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Updated: May 5, 2026
Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells
Published on: August 23, 2014
Genome-wide screening in human embryonic stem cells identifies genes and pathways involved in the p53 pathway
Amir Haddad1,2, Tamar Golan-Lev1,2, Nissim Benvenisty1,2
1Department of Genetics, The Alexander Silberman Institute of Life Sciences, the Hebrew University of Jerusalem, 91904, Jerusalem, Israel.
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.
Insights
This study identified novel pathways, including the Hippo pathway and TRIP12, that regulate the tumor suppressor p53. Understanding these mechanisms is key for developing new cancer treatments.
Area of Science:
- Cancer Biology
- Molecular Oncology
- Genomics
Background:
- The p53 tumor suppressor is crucial for DNA damage response and genome stability.
- Mutations in p53 occur in approximately half of human cancers.
- Understanding the p53 pathway is vital for advancing cancer therapy and diagnostics.
Purpose of the Study:
- To identify genes and pathways that confer resistance to p53 upregulation.
- To investigate novel regulators of the p53 pathway.
Main Methods:
- Genome-wide CRISPR-Cas9 loss-of-function screening using Nutlin-3a.
- Bioinformatics analysis to identify enriched pathways.
- Cell survival assays and RNA-sequencing (RNA-seq) for validation.
Main Results:
- Identified heparan sulfate glycosaminoglycan biosynthesis, diphthamide biosynthesis, and the Hippo pathway as significantly enriched.
- TRIP12 was highlighted as a key gene.
- TRIP12 is essential for the p53-dependent transcription of pro-apoptotic genes.
Conclusions:
- Discovered two novel pathways involved in p53-mediated growth restriction.
- Elucidated an interaction between the Hippo and p53 pathways.
- Demonstrated TRIP12's significant role in modulating p53 transcriptional activity.
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