Scientists have known for decades that the protein p53 is critical for safeguarding against genetic damage and the onset of cancer. Many tumors exhibit mutations that undermine p53’s ability to regulate the genes responsible for activating DNA repair and halting cell division. While p53 also appears to be an important controller of other cellular processes, identifying the additional genes that p53 targets remains challenging.
Now, Ee Chee Ren and colleagues at the A*STAR Singapore Immunology Network have developed an assay that could accelerate identification of the genes that p53 regulates1. p53 binds target genes directly via a family of closely related DNA sequences known as p53 response elements (p53REs). These are short DNA sequences found in a gene’s promoter region that bind specific proteins that regulate gene transcription (see image).
Prior searches for p53 target genes were informed by only limited knowledge of the interaction between p53 and DNA; these studies compared changes in gene expression in the presence or absence of normal, or ‘wild-type’, p53, but without validating the genes that appeared to be affected or their physical association with p53. However, Ren and colleagues were able to use p53RE-defining sequence elements that they had previously identified2 to design a bioluminescence-based assay capable of accurately determining whether genes are subject to p53-mediated regulation.
Ren’s team assembled a panel of 16 p53 proteins with slight structural differences by combing a database of DNA sequences from cancer patients and identifying sequence changes that affected the amino-acid makeup of p53. This allowed them to distinguish variants that exhibited similar p53RE binding and gene activation to wild-type p53 from those that were nonfunctional.
Importantly, they found that similarly classified p53 variants exhibited the same DNA binding and gene activation profiles as each other, enabling the effects of p53 to be determined from multiple, parallel experiments. “We developed an efficient screening approach that can examine putative p53 target genes based on the signature obtained with these 16 variants, instead of relying on only one test with just the wild-type p53,” explains Ren.
The team’s assay proved far more efficient at accurately identifying known p53 target genes than standard experimental techniques, confirming its effectiveness as a discovery tool. The researchers demonstrated this by using their panel to identify nearly 600 new target genes of p53 that affect diverse cellular functions. “Our results show that the reach of p53 is far wider than previously believed, and that it participates in many normal physiological processes of the cell,” says Ren.
The A*STAR-affiliated researchers contributing to this research are from the Singapore Immunology Network
- Wang, B., Niu, D., Lam, T. H., Xiao, Z. & Ren, E. C. Mapping the p53 transcriptome universe using p53 natural polymorphs. Cell Death & Differentiation 21, 521–532 (2014). | article
- Wang, B., Xiao, Z. & Ren, E. C. Redefining the p53 response element. Proceedings of the National Academy of Sciences USA 106, 14373–14378 (2009). | article
Reproduced, with permission, from Ref. 1 © 2014 B. Wang et al.