NEW YORK (GenomeWeb) – Researchers at the Hebrew University in Jerusalem, Israel have built a reference library of essential and growth-restriction genes in haploid human pluripotent stem cells (hPSCs) that they believe will reveal key aspects of cellular essentiality.
In a study published yesterday in Nature, the researchers used a recently discovered group of haploid hPSCs to generate a genome-wide CRISPR-Cas9-based loss-of-function screen on karyotypically normal haploid hPSCs in order to define genes essential for normal growth and survival, as well as genes that restrict cell growth.
Examining the cellular localization of cell-essential genes, the team found an intrinsic bias of essentiality in different components of the cell, with nuclear and mitochondrial compartments showing high proportions of essential genes.
The researchers, therefore, believe that this bias in cellular localization may suggest different roles for essential genes in cell growth regulation.
The researchers noticed that genes responsible for autosomal recessive (AR) disorders that exhibit a growth-retardation phenotype were significantly enriched. Focusing on Fanconi anemia, they identified 14 genes associated with mutations causing the disease that were essential in human embryonic stem cells (hESCs). They, therefore, believe that the phenotype of growth retardation linked to AR disorders may begin at very early stages of embryogenesis. In addition, ESCs may provide a more suitable model to study the phenotypes of developmental human disorders.
The team also uncovered two opposing roles for tumor suppressor genes, namely the essential and growth-restricting genes.
Functional categorization of the hESC-specific essentialome revealed that hPSC-enriched essential genes mainly encoded transcription factors and proteins linked to DNA repair and the cell cycle, demonstrating that a quarter of the nuclear factors are crucial for normal growth.
“When combined with … gene expression analyses, our essential gene screen in hESCs reveals that the majority of these pluripotency-associated TFs are dispensable for the growth and survival of hESCs,” the authors noted in the study.
The team identified seven essential transcription factors with enriched expression in hESCs, including SALL4, POU5F1, PRDM14, NANOG, FOXB1, MYBL2 and MYCN.
The investigators also identified growth-restricting genes, highlighting the role of the P53-mTOR pathway. They then analyzed the group of growth-restricting genes in hESCs, identifying TP53 and PTEN as the highest scoring tumor suppressor genes. Among 14 distinct P53 target pathways, the team found an enrichment of highest scoring growth-restricting genes of hESC in the branch inhibiting the IGF1/mTOR pathway.
Attempting to validate mTOR’s direct involvement in hESC growth involvement, the team treated hESC with rapamycin, a mTOR inhibitor, in the presence and absence of IGF1. Blocking mTOR caused dramatic growth inhibition of hESCs, while adding IGF1 failed to save the inhibition, indicating that IGF1 acts upstream of mTOR. The results indicate that the selective advantage of TP53 mutations might be overridden by providing chemical mTOR-activators to prevent overgrowth of TP53 mutations.
Overall, the team believes it has constructed an atlas of crucial and growth-restricting genes in hPSCs, highlighting key elements of cellular essentiality.
The researchers believe that future studies may focus on whether MYBL2 and MYCN can replace c-MYC in reprogramming factor cocktails to yield more authentic induced PSCs. In addition, the authors noted that the work “lays the ground for future studies investigating a broad range of genes essential to human pluripotency, growth regulation in hPSCs, and disease modeling using hPSCs.”