BMC Genomics 2007, 8:139 http://www.biomedcentral.com/1471-2164/8/139 FWigeubrLeog9o multiple sequence alignment of p53 binding sites WebLogo multiple sequence alignment of p53 binding sites. Individual base letter height indicates level of conservation within each binding site position. A) Consensus sequence of p53-induced targets shown in Figures 4 and 5. B) Consensus sequence of p53-repressed targets shown in Figure 6. is suggested by the reduction of neurite outgrowth observed during p53 knockdown studies described here, which is consistent with previous work showing similar findings using a temperature-sensitive p53 mutant [39,40]. However, we demonstrate p53-dependence for target gene transcription using three PC12 cell lines with varying levels of shRNA-mediated p53 silencing, further enabling inference into the transcriptional sensitivity of each target gene to reduced levels of p53 protein. The 7- day time point of NGF treatment was selected for further analysis to explore p53 transcriptional activity at the time of highest p53 protein accumulation representing a stable timeframe of p53-DNA occupancy following NGF expo- sure. As the ChIP-cloning approach utilized here is essen- tially a snapshot of p53-DNA occupancy following 7 days of NGF exposure, it is interesting to consider that the pop- ulation of p53-regulated gene targets may vary over time following NGF treatment. Here we describe a number of novel p53-occupied genomic sites within the NGF-differentiated Rattus nor- vegicus neuronal PC12 cell line, and further identify corre- sponding genes regulated by p53 binding to these regions. The transcriptional targets described here represent entic- ing mechanisms through which PC12 cellular differentia- tion may be controlled by activated p53. Particularly interesting is the regulation of wnt7b and the tfcp2l4 grainyhead homolog gene. Transcriptional regulation of Wnt pathway genes by p53 has been described for several tqFoRigxTiuc-rPeeCxR1po0esxuprreession analyses of p53 targets following geno- qRT-PCR expression analyses of p53 targets following geno- toxic exposure. A) mRNA expression levels of known p53 targets comparing naïve, 7-day NGF (50 ng/mL), 6 h 5-fluor- ouracil (375 µM) and 6 h bleomycin (10 µg/mL) exposure. Expression was measured via real time PCR and normalized to naïve expression levels using 2-∆∆ct where significance at p ≤ 0.05 is designated by an asterisk (*). Error bars represent standard deviation. B) Same as in Panel A, depicting genes whose expression levels were not regulated in the same manner between NGF treatment and genotoxic treatment. C) Same as in Panel A, depicting genes with comparable regu- lation between NGF treatment and genotoxic exposure. factors, including the negative regulator Dickkopf-1 [59], the transcription factor Tcf-4 [60] and beta-catenin [61]. Until now, direct DNA binding and transcriptional regu- lation of Wnt genes themselves has not been previously reported for p53. Wnt7b functional activity is necessary for dendritic development and neuronal connectivity in hippocampal neurons [62], consistent with the general- ized lack of neurite extension observed in NGF-treated p53 knockdown PC12 cells in this study. The transcrip- tional regulation of wnt7b by p53 may be extensible to other cell types. For example, macrophage-derived wnt7b has been reported as an apoptotic initiator in vascular endothelial cells of the developing eye [63], raising the Page 11 of 17 (page number not for citation purposes)