Genome-wide CRISPR screen identifies noncanonical NF-κB signaling as a regulator of density-dependent proliferation.
Genome-wide CRISPR screen identifies noncanonical NF-κB signaling as a regulator of density-dependent proliferation.
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DOI：
10.7554/elife.63603
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发表时间：
2020-11-13
期刊：
影响因子：
7.7
通讯作者：
Macara IG
中科院分区：
文献类型：
--
作者：
Fomicheva M;Macara IG
上皮细胞具有维持正常组织形态发生和稳态的适当细胞密度的内在机制。这些机制的缺陷可能导致增生和癌症的发生。为了鉴定调节小鼠乳腺上皮细胞密度依赖性增殖的基因，我们开发了一种基于荧光泛素化细胞周期指示剂的荧光激活细胞分选试验，该细胞周期指示剂用不同的荧光团标记细胞周期的不同阶段。使用这种强大的检测方法，我们进行了全基因组CRISPR/Cas9敲除筛选，选择在低密度下正常增殖但在高密度下继续分裂的细胞。出乎意料的是，其中一个最受欢迎的是Traf 3，它是NF-κB信号的负调节因子，以前从未与密度依赖性增殖有关。我们证明Traf 3的缺失特异性激活非经典NF-κB信号。这反过来又触发了先天免疫应答，并通过阻断进入静止状态而独立于已知的密度依赖性增殖机制（包括雅普/TAZ信号传导和细胞周期蛋白依赖性激酶抑制剂）驱动细胞分裂。
Epithelial cells possess intrinsic mechanisms to maintain an appropriate cell density for normal tissue morphogenesis and homeostasis. Defects in such mechanisms likely contribute to hyperplasia and cancer initiation. To identify genes that regulate the density-dependent proliferation of murine mammary epithelial cells, we developed a fluorescence-activated cell sorting assay based on fluorescence ubiquitination cell cycle indicator, which marks different stages of the cell cycle with distinct fluorophores. Using this powerful assay, we performed a genome-wide CRISPR/Cas9 knockout screen, selecting for cells that proliferate normally at low density but continue to divide at high density. Unexpectedly, one top hit was Traf3, a negative regulator of NF-κB signaling that has never previously been linked to density-dependent proliferation. We demonstrate that loss of Traf3 specifically activates noncanonical NF-κB signaling. This in turn triggers an innate immune response and drives cell division independently of known density-dependent proliferation mechanisms, including YAP/TAZ signaling and cyclin-dependent kinase inhibitors, by blocking entry into quiescence.