Department of Biochemistry, University of Washington, Seattle, WA, 98195, USA.
Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, 98109, USA.
Nat Commun. 2019 Feb 7;10(1):632. doi: 10.1038/s41467-018-08020-0.
To reveal how cells exit human pluripotency, we designed a CRISPR-Cas9 screen exploiting the metabolic and epigenetic differences between naïve and primed pluripotent cells. We identify the tumor suppressor, Folliculin(FLCN) as a critical gene required for the exit from human pluripotency. Here we show that FLCN Knock-out (KO) hESCs maintain the naïve pluripotent state but cannot exit the state since the critical transcription factor TFE3 remains active in the nucleus. TFE3 targets up-regulated in FLCN KO exit assay are members of Wnt pathway and ESRRB. Treatment of FLCN KO hESC with a Wnt inhibitor, but not ESRRB/FLCN double mutant, rescues the cells, allowing the exit from the naïve state. Using co-immunoprecipitation and mass spectrometry analysis we identify unique FLCN binding partners. The interactions of FLCN with components of the mTOR pathway (mTORC1 and mTORC2) reveal a mechanism of FLCN function during exit from naïve pluripotency.
为了揭示细胞如何退出人类多能性，我们设计了一种 CRISPR-Cas9 筛选方法，利用原始和初始多能细胞之间的代谢和表观遗传差异。我们确定肿瘤抑制因子 Folliculin(FLCN)是退出人类多能性所必需的关键基因。在这里，我们表明 FLCN 敲除 (KO) hESCs 保持原始多能状态，但不能退出状态，因为关键转录因子 TFE3 仍然在核内活跃。在 FLCN KO 退出测定中上调的 TFE3 靶标是 Wnt 途径和 ESRRB 的成员。用 Wnt 抑制剂处理 FLCN KO hESC，但不是 ESRRB/FLCN 双突变体，可挽救细胞，使其退出原始状态。通过共免疫沉淀和质谱分析，我们鉴定了独特的 FLCN 结合伙伴。FLCN 与 mTOR 途径（mTORC1 和 mTORC2）组件的相互作用揭示了 FLCN 在退出原始多能性过程中的功能机制。
