Folliculin regulates mTORC1/2 and WNT pathways in early human pluripotency

It is important to understand how cells can maintain and exit human pluripotency. We exploited the metabolic and epigenetic differences between naïve and primed pluripotent cells to design a CRISPR-Cas9 screen for identifying genes that promote the exit from naïve pluripotency. Among the known and novel regulators of this early step of human development, we identified the tumor suppressor folliculin (FLCN). Flcn is important for implantation of the mouse embryo into the uterus and has been shown to regulate the exit of pluripotency in mouse through activation of Esrrb. However, the function of FLCN is unknown in human embryonic stem cells (hESC). Knock-out (KO) of FLCN revealed that it is not essential to maintain the naïve pluripotent state but is required for the exit of that state, in part by controlling the localization of the transcription factor TFE3. While mainly found in the cytoplasm of cells exiting the naïve state, FLCN KO resulted in TFE3 nuclear localization. TFE3 targets up-regulated in FLCN KO exit assay were members of Wnt pathway and ESRRB. Treatment of FLCN KO hESC with a Wnt inhibitor rescued the phenotype and allowed the cells to exit the naïve state. In contrast, the lack of rescue in ESRRB/FLCN double KO lines suggested that ESRRB was not responsible for the FLCN mutant phenotype. Using co-immunoprecipitation and mass spectrometry analysis we identified unique FLCN binding partners, in addition to known FLCN interactors, at various stages of pluripotency. The interactions of FLCN with components of the mTOR pathway (mTORC1 and mTORC2) revealed a mechanism of FLCN function during exit from naïve pluripotency. RNA-seq of 2 biological replicates in 6 conditions: Elf 2iLIF, Elf 2iLIF FLCN KO, Elf 2iLIF FLCN AAVS1-FLCN-GFP no Dox, Elf 2iLIF FLCN KO AAVS1-FLCN-GFP with Dox, Elf FLCN KO in TeSR 7 day, and Elf in TeSR 7 day; RNA-seq of Elf FLCN KO treated with IWP2 and DMOS; ChIP-seq of H3K27me3 for NNMT CRISPR mutant 6.2.4