BMC Genomics 2007, 8:139 http://www.biomedcentral.com/1471-2164/8/139 Evidence suggests a functional role for p53 in differentia- tion and development, particularly within the nervous system. While initial p53-null mouse models were described as phenotypically normal at birth with mark- edly increased tumor development later in life [8,9], later studies demonstrated a proportion of p53-/- mice had defects in neural tube closure resulting in exencephaly [10,11]. In vivo measurements in p53-responsive lacZ transgenic mice demonstrated elevated p53 transcrip- tional activity in the embryonic and early gestational nerv- ous system [12], while high levels of p53 expression have been described throughout the chick, rat, mouse and Xenopus embryo [13-17] through midgestation. p53 func- tional activity has been further implicated in the differen- tiation of neuronal precursors [10,11,16] and is transcriptionally active within the nucleus of differentiat- ing primary hippocampal neurons and oligodendrocytes [18], while p53 is inactive and localized to the cytoplasm of postmitotic differentiated sympathetic neurons [19]. p53 transcriptional activity may be further involved in the differentiation of other non-neuronal cell types. Tran- scriptional activity of p53 plays a direct role in embryonic stem cell differentiation by suppression of the Nanog gene [20], and can directly induce Xenopus homebox gene expression [21]. Involvement of p53 has also been shown in spermatogenesis [22], eye development [23] renal development [24], osteogenesis [25], immune develop- ment [26], lung development [27] and muscle differenti- ation [28]. Despite data supporting the transcriptional activity of p53 protein in differentiation, few genetic tar- gets have been described and fewer still within a neuronal context. Discovery of non-genotoxic mechanisms through which p53 protein may be activated within tumor cells harbor- ing wild-type p53 remains an important therapeutic objective, both in effort to alleviate traditional off-target chemotherapeutic side-effects and to prevent the unpre- dictable mutagenicity characteristic of these therapies. Wild-type p53 can be activated in several malignancies by treatment with nerve growth factor [29] or pharmacologic agents [30-33], occurring through mechanisms sparing cells from the concomitant genotoxicity following chem- otherapy. Recent evidence demonstrates that while p53 transcriptional regulation of target genes may be stimulus- dependent (e.g. DNA damage), promoter occupancy is generalized and may occur independently of the activat- ing genotoxic stimulus [34]. Whether this paradigm is also applicable to nongenotoxic p53 stimuli compared to DNA damage-induced p53 activation is unclear. NGF- mediated p53 activation mainly results in decreased pro- liferative ability of tumor cells and not apoptosis [29], raising the question of whether NGF-regulated p53 activ- ity is distinguishable from other means of p53 activation like DNA damage. While the mechanism of NGF-induced p53 activation is uncertain, inquiry into the transcrip- tional program following such activation is warranted to discern possible receptor-dependent p53 transactivational selectivity compared to genotoxic agents. In the current study, the p53 transcriptional program was studied during nerve growth factor-induced differentia- tion of ectoderm-derived rat PC12 pheochromocytoma cells. A global chromatin immunoprecipitation-based screen was used to identify genetic elements bound to and regulated by p53. Treatment of PC12 cells with NGF induces differentiation into neuron-like cells marked by cell cycle arrest and neurite extension [35] along with marked changes in gene expression [36] and signaling pathway activity [37]. NGF-responsive p53 activity [38] has a functional contribution towards NGF-mediated PC12 differentiation [39-41], but a role in gene-specific transactivation has not yet been established. We hypothe- sized that marked accumulation of p53 protein within dif- ferentiating PC12 cells would be accompanied by concomitant transcriptional regulation of genes involved in differentiation and cell cycle arrest. Furthermore, we hypothesized that genomic elements regulated by p53 during neuronal differentiation may be unique from those regulated during genotoxic stress and apoptosis. We now report that p53 protein is transcriptionally activated in NGF-mediated neuronal differentiation and describe its binding to a number of novel and previously unre- ported genomic regions as well as the transcriptional out- come of this binding. We identify and validate genomic targets of p53 activity within the differentiating neuron- like cell, and demonstrate selectivity between NGF-acti- vated versus genotoxicant-activated p53 transcriptional activity in a select number of p53 target genes. Results NGF induces activation of p53 protein within PC12 cells PC12 morphologic changes and neurite extension consist- ent with differentiation were visible within 12 hours of NGF addition to the media and became marked over the course of 7 days [See Additional file 1; Figure 1A]. NGF- dependent differentiation after 7 days treatment in PC12 cells was evidenced by expression of the dopamine trans- porter, a marker for dopamine generation [42] and trans- port, and decreased nestin immunoreactivity, indicating loss of undifferentiated phenotype [43] [See Additional file 1; Figure 1B]. In agreement with previous reports [44], cell cycle analysis by flow cytometry using FITC-BrdU labeling of total and newly synthesized DNA, respectively, revealed the population of cells within S phase was decreased to ~1% of the total cell population within 96 hours of NGF treatment [See Additional file 1; Figure 2A]. Reduction of S phase cells was accompanied by a concom- itant increase in the number of cells within G1 phase after 7 days of NGF exposure [See Additional file 1; Figure 2B]. Page 2 of 17 (page number not for citation purposes)