The role of GCN2 in human induced pluripotent stem cell maintenance, differentiation and endothelial cell function

PROJECT SUMMARY The serine/threonine protein kinase GCN2 (General control nonderepressible 2) is an amino acid sensor that coordinates mRNA translation (also known as protein synthesis) in response to amino acid availability through phosphorylation of eIF2. Despite extensive studies of the GCN2-eIF2 pathway in the context of cellular stress, the importance of this pathway in early embryogenesis and differentiation has not been established. The focus of this research is to uncover the role of GCN2-dependent translational control in mediating the effect of amino acid deprivation stress on human induced pluripotent stem cells (hiPSCs), hiPSC to endothelial cell (EC) differentiation and hiPSC-EC function. Our preliminary data show that the expression of several key pluripotent factors is reduced in CRISPR-engineered GCN2 knock out (GCN2 KO) hiPSCs compared to isogenic control hiPSCs. We also observed that hiPSCs undergo a profound increase in p-eIF2 at an early stage of differentiation toward endothelial cells. Additionally, two downstream targets of p-eIF2, ATF4 and GADD34, were induced on day 3 of differentiation. Given these findings, I hypothesize that GCN2 regulates hiPSCs, hiPSC to EC differentiation and hiPSC-EC function through phosphorylation of eIF2. The proposed experiments will fully assess the self-renewal and EC differentiation potential of GCN2 KO hiPSCs using cellular and biochemical strategies. Importantly, ribosome footprinting, a high-throughput translatome technique, will be used to identify downstream translational targets of the GCN2-eIF2α pathway that regulate hiPSC-EC function in response to amino acid stress. The identified GCN2 targets will be functionally assessed for their role in endothelial cell differentiation and function. The results of this research will provide insight into new candidates for treating endothelial-related diseases.