Genome-wide screening in human embryonic stem cells identifies genes and pathways involved in the p53 pathway.

Abstract

Background: The tumor suppressor protein, p53, which is mutated in half of human tumors, plays a critical role in cellular responses to DNA damage and maintenance of genome stability. Therefore, increasing our understanding of the p53 pathway is essential for improving cancer treatment and diagnosis.

Methods: This study, which aimed to identify genes and pathways that mediate resistance to p53 upregulation, used genome-wide CRISPR-Cas9 loss-of-function screening done with Nutlin-3a, which inhibits p53-MDM2 interaction, resulting in p53 accumulation and apoptotic cell death. We used bioinformatics analysis for the identification of genes and pathways that are involved in the p53 pathway and cell survival assays to validate specific genes. In addition, we used RNA-seq to identify differentially expressed p53 target genes in gene knockout (KO) cell lines.

Results: Our screen revealed three significantly enriched pathways: The heparan sulfate glycosaminoglycan biosynthesis, diphthamide biosynthesis and Hippo pathway. Notably, TRIP12 was significantly enriched in our screen. We found that TRIP12 is required for the p53-dependent transcription of several pro-apoptotic genes.

Conclusion: Our study has identified two novel pathways that play a role in p53-mediated growth restriction. Moreover, we have highlighted the interaction between the Hippo and the p53 pathways. Interestingly, we have shown that TRIP12 plays an important function in the p53 pathway by selectively affecting its role as a transcription factor.