And apoptotic genes as observed by steady state RNA measurements.International evaluation of p53 effects on RNA synthesis vs RNA steady state levelsThe worldwide p53 transcriptional response has been previously investigated using measurements of RNA steady state levels (i.e., microarray profiling) and p53 chromatin binding (e.g., ChIP-seq). Meta-analysis of four recent reports applying this method indicates that 1200 genes are putative direct targets of p53 transactivation, but only 26 are widespread in between the 4 research (Figure 2– figure supplement 1A,B; Supplementary file 2) (Nikulenkov et al., 2012; Menendez et al., 2013; Schlereth et al., 2013; Wang et al., 2013). Additionally, these studies suggest 80 genes that might be straight repressed by p53, yet none are shared amongst any two research (Figure 2– figure supplement 1A,B; Supplementary file 2). To be able to investigate how GRO-seq evaluation of the instant p53 transcriptional response would compare to a international evaluation of RNA steady state levels, we performed a microarray analysis of HCT116 p53 ++ cells just after 12 hr of Nutlin treatment, a time point 
similar to that employed in the prior studies. Numerous crucial observations arise from this comparison. Very first, there’s a clear lack of overlap involving the two analyses (Figure 2A). Among the Tartrazine induced genes identified by the two experimental platforms, only 102 are frequent. 291 genes are known as as induced by the microarray experiment only. This group would incorporate genes whose transcription PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/21352867 may be stimulated at later time points by means of indirect mechanisms, but may possibly also incorporate true direct p53 target genes that need larger levels of p53 to become activated. By way of example, we noted that the canonical p53 target gene GADD45A fell in this group, as its transcription was mildly induced at 1 hr and thus fell below our statistical cut-off. Interestingly, 72 genes were identified as induced by GRO-seq only, despite the truth that the microarrays utilized harbored multiple probes against these mRNAs. The feasible explanations for this obtaining are discussed below. Second, microarrays detect 324 genes repressed upon 12 hr of Nutlin treatment, none of which were called as repressed by GRO-seq. The mechanism of p53-mediated gene repression remains debated in the field. Multiple independent ChIP-seq research concur in that p53 binds weakly and very distally to these gene loci whose mRNAs are downregulated in the steady state level, and that the p53REs discovered at these sites match poorly towards the consensus DNA sequence (Nikulenkov et al., 2012; Menendez et al., 2013; Schlereth et al., 2013; Wang et al., 2013). Utilizing seven different obtainable international ChIP datasets derived from HCT116 and two other cell lines, we produced a collection of high self-assurance p53 binding events to analyze p53 binding inside the vicinity in the a variety of gene groups (`Materials and methods’). Practically 40 in the 198 genes induced by GRO-seq harbor a p53 binding occasion within 25 kb, drastically more than expected from random occurrence (p=1e-48, Hypergeometric test) (Figure 2B). Among the genes induced by microarray only, nearly 15 harbored p53 binding within 25 kb, nonetheless considerably greater than expected by likelihood (p=8e-11), which suggests that a few of these genes can be correct direct targets activated at later time points. Most importantly, genes regarded as as repressed by the microarray profiling show little p53 binding inside 25 kb, barely above what exactly is anticipated by opportunity (p=3e-2), suggesting that the repression.
