The p53 tumor suppressor is a sequence-specific pleiotropic transcription factor that coordinates cellular responses to DNA damage and stress, initiating cell-cycle arrest or triggering apoptosis. Although the human p53 binding site sequence (or response element [RE]) is well characterized, some genes have consensus-poor REs that are nevertheless both necessary and sufficient for transactivation by p53. Identification of new functional gene regulatory elements under these conditions is problematic, and evolutionary conservation is often employed. We evaluated the comparative genomics approach for assessing evolutionary conservation of putative binding sites by examining conservation of 83 experimentally validated human p53 REs against mouse, rat, rabbit, and dog genomes and detected pronounced conservation differences among p53 REs and p53-regulated pathways. Bona fide NRF2 (nuclear factor [erythroid-derived 2]-like 2 nuclear factor) and NFκB (nuclear factor of kappa light chain gene enhancer in B cells) binding sites, which direct oxidative stress and innate immunity responses, were used as controls, and both exhibited high interspecific conservation. Surprisingly, the average p53 RE was not significantly more conserved than background genomic sequence, and p53 REs in apoptosis genes as a group showed very little conservation. The common bioinformatics practice of filtering RE predictions by 80% rodent sequence identity would not only give a false positive rate of ∼19%, but miss up to 57% of true p53 REs. Examination of interspecific DNA base substitutions as a function of position in the p53 consensus sequence reveals an unexpected excess of diversity in apoptosis-regulating REs versus cell-cycle controlling REs (rodent comparisons: p < 1.0 e−12). While some p53 REs show relatively high levels of conservation, REs in many genes such as BAX, FAS, PCNA, CASP6, SIVA1, and P53AIP1 show little if any homology to rodent sequences. This difference suggests that among mammalian species, evolutionary conservation differs among p53 REs, with some having ancient ancestry and others of more recent origin. Overall our results reveal divergent evolutionary pressure among the binding targets of p53 and emphasize that comparative genomics methods must be used judiciously and tailored to the evolutionary history of the targeted functional regulatory regions.
The p53 tumor suppressor is a transcription factor that coordinates cellular responses to DNA damage and stress, initiating cell-cycle arrest or triggering apoptosis. Evolutionary conservation is often employed to separate the functional “wheat” from the nonfunctional “chaff” when identifying binding sites of transcription factors like p53. We evaluated evolutionary conservation of 83 experimentally validated human p53 binding sites against mouse, rat, rabbit, and dog genomes, and similarly examined binding sites for two other transcription factors as controls, NRF2 (nuclear factor [erythroid-derived 2]-like 2 nuclear factor) and NFκB (nuclear factor of kappa light chain gene enhancer in B cells), which direct oxidative stress and innate immunity responses, respectively. NRF2 and NFκB binding sites both exhibited high interspecific conservation, indicative of purifying natural selection, but surprisingly, human p53 response elements on average displayed a lack of conservation. Thus conservation is not useful in the prediction of p53 binding sites. After grouping p53 REs by gene ontology, we observed that binding sites in cell-cycle genes like CDKN1A displayed high conservation, while p53 binding sites in apoptosis and DNA repair genes showed an unexpected excess of diversity and very little homology with rodent sequences. Overall these results reveal divergent evolutionary pressure among the binding targets of p53 and suggest caution in generalizing about the similarity of regulation of the p53 pathway between humans and rodents.