Sequence Variation and p53 DNA Binding Figure 5. Evaluation of p53 binding to response element SNP alleles. Binding for predicted strong (Dark bars) and weak (open bars) alleles of the polymorphic sequence is shown relative to p21 RE binding (100%). Each bar represents the mean of 3 experiments carried out on different days and performed in triplicate (error bars represent SD). Positive and negative control oligonucleotides (left side) were included in each experiment and each sample was tested with a negative control extract (data not shown). This experiment was replicated using p53 extracts generated from a second tissue type (U2OS cells, Figure S8). These results are in close agreement with evaluation of these SNP alleles when cloned into a luciferase construct and transfected into SaOS2 cells expressing p53 (Tomso et al. [5]). doi:10.1371/journal.pgen.1000462.g005 Several recent studies have identified SNPs in p53 REs and evaluated their impact on expression or binding [5,30]. The present results suggest that MAPD can be useful for assessing the magnitude of p53 binding to newly discovered candidate sequences and also for evaluating the impact of SNPs or mutations in those sequences. Whole genome association studies frequently identify SNPs in gene regulatory regions containing putative TFBSs; assessing TF binding using the approach outlined here is one practical way to evaluate the effects of regulatory region SNPs. One issue in developing good prediction of TFBSs has been the lack of quantitative binding data. We show that integrating binding information from site-directed mutagenesis of a p53 consensus sequence into a computational binding site model improves prediction for a second set of REs. We used MAPD to investigate sequence-specific binding between p53 protein and a number of known and well-studied REs in the p53 pathway. Various in vitro assays have suggested that p53 has a higher affinity for REs in cell-cycle arrest and DNA repair genes than the REs in apoptosis genes [4,24]. Similar observations were obtained in vivo using a yeast-based system [22,23], and the present results are in agreement. This is also supported by Riley et al, who report that apoptosis-related p53 REs differ more from consensus binding sequence than cell cycle- related REs. In addition an evolutionary analysis [43] indicates that p53 RE sequences in human apoptosis genes tend to diverge considerably from those of rodents—although some weak binding REs do demonstrate strong evolutionary conservation. We observed a strong correlation between binding to target p53 REs and p53-driven luciferase gene expression in mammalian cells (Figure 6A). We have also seen that binding correlates with expression for the p21 RE when constructed with variable numbers of nucleotides between p53 half-sites [44]. These correlations suggest that our in vitro binding assay using p53 in nuclear extracts may be a surrogate for some in vivo transactivation studies. However, further work to evaluate binding to full length native promoter sequence with direct comparisons to chromatin IP and expression is needed [45,46]. The importance of p53 binding has been highlighted by Shaked et al al [34] who reported that p53 activation status and RE occupancy differ between tumor cell lines and normal primary cells. Our approach, assessing binding with nuclear extracts, can be used with tumor lines, EBV transformed lymphoblasts or normal primary cells. This will allow quantitative evaluation of the impact of post-translationally modified and activated wt p53 on binding to target gene REs as in Figure 6, or under various stress and DNA-damaging situations as in Shaked et al [34]. Given that p53 mutations are the most common genetic changes in cancer and are a major determinant of treatment outcome [35,36], the MAPD system may be useful in assessing the impact of mutations on p53 RE interactions (Figure 6B). Since radiation and alkylating agents are primary therapies in cancer treatment, understanding how exposures affect p53 activation and PLoS Genetics | www.plosgenetics.org 7 May 2009 | Volume 5 | Issue 5 | e1000462