p53 Pulses Diversify Target Gene Expression Dynamics in an mRNA Half-Life- Dependent Manner and Delineate Co-regulated Target Gene Subnetworks  Joshua R. Porter, Brian E. Fisher, Eric Batchelor  Cell Systems  Volume 2, Issue 4, Pages 272-282 (April 2016) DOI: 10.1016/j.cels.2016.03.006 Copyright © 2016 Elsevier Inc. Terms and Conditions

Cell Systems 2016 2, 272-282DOI: (10.1016/j.cels.2016.03.006) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 p53 Target Genes Show Different Temporal Patterns of Expression in Response to DNA DSBs (A) Clustering analysis of gene expression time courses (n = 4, averaged, filtered) identifies groups of genes that show pulsing and rising expression patterns. Gene expression level is shown relative to the maximum and minimum expression levels of each gene. Fold differences between maximum and minimum expression for each gene are shown in parentheses next to each gene name. Genes with fold differences of at least 1.5 are shown in bold. Gene function (Table S1) is indicated by the colored box on the right. +, positive effect in the specified functional pathway; −, negative effect. (B–J) Sample gene expression time courses (averaged, unfiltered) for three genes with strongly pulsing expression (B–D), three with weakly pulsing expression (E–G), and three with rising expression (H–J). Error bars represent SEM (n = 4). See also Figure S1 and Data S1. Cell Systems 2016 2, 272-282DOI: (10.1016/j.cels.2016.03.006) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Gene Expression Dynamics Correlate with mRNA Decay Rate (A) A simple model predicts that pulses of p53 expression in a single cell can generate different target gene expression dynamics for different mRNA decay rates. For target 1, the mRNA decay rate kd = 1.0 hr−1; for target 2, kd = 0.18 hr−1, the frequency of p53 pulsing; for target 3, kd = 0.01 hr−1. (B) A model of p53 dynamics and target gene expression in a population of cells fits measurements of p53 target gene expression (Figure 1). Model fits are shown for representative target genes with strongly pulsing (BTG2), weakly pulsing (C12orf5/TIGAR), and rising (DDB2) expression dynamics. Error bars on gene expression measurements represent SEM (n = 4). (C) Measured mRNA decay rates are predictive of p53 target gene expression dynamics. mRNA decay rates for nascent transcripts were measured for the indicated genes in MCF-7 p53-Venus cells in response to treatment with 400 ng/mL NCS for 3 hr. The pulsatility index was calculated for each target gene by fitting the model of population-level target gene expression dynamics to measurements of the same (Figure 1). Pulsatility index is correlated with mRNA decay rate (Spearman’s ρ = 0.69, p = 4.9 × 10−6). See also Figure S2 and Data S2. Cell Systems 2016 2, 272-282DOI: (10.1016/j.cels.2016.03.006) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Changing the p53 Pulse Frequency Changes the Set of Genes Showing Pulsatile Expression (A) p53 pulses occur with a period of ∼5.5 hr in response to DNA DSBs. p53 pulses with a period of 3 hr were generated by periodic treatment of cells with 5 μM Nutlin-3. (B) p53 target genes BTG2 and PPM1D (WIP1), which have strongly pulsing expression dynamics in response to DNA DSBs, retained pulsatile expression in response to periodic Nutlin-3 treatment. (C) C12orf5 (TIGAR) and FAS, which have weakly pulsing expression dynamics in response to DNA DSBs, switched to rising expression in response to periodic Nutlin-3 treatment. (D) DDB2 and RRM2B, which have rising expression dynamics in response to DNA DSBs, retained rising expression in response to periodic Nutlin-3 treatment. The pulsatility index (PI) for each expression profile is indicated. Error bars represent SEM (n = 4 NCS-treated samples, n = 3 Nutlin-3-treated samples). See also Figure S3. Cell Systems 2016 2, 272-282DOI: (10.1016/j.cels.2016.03.006) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Single-Cell Transcriptional Profiling Shows that p53 Target Genes Are Co-regulated in Subnetworks in Response to DNA DSBs (A) MCF-7 cells expressing p53-Venus or with p53 knocked down (sh-p53) were treated with NCS, then sorted by FACS at specific times into lysis buffer. mRNA of 95 genes was reverse-transcribed, and the resulting cDNA was preamplified. Presence of a sorted cell was verified by measuring GAPDH amplification by real-time PCR. In samples passing this test, expression of 95 target genes was measured by real-time PCR. Measurements were normalized by the biological size factor for each cell (Experimental Procedures). (B–E) Examples of gene pairs with different correlation coefficients (Kendall’s τ), representing relatively strong positive (B), strong negative (C), weak positive (D), and weak negative (E) correlations. Each point represents normalized gene expression in a single cell. All units are molecules × RT efficiency (ERT). P values test the null hypothesis of no correlation. (F–J) Network diagrams of correlations between expression of genes (p < 0.005 in each of two biological replicates) in MCF-7 p53-Venus cells treated with NCS. Networks are shown for untreated cells (F) or cells sorted during the first (G), second (H), third (I), or fifth (J) pulse of p53. Each node in the network represents a gene, whose identity is shown in (K). Red and blue edges represent positive and negative correlations, respectively. Line thickness indicates strength of correlation as measured by Kendall’s τ. Node size represents the sum of correlations for a given gene. (K) Network diagram showing total of gene expression correlations for all time points (F–J). Green and orange regions represent sets of genes whose members are positively correlated with at least two other member genes and negatively correlated with nonmember genes based on analysis of all time points. (L–P) Network diagrams of correlations between expression of genes in MCF-7 sh-p53 (p53 knockdown) cells treated with NCS. Networks are shown for untreated cells (F) or cells sorted at times (M–P) corresponding to those in (G–J). Each node represents a gene, whose identity is shown in (Q). (Q) Network diagram showing total of gene expression correlations for all time points (L–P). Orange region represents a set of genes whose members are positively correlated with at least two other member genes and negatively correlated with nonmember genes on the basis of analysis of all time points. See also Figure S4 and Data S3. Cell Systems 2016 2, 272-282DOI: (10.1016/j.cels.2016.03.006) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Sustained High p53 Expression Changes Patterns of Target Gene Co-regulation (A and B) Network diagrams of genes with correlated expression as determined by single-cell transcriptional profiling were generated as in Figure 4 for MCF-7 p53-Venus cells (A) treated with NCS for 14 hr and (B) treated with NCS followed by sequential doses of Nutlin-3 designed to hold p53 at a high level. Green and orange regions represent sets of genes whose members are positively correlated with each other and negatively correlated with nonmember genes. See also Figure S5. Cell Systems 2016 2, 272-282DOI: (10.1016/j.cels.2016.03.006) Copyright © 2016 Elsevier Inc. Terms and Conditions