BMC Genomics 2007, 8:139 http://www.biomedcentral.com/1471-2164/8/139 IFdiegnutrifeica3tion of p53-occupied genomic sites Identification of p53-occupied genomic sites. A) Schematic of chromatin immunoprecipitation (anti-p53, FL-393) cloning strategy. See methods for more detail. B) Location of tags containing putative p53 binding sites relative to known genes within the rat genome. Binding sites scoring 90 or above out of 100 using the p53MH algorithm were included in this anal- ysis. C) A hypothetical 100 kb gene map is used to show the relative location of 364 of experimentally determined p53 site-containing tags that achieved scores of ≥ 90 with the p53MH predictive binding algorithm. Data show that p53 site-containing tags were found in the upstream 5', down- stream 3' and intergenic gene regions. p53 binding scores alone may pose a constraint on puta- tive regulated genes because p53-mediated transrepres- sion can occur in both the absence of a consensus binding site [48,49] or by binding to sites sufficiently distinct from the consensus sequence that fall below our accepted p53MH binding site prediction level [50]. A preponder- ance of genes whose known functions may be relevant in neuronal differentiation also factored into our selection of tags and clusters for validation. We selected 100 tags for ChIP-based validation based on criteria of ontological classification (i.e. GO terms related to development, dif- ferentiation or morphogenesis), known function of asso- ciated genes, or p53 binding site score in order to identify candidate p53-regulated targets with a role in PC12 differ- entiation. ChIP analysis demonstrated significantly ele- vated (p ≤ 0.05) p53 occupancy in an NGF-dependent manner for roughly 30% of the 100 studied validation tar- gets. Those targets to which p53 binding was significantly increased in NGF-treated cells are shown in Figures 4, 5, 6, 7, 8. As expected, reproducible binding was demonstrated to promoters of known p53-inducible target genes shown in Figures 4 and 5 including p21Waf1/Cip1, MDM2 and cyc- lin G1 (Figure 4). In addition to NGF-responsive genomic p53 binding sites, roughly 40% of targets showed signifi- cant p53-binding independent of NGF treatment [See Additional file 1; Table 3]. These targets were significantly enriched in ChIP validation when compared to negative control regions, although no NGF-responsive changes were noted in p53 binding. Absence of NGF-dependent 53 binding to these regions precluded further analysis since they were beyond the objectives of this study. While these genomic locations were ChIP-enriched, cloned and demonstrated to be enriched once more in a validation ChIP experiment, further study will be necessary to dem- onstrate p53-dependent transcription. In order to determine the transcriptional outcome of p53 binding to NGF-responsive target regions described above, RT-PCR was performed to compare mRNA levels for indicated targets among naïve and differentiated PC12 cells (Figures 4, 5, 6, 7, 8). p53-dependence on target gene expression was verified using three stable anti-p53 shRNA-expressing PC12 lines with varying p53 knock- down efficiencies. Use of the three shRNA clones formed a gradient of p53 mRNA and protein expression (Figure 2A), where p53#3low had the lowest level of expression and p53#2mid & p53#4mid were both reproducibly higher with a moderate level of p53 expression. Including known targets, a total of 14 genes were p53-induced in differentiating PC12 cells. Importantly, we discovered ten novel, p53-inducible targets regulated in an NGF-respon- sive manner (Figures 4 and 5), including the sesn3 and nme1 targets recently described to be p53 regulated either by association or indirect experimental evidence [51,52]. In addition, the dusp5 gene was an NGF-inducible p53 target, recapitulating earlier reports of this gene being p53-regulated [53]. Further, we report four novel p53 gene targets that demonstrated a p53-dependent repres- sion (Figure 6). These included the pkcbpb15/trib3 locus, where transcripts of both genes were elevated in p53- silenced cells. We also demonstrate eleven p53 occupied regions where transcriptional changes in nearby genes during NGF-mediated differentiation could not be accred- ited solely to p53 activity in the study (Figures 7 and 8), including the previously described p53 targets snk [54] and nck2 [5]. Additionally, new p53-occupied binding sites were identified for the MDM4 and dyrk3 genes, for Page 5 of 17 (page number not for citation purposes)