Genome-wide screening in human embryonic stem cells to identify genes involved in the TP53 pathway
2Department of Genetics, The Hebrew University of Jerusalem, Israel
p53 is a tumor suppressor protein involved in the cellular response to a diverse set of cellular stresses. It regulates many cell responses such as apoptosis and cell cycle arrest and it is frequently mutated in human tumors. We treated genome-wide CRISPR-Cas9 loss-of-function library cells with Nutlin-3a, which leads to the accumulation of the p53 protein in cells and therefore it is toxic. Out of 736 genes, which were statistically significant in the screen, 177 genes were enriched, meaning that their mutation assisted in overcoming p53-induced apoptosis. As expected, p53 has the highest score and is the most highly significant gene in the screen. Moreover, several genes involved in canonical p53 pathways, such as PMAIP1, BAX and PTEN, are significantly enriched. In agreement, a gene set enrichment analysis conducted using our screen results shows that gene sets involving p53, DNA damage, and apoptotic signaling are highly enriched. Moreover, genes involved in the Hippo KEGG pathway are statistically significant in our screen. Additionally, 27 genes expressing ubiquitin ligases or deubiquitinases are statistically significant, 11 of which are enriched. Thus, the deubiquitinase - USP28 gene and the E3 Ubiquitin ligase - TRIP12 were chosen for further validation. CRISPR-Cas9 was used to produce knock-out cell lines for each of these genes, and survival assays showed reduced sensitivity to Nutlin-3a, thereby verifying the results obtained in the screen. This study sheds light on new insights regarding p53-mediated apoptosis and may prove useful in finding new therapeutic targets in cancer treatments.