The authors performed a large-scale screen in which they transfected mouse ES cells maintained in a 2i culture medium — that is, a medium containing two small-molecule inhibitors known to repress exit from pluripotency — with siRNAs and then selected for resistance to differentiation upon 2i withdrawal. Several genes were implicated in the onset of differentiation; the authors focused on the gene encoding the tumour suppressor folliculin (FLCN), as it had not been previously linked to pluripotency, and because loss of Flcn causes early embryonic lethality. The authors reasoned that embryonic lethality could arise because cells fail to initiate differentiation during early development. In support of this, they observed that Flcn-knockdown cells were resistant to differentiation even in a medium supplemented with differentiation-promoting factors. Notably, another hit in the screen was tuberous sclerosis complex 2 (TCS2), a negative regulator of mammalian target of rapamycin (mTOR), which had previously been linked to FLCN.
FLCN regulates the nuclear translocation of the bHLH transcription factor TFE3 in human kidney cells and mouse embryonic fibroblasts, so Smith and colleagues investigated whether TFE3 might also be a target of FLCN in mouse ES cells. TFE3 was present in both the nucleus and the cytoplasm of ES cells maintained in 2i but was rapidly restricted to the cytoplasm when differentiation was induced by withdrawal of the 2i culture medium. However, when Flcn was knocked down, TFE3 remained in the nucleus. Similarly, the absence of two FLCN-interacting proteins, FNIP1 and FNIP2, also promoted nuclear localization of TFE3, indicating that FLCN protein complexes drive cytoplasmic relocalization of TFE3 during differentiation. Moreover, TFE3 localized to the nucleus upon knockdown of TSC2, consistent with negative regulation of FLCN by mTOR.
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