) The percentages of genes that contain at least one p53 peak or S18P peak in each region are shown. p53 targets are divided into the activated group and the repressed group. A randomly selected group serves as a comparison. The statistical analyses are in Table S3. (C) Heatmaps showing the occupancy of p53 and S18P (log2 transformed tags/10 million) in the promoter region of each p53 target. Red, strong binding; yellow, weak binding; white, no binding. Genes are rank-ordered according to the fold change of expression (represented by a color bar on the left side). A walking average of total tags in the entire promoter-proximal region for each p53 target (red line) and randomly selected gene (blue line) are shown on the left. (D) Quantile-based analysis reveals the linkage of promoter binding of p53 with expression induction for p53-activated genes but not for the repressed genes. Quantiles are described in (C). Welch t test is performed. (E) Genome-wide averages (tags/10 million) of p53 and S18P occupancy on p53-activated (2070), p53-repressed (1627) and randomly selected genes. Gene body region is split into 100 bins. See also Tables S3 and S4. Molecular Cell 2012 46, 30-42DOI: (10.1016/j.molcel.2012.01.020) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 p53-Repressed Genes Have a Higher Tendency to Have Distal p53 Binding than the Activated Genes (A) Density plot of p53 binding at the three regions in Adr-treated mES cells. 3976 genes are rank-ordered by fold induction and tabulated into 37 bins. The percentage of genes with at least one p53 binding site in the indicated region is calculated for each bin and plotted against the bin number. A trend line is shown. (B) Genomic views of p53 and S18P occupancy at four transcription factor loci: Nanog, Sox2, Oct4, and Sall4. (C) p53-repressed genes have a higher percentage of p53 binding at the distal region than the activated genes. See also Figure S3 and Table S4. Molecular Cell 2012 46, 30-42DOI: (10.1016/j.molcel.2012.01.020) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 Interference with the Distal Enhancer Activity by p53 Binding Is One of the Mechanisms Underlying p53-Mediated Repression (A) Luciferase-based enhancer test. Immunoblotting shows that p53 wild-type and R175H mutant are expressed at a comparable level in p53−/− mES cells. Cdkn1a-Luc is used as a positive control. Luminescence signal of a luciferase vector containing neither a promoter nor an enhancer (empty reporter) is set to 1. Fold induction is defined as the luciferase activity of a reporter compared to that of the empty reporter. (B) Pairs of the minimal promoter and enhancer of the Nanog or Utf1 genes are tested for repression by p53. (C) Testing the enhancer activity and p53-mediated activation or repression of another p53 binding site, noted as dr (distal region). (D) Enhancer test to study the effect of enhancer binding of p53 on the activation of p53-activated genes: Mdm2 and Btg2. (E) DNA sequences of putative p53 response elements in the enhancer regions of the Nanog and Utf1 genes. Nucleotides in red indicate p53 binding half sites. Those with underlines are spacers between the two half sites. Nanog_en_del represents Nanog enhancer with the spacer being deleted. Similar derivation is used for Utf1_en_del. (F) Assessing the length effect of the spacer between the two half sites on p53-mediated repression of the Nanog (left) and Utf1 gene (right). For all panels, error bars are SEM, n = 2–4; ∗p < 0.05; ∗∗p < 0.01. pr, promoter; en, enhancer; Nanog_pr_en, a reporter with Nanog promoter and enhancer. Similar nomenclature is used for others. Molecular Cell 2012 46, 30-42DOI: (10.1016/j.molcel.2012.01.020) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 p53-Activated Genes Are Linked to ES Cell Differentiation, and p53-Repressed Genes Are Associated with ES Cell Status (A) Gene Set Enrichment Analysis (GSEA) showing the association of p53-activated genes and p53-repressed genes with the genes associated with the ES, differentiated ES, MEF, and iPS cells. (B) Venn diagram showing the overlap between p53-repressed genes and the ES cell module (Kim et al., 2010). (C) Gene expression levels (log2) of p53-activated genes and p53-repressed genes. (D) Heatmap showing the levels (log2) of histone modifications, H3K4me3, H3K27me3, and H3K79me2, as well as Pol II, on p53 targets. Genes are rank-ordered according to the fold induction. A walking average of the total tags in the promoter-proximal region for each gene is shown on the right side. Arrow indicates the TSS. See also Figure S4 and Table S5. Molecular Cell 2012 46, 30-42DOI: (10.1016/j.molcel.2012.01.020) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 7 p53 Regulates mES Cell Differentiation and Pluripotency (A) Alkaline phosphatase staining assay to measure the self-renewal of mES cells untreated or treated with Adr for 8 hr. (B) Design of the embryoid body formation assay to test the effect of DNA damage on the pluripotency of mES cells. (C) Real-time PCR analysis of markers for the three germ layers. (D) A model showing the dual functions of p53 in regulating ES cell differentiation. See also Figure S5. For all panels, error bars are SEM, n ≥ 3. Molecular Cell 2012 46, 30-42DOI: (10.1016/j.molcel.2012.01.020) Copyright © 2012 Elsevier Inc. Terms and Conditions