C3G Regulates STAT3, ERK, Adhesion Signaling, and Is Essential for Differentiation of Embryonic Stem Cells.

C3G (RAPGEF1), engaged in multiple signaling pathways, is essential for the early development of the mouse. In this study, we have examined its role in mouse embryonic stem cell self-renewal and differentiation. C3G null cells generated by CRISPR mediated knock-in of a targeting vector exhibited enhanced clonogenicity and long-term self-renewal. They did not differentiate in response to LIF withdrawal when compared to the wild type ES cells and were defective for lineage commitment upon teratoma formation in vivo. Gene expression analysis of C3G KO cells showed misregulated expression of a large number of genes compared with WT cells. They express higher levels of self-renewal factors like KLF4 and ESRRB and show high STAT3 activity, and very low ERK activity compared to WT cells. Reintroduction of C3G expression in a KO line partially reverted expression of ESRRB, and KLF4, and ERK activity similar to that seen in WT cells. The expression of self-renewal factors was persistent for a longer time, and induction of lineage-specific markers was not seen when C3G KO cells were induced to form embryoid bodies. C3G KO cells showed poor adhesion and significantly reduced levels of pFAK, pPaxillin, and Integrin-β1, in addition to downregulation of the cluster of genes involved in cell adhesion, compared to WT cells. Our results show that C3G is essential for the regulation of STAT3, ERK, and adhesion signaling, to maintain pluripotency of mouse embryonic stem cells and enable their lineage commitment for differentiation.