Mapping the p53 transcriptome universe using p53 natural polymorphs

Abstract

The tumor suppressor p53 has defined roles in varied cellular processes including apoptosis and DNA repair. While conventional genomic approaches have suggested a large number of p53 targets, there is a need for a systematic approach to validate these putative genes. We developed a method to identify and validate p53's transcriptional behavior by utilizing 16 non-synonymous p53 single-nucleotide polymorphism (SNP) variants. Five SNPs located within the DNA-binding domain of p53 were found to be functionally null, whereas the other 11 SNPs were p53WT-like in behavior. By integrating p53 ChIP-seq analysis with transcriptome data from the p53 SNP variants, 592 genes were identified as novel p53 targets. Many of these genes mapped to previously less well-characterized aspects of p53 function, such as cell signalling, metabolism, central nervous system, and immune system. These data provide pivotal insights into the involvement of p53 in diverse pathways of normal physiological processes and open new avenues for investigation of p53 function. Cell Death and Differentiation (2014) 21, 521-532; doi:10.1038/cdd.2013.132; published online 27 September 2013The tumor suppressor p53 acts as a master regulator of cellular stress responses through transcriptionally regulating specific target genes involved in diverse cellular functions including cell cycle arrest, apoptosis, and DNA repair. 1 With the aid of large-scale screening techniques, it has become clear that the scope of p53 functions is much broader than previously thought, 2,3 expanding to cellular and physiological processes such as metabolism 4 and development. 5 To have a better understanding of this important tumor suppressor, we must be able to study the full complexity of its downstream effects and this requires the efficient identification and validation of genes directly regulated by p53. To date, there are fewer than 150 validated p53 target genes 6-8 and at the same time validation studies have been slow, which is a limitation on the progress in this field.In order to be regulated by p53 a gene must possess the appropriate cognate response element (RE) within its regulatory region. However, our previous study has shown that simple sequence motif identification is not sufficient, as the p53RE exhibits variable binding kinetics that are dependent upon the dinucleotide core 'CWWG' combinations. 9 These variables often result in spurious outcomes and hence we are in need of more robust approaches to accurately identify p53 transcriptional networks.Here, we describe an unbiased method to review 135 reported p53 target genes and at the same time identify new p53 targets on a genome-wide scale. We utilized wild-type (WT) p53 together with 16 allelic replicates comprising all reported non-synonymous single-nucleotide polymorphism (SNP) variants of p53. As the transcriptional functions of these 16 SNPs have not been well studied, we first developed a customized luciferase assay with promoter reporter vectors encompassing all combinatorial forms of the dinucleotide ...

A Comprehensive and High-Resolution Genome-wide Response of p53 to Stress

“…Thousands of putative p53-bound locations have been reported across the human genome (Botcheva et al, 2011; Cawley et al, 2004; Menendez et al, 2013; Nikulenkov et al, 2012; Schlereth et al, 2013; Smeenk et al, 2011; Smeenk et al, 2008; Wang et al, 2014; Wei et al, 2006; Yu et al, 1999; Zhao et al, 2000). However, confidence in such locations is limited by assay sensitivity (signal:noise) that tends to detect the most highly occupied regions, and assay resolution having positional uncertainty of several hundred base-pair rather than pin-pointing its exact location.…”

Identification of a core TP53 transcriptional program with highly distributed tumor suppressive activity

“…These observations triggered a flurry of activity to identify new TP53 target genes involved in tumor suppression. Several studies employed a combination of chromatin occupancy assays and steady-state RNA expression measurements to generate lists of genes that are bound by TP53 within arbitrary distances from their promoters and that show changes in mRNA expression at various times after TP53 activation (Wei et al 2006;Li et al 2012a;Nikulenkov et al 2012;Kenzelmann Broz et al 2013;Menendez et al 2013;Schlereth et al 2013;Wang et al 2014). However, independent meta-analyses revealed very little overlap among the catalogs of TP53 target genes obtained by these different research teams (Allen et al 2014;Verfaillie et al 2016;Fischer 2017).…”

Expanding the reach of the p53 tumor suppressor network

“…How p53 controls such essential processes to suppress tumorigenesis is primarily based on its ability to transcriptionally regulate an ever-expanding series of downstream target genes. The study by Wang et al 2 has taken an integrative genomic approach using chromatin immunoprecipitation-coupled sequencing (ChIP-seq) and transcriptome analyses to identify an extensive panel of new target genes that may mediate established p53 activities and less well-characterized p53 functions involved in cell signaling, metabolism, motility and immunity. These findings open new opportunities for advancing our understanding of how the p53 network protects against oncogenesis.…”

“…The results of this study have been compiled at the IARC TP53 Database and serve as a good first check for whether a mutation identified in a tumor is functional or not. The present study by Wang et al 2 took a similar, but more physiological approach in mammalian cells. Wild-type p53 (WTp53) and 16 naturally occurring variants resulting from non-synonymous SNPs were evaluated for transactivation of the p53 response element (RE) and all combinations of the CWWG core motif, as well as several established promoter reporters (e.g., p21 Cip1 , PLK3 and RNF144B).…”

“…It is well known that p53 controls cell cycle, apoptosis, angiogenesis and senescence (e.g., orbits closer to the master regulator TP53). The current work by Wang et al 2 identifies not only an extensive panel of new p53 target genes involved in these processes, but also less-wellunderstood TP53 activities impacting fertility, cell motility, immunity and human diseases, such as diabetes and rheumatoid arthritis (orbits further and further away)…”