Genome-scale screens identify JNK/JUN signaling as a barrier for pluripotency exit and endoderm differentiation

Human embryonic and induced pluripotent stem cells (hESCs/hiPSCs) hold great promise for cell-based therapies and drug discovery. However, homogeneous differentiation remains a major challenge, highlighting the need for understanding developmental mechanisms. We performed genome-scale CRISPR screens to uncover regulators of definitive endoderm (DE) differentiation, which unexpectedly uncovered five JNK/JUN family genes as key barriers of DE differentiation. The JNK/JUN pathway does not act through directly inhibiting the DE enhancers. Instead JUN co-occupies ESC enhancers with OCT4, NANOG and SMAD2/3, and specifically inhibits the exit from the pluripotent state by impeding the decommissioning of ESC enhancers and inhibiting the reconfiguration of SMAD2/3 chromatin binding from ESC to DE enhancers. Therefore, the JNK/JUN pathway safeguards pluripotency from precocious DE differentiation. Direct pharmacological inhibition of JNK significantly improves the efficiencies of generating DE and DE-derived pancreatic and lung progenitor cells, highlighting the potential of harnessing the knowledge from developmental studies for regenerative medicine. Overall design: We performed genome-wide CRISPR screen using GeCKO and Brunello libraries in hESC to identify regulators of definitive endoderm differentiation. We identified JNK signaling as an inhibitory pathway for definitive endoderm differentiation. We performed RNA-seq, scRNA-seq, ATAC-seq and ChIP-seq to study the role of JNK pathway in DE differentiation.