Abstract
p53 is the most frequently mutated tumor suppressor gene in human neoplasia and encodes a transcriptional coactivator. Identification of p53 target genes is therefore key to understanding the role of p53 in tumorigenesis. To identify novel p53 target genes, we first used a comparative genomics approach to identify p53 binding sequences conserved in the human and mouse genome. We hypothesized that potential p53 binding sequences that are conserved are more likely to be functional. Using stringent filtering procedures, 32 genes were newly identified as putative p53 targets, and their responsiveness to p53 in human cancer cells was confirmed by reverse transcription-PCR and real-time PCR. Among them, we focused on the vitamin D receptor (VDR) gene because vitamin D3 has recently been used for chemoprevention of human tumors. VDR is induced by p53 as well as several other p53 family members, and analysis of chromatin immunoprecipitation showed that p53 protein binds to conserved intronic sequences of the VDR gene in vivo. Introduction of VDR into cells resulted in induction of several genes known to be p53 targets and suppression of colorectal cancer cell growth. In addition, p53 induced VDR target genes in a vitamin D3-dependent manner. Our in silico approach is a powerful method for identification of functional p53 binding sites and p53 target genes that are conserved among humans and other organisms and for further understanding the function of p53 in tumorigenesis.
Highlights
The transcriptional coactivator p53 binds to DNA in a sequence-specific manner and induces transcription of a variety of genes involved in cell cycle regulation, apoptosis, inhibition of angiogenesis, and DNA repair [1, 2]
We found that comparison of the p53 binding sequences between human and mouse is highly useful for identifying functional p53 response elements (p53RE)
In several known p53 target genes, the sequences surrounding the p53 binding sites are highly conserved among different species, suggesting that other unknown elements are necessary for p53 protein to bind to p53REs and function as a transcriptional coactivator
Summary
The transcriptional coactivator p53 binds to DNA in a sequence-specific manner and induces transcription of a variety of genes involved in cell cycle regulation, apoptosis, inhibition of angiogenesis, and DNA repair [1, 2]. Several approaches have been used to successfully identify genes induced by p53 including differential display, representational difference analysis, cDNA microarrays, and serial analysis of gene expression [3,4,5,6]. A complete data set of functional p53REs in the human genome is not yet available. Using a computational approach, coding regions, regulatory elements, and noncoding RNA conserved in different vertebrates have been identified [10]. We hypothesized that both known and novel functional p53REs important for p53 function are likely to exhibit greater sequence conservation than nonfunctional sequences [11]. A comparative genomic analysis of putative p53 binding sequences may be a way to identify the downstream mediators of p53 function
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