A pooled single-cell genetic screen identifies regulatory checkpoints in the continuum of the epithelial-to-mesenchymal transition.,Nature Genetics

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A pooled single-cell genetic screen identifies regulatory checkpoints in the continuum of the epithelial-to-mesenchymal transition.
Nature Genetics ( IF 31.7 ) Pub Date : 2019-09-02 , DOI: 10.1038/s41588-019-0489-5
José L McFaline-Figueroa ₁ , Andrew J Hill ₁ , Xiaojie Qiu _{1,

2} , Dana Jackson ₁ , Jay Shendure _{1,

3,

4,

5} , Cole Trapnell _{1,

3,

5}

Affiliation

Integrating single-cell trajectory analysis with pooled genetic screening could reveal the genetic architecture that guides cellular decisions in development and disease. We applied this paradigm to probe the genetic circuitry that controls epithelial-to-mesenchymal transition (EMT). We used single-cell RNA sequencing to profile epithelial cells undergoing a spontaneous spatially determined EMT in the presence or absence of transforming growth factor-β. Pseudospatial trajectory analysis identified continuous waves of gene regulation as opposed to discrete 'partial' stages of EMT. KRAS was connected to the exit from the epithelial state and the acquisition of a fully mesenchymal phenotype. A pooled single-cell CRISPR-Cas9 screen identified EMT-associated receptors and transcription factors, including regulators of KRAS, whose loss impeded progress along the EMT. Inhibiting the KRAS effector MEK and its upstream activators EGFR and MET demonstrates that interruption of key signaling events reveals regulatory 'checkpoints' in the EMT continuum that mimic discrete stages, and reconciles opposing views of the program that controls EMT.

中文翻译：

汇集的单细胞遗传筛选可识别上皮-间质转化连续体中的调节检查点。

将单细胞轨迹分析与汇总遗传筛选相结合可以揭示指导发育和疾病中细胞决策的遗传结构。我们应用这种范例来探测控制上皮间质转化（EMT）的遗传回路。我们使用单细胞 RNA 测序来分析在存在或不存在转化生长因子-β 的情况下经历自发空间确定的 EMT 的上皮细胞。伪空间轨迹分析确定了基因调控的连续波，而不是EMT的离散“部分”阶段。 KRAS 与上皮状态的退出和完全间充质表型的获得有关。一项汇集的单细胞 CRISPR-Cas9 筛选鉴定了 EMT 相关受体和转录因子，包括 KRAS 调节因子，KRAS 的缺失阻碍了 EMT 的进展。抑制 KRAS 效应器 MEK 及其上游激活剂 EGFR 和 MET 表明，关键信号传导事件的中断揭示了 EMT 连续体中模仿离散阶段的监管“检查点”，并调和了控制 EMT 程序的相反观点。

更新日期：2019-09-03

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