Fig. 5: Identification of new p53 distal target genes through enhancer-promoter interactions.

A Overlap between p53 distal target genes at Nut 1 h and Nut 10 h time points. In parentheses are the number of p53 distal target genes which were primed (already interacting with a distal p53-bound enhancer prior to p53 activation) (i.e., Nut 0 h). Distal target genes are those genes engaging in PCHi-C interaction with a p53-bound enhancer. Nut 0 h refers to control cells without p53 activation. B Schematic representation of our definition of p53 target genes. “Proximal” target genes are those with p53 binding at their promoter (i.e., −1000/+200 nucleotides from the TSS). “Nearest” genes are those found closest in linear proximity to a p53-bound enhancer. “Distal” target genes are those found to interact with a distal p53-bound enhancer (defined using PCHi-C data). C Density distribution of the distance between the midpoint of a p53-bound enhancer and the transcription start site of the “nearest” gene (gray), or the transcription start site of the “distal” target gene linked by PCHi-C after 1 h (green) or 10 h (purple) of p53 activation. D Gene set enrichment analysis of gene sets from the Molecular Signatures Database (MSigDB) with significant enrichment (p-adj <0.05) in one or more p53 target gene groups. Bubble size indicates the number of genes found enriched for a given gene set, and green to yellow corresponds to the decreasing adjusted p value for enrichment. p values were calculated using a hypergeometric distribution test and adjusted for multiple comparisons using Benjamini-Hochberg (BH). E Differential H3K27ac between Nut 1 h and Nut 0 h time points (log2FC) at p53-bound regulatory elements (enhancer and promoter) and genome-wide (control). Genomic regions from p53-bound regulatory elements are excluded from genome-wide analysis. Two-sided Mann-Whitney test was used to test whether the mean H3K27ac log2FC differed significantly between groups using a p value cutoff of 0.05 (star). Sample sizes are respectively 175, 82 and 476,760, for enhancer, promoter and control regions. F Differential H3K27ac between Nut 1 h and Nut 0 h time points (log2FC) at promoter elements of proximal and distal p53 target genes, and promoter elements of non-targeted genes (control). Two-sided Mann-Whitney test was used to test whether the mean H3K27ac log2FC differed significantly between groups using a p value cutoff of 0.05 (star). G Differential expression between Nut 1 h and Nut 0 h time points (log2FC) of proximal and distal p53 target genes and non-targeted genes (control). Two-sided Mann-Whitney test was used to test whether the mean H3K27ac log2FC differed significantly between groups using a p value cutoff of 0.05 (star). F–G Sample sizes are respectively 191, 82 and 14,233, for distal, proximal and control genes. H, I and J. As in (E, F and G), respectively, but considering target genes controlled by p53 at 10 h of activation. H Sample sizes are respectively 184, 77 and, 189,993, for enhancer, promoter and control regions. I, J Sample sizes are respectively 226, 77 and 13,803, for distal, proximal and control genes. E–J The boxes show the interquartile range (IQR), the central line represents the median, the whiskers add 1.5 times the IQR to the 75 percentile (box upper limit) and subtract 1.5 times the IQR from the 25 percentile (box lower limit). Significance calculated with two-sided Mann–Whitney test. The star indicates p values below 0.05. K Relative nascent mRNA expression (fold change) of p53 distal target genes along p53 activation. A one-tailed Student’s t test was used to test whether relative expression differed significantly (p value  < 0.05) between adjacent time points for each gene (star). A linear mixed model was used to test whether relative expression differed significantly between adjacent time points, globally. For this, relative expression was averaged between the three replicates at each time point for each gene (p value: 6.5e-14 after 1 h, and 4.3e-13 after 10 h. linear mixed model see methods). Error bars correspond to one standard deviation of the mean. L Interaction landscape of PLK2’s gene promoter (blue shade) showing stable interactions (arcs) with a p53-bound enhancer (red asterisk and pink shade) located 966 kb downstream. H3K27ac profiles along p53 activation are represented at the top. Peak tips colored in black represent a peak going over the scale limit. Arrows symbolize gene placement and orientation along the genomic window.