Lysosomal signaling licenses embryonic stem cell differentiation via inactivation of Tfe3

Self-renewal and differentiation of pluripotent murine embryonic stem cells (ESCs) is regulated by extrinsic signaling pathways. It is less clear whether cellular metabolism instructs developmental progression. In an unbiased genome-wide CRISPR/Cas9 screen, we identified components of a conserved amino acid-sensing pathway as critical drivers of ESC differentiation. Functional analysis revealed that lysosome activity, the Ragulator protein complex, and the tumor-suppressor protein Folliculin enable the Rag GTPases C and D to bind and seclude the bHLH transcription factor Tfe3 in the cytoplasm. In contrast, ectopic nuclear Tfe3 represses specific developmental and metabolic transcriptional pro- grams that are associated with peri-implantation development. We show differentiation-specific and non-canonical regulation of Rag GTPase in ESCs and, importantly, identify point mutations in a Tfe3 domain required for cytoplasmic inactivation as potentially causal for a human developmental disorder. Our work reveals an instructive and biomedically relevant role of metabolic signaling in licensing embryonic cell fate transitions. Mouse embryonic stem and differentiated cell mRNA profiles of wild type, Flcn KO, LT1 KO and ectopic nuclear Tfe3 cell lines were generated by deep sequencing, in duplicates, using Illumina HiSeq2500.