BMC Genomics 2007, 8:139 http://www.biomedcentral.com/1471-2164/8/139 p53 protein levels were significantly elevated in response to NGF treatment in PC12 cells as previously described by others [44,45]. Accumulation of p53 protein was detecta- ble at 8 hours following NGF exposure and remained highly elevated over the course of 7 days (Figure 1A). Phosphorylation of p53 protein at serine 15, which is typ- ically needed for transactivation, began at about 8 hours after NGF treatment and remained highly phosphorylated throughout 7 days (Figure 1A). Accumulation of p53 pro- tein can be attributed to protein stabilization since levels of p53 mRNA remained unchanged over a 7-day course of NGF treatment (Figure 1B). Virtually all p53 protein was localized within the nucleus in 7 day NGF-differentiated cells, as determined by immunoblot of nuclear and cyto- plasmic fractions (data not shown). Accumulated p53 protein was transcriptionally active, as demonstrated by luciferase expression using the p53TA-luc reporter vector in differentiated compared to naïve cells (Figure 1C). The role of p53 activity in differentiation of NGF treated PC12 cells was investigated in anti-p53 shRNA-expressing PC12 cells (Figure 2A). Three stable PC12 lines expressing different anti-p53 shRNAs were created to observe p53- dependent function in PC12 differentiation. p53 RNA silencing resulted in reduced p53 protein levels, with p53#3low having the lowest levels, followed by p53#2mid and p53#4mid. The p53#4mid cell line repro- ducibly expressed p53 protein levels approaching those observed in the wild-type cell. In combination, these three shRNA cell lines formed a gradient of p53 protein expres- sion compared to the wild-type cell. While no apparent morphological differences were noted in the shRNA expressing cell lines maintained in the naïve state, NGF differentiation of these cells demonstrated reduced early neurite extension compared to their wild-type counter- parts. Within 3 days of NGF exposure, p53#3low cells had markedly reduced neurite outgrowth compared to wild- type cells (Figure 2B), lasting up to 7 days (data not shown). These results agree with prior work showing dis- ruption of morphological differentiation following both dominant-negative and temperature-sensitive p53 trans- fection into PC12 cells [18,39,46]. Chromatin immunoprecipitation and cloning of ChIP- generated tags p53 DNA binding was studied in seven-day differentiated PC12 cells via chromatin immunoprecipitation (ChIP) analysis. Preliminary assay validation demonstrated p53 binding to a known, well-defined binding site within the p21Waf1/Cip1 promoter at a 2-fold greater level in differenti- ated versus naïve cells [See Additional file 1; Figure 3]. Greater than 10-fold increased p53 occupancy at the p21Waf1/Cip1 proximal binding site was detected with FL- 393 immunoprecipitating antibody compared to IgG alone, confirming antibody specificity [See Additional file 1; Figure 3]. Having validated p53 occupancy to a known gene promoter, ChIP-enriched DNA from differentiated PC12 cells was amplified, concatemerized and cloned. Cloned concatemers were sequenced and separated, resulting in 7,184 individual DNA tags representative of putative p53-occupied locations within the PC12 genome [See Additional file 1; Table 1]. Tags were aligned to the Rattus norvegicus genome and locations with >1 tag map- ping within a 2 kb area ("clusters") were identified. Clus- ter chromosomal coordinates and sequences have been compiled and organized [see Additional File 2]. Sequence data were obtained for 500 bp upstream and downstream of each tag, single or clustered, and tags were recompiled to represent the original p53-bound ChIP-enriched frag- ments. Since the number of sequenced tags were non-sat- urating compared to the total population of p53-occupied fragments, both clusters and single tags were potentially informative. Tags representing putative p53-occupied genomic fragments were found to span the entire Rattus norvegicus genome in addition to known p53 binding regions within the MDM2, cyclin G1 and other known tar- get promoters, validating this experimental approach for the study of other novel putative targets. Recompiled tag sequences were analyzed for predicted p53 binding sites using the p53MH algorithm [47] and sorted according to both binding site score and location relative to nearby genetic elements. Stringency for p53 binding site probability score was set at a cutoff of 90 (out of 100) as determined by the p53MH algorithm using likelihood ratio scoring and a 14 base gap size limit between half sites. Results using these criteria demon- strated 364 p53 binding sites within tag sequences located in upstream 5', downstream 3' and intergenic gene regions. Of those tags with a p53MH binding site score ≥ 90 (out of 100), 56% mapped to within a gene, 25% to the upstream 5' region and 19% to the downstream 3' region (Figure 3B). Observed p53-occupied sites in the PC12 genome were visualized by plotting normalized tag alignment locations relative to a hypothetical gene size of 100 kb (Figure 3C). Scored p53 binding sites clustered to both the 5' and 3' gene regions, while intergenic binding sites were observed throughout the entire length of the gene. Validation of p53 occupancy and transcriptional regulation We selected a total of 100 clusters and single tags com- bined for ChIP-based validation of p53 occupancy based on either the presence of a putative p53 binding site as determined by the p53MH algorithm, or tag location rel- ative to a genomic element of interest. Since tag sequenc- ing and identification were not performed to saturation we considered individual tags and clusters as potentially informative. We recognized that selection based on high Page 4 of 17 (page number not for citation purposes)