Coupling shRNA screens with single-cell RNA-seq identifies a dual role for mTOR in reprogramming-induced senescence.
Coupling shRNA screens with single-cell RNA-seq identifies a dual role for mTOR in reprogramming-induced senescence.
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DOI：
10.1101/gad.297796.117
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发表时间：
2017-10-15
影响因子：
10.5
通讯作者：
Gil J
中科院分区：
文献类型：
--
作者：
Aarts M;Georgilis A;Beniazza M;Beolchi P;Banito A;Carroll T;Kulisic M;Kaemena DF;Dharmalingam G;Martin N;Reik W;Zuber J;Kaji K;Chandra T;Gil J
Aarts等人开发了一种创新方法，该方法将单细胞RNA测序与表达OCT 4、SOX 2、KLF 4和cMYC的原代人成纤维细胞中的shRNA筛选相结合，以研究所鉴定候选物的作用机制。这种方法揭示了衰老的调节是一种新的方式，雷帕霉素（mTOR）的机制靶点影响重编程。转录因子OCT 4、SOX 2、KLF 4和cMYC（OSKM）的表达将体细胞重编程为诱导多能干细胞（iPSC）。重编程是一个缓慢而低效的过程，这表明存在对抗细胞命运转换的保护机制。其中一个机制是衰老。为了鉴定重编程诱导衰老的调节剂，我们在表达OSKM的原代人成纤维细胞中进行了全基因组shRNA筛选。在筛选中，我们确定了OSKM诱导衰老的新介质，并验证了先前涉及的基因，如CDKN 1A。我们开发了一种创新的方法，将单细胞RNA测序（scRNA-seq）与shRNA筛选相结合，以研究所识别候选物的作用机制。我们的数据揭示了衰老的调节是一种新的方式，雷帕霉素（mTOR）的机制靶点影响重编程。一方面，mTOR抑制减弱了细胞周期蛋白依赖性激酶（CDK）抑制剂（CDKI）的诱导，包括p16 INK 4a、p21 CIP 1和p15 INK 4 b，从而阻止了OSKM诱导的衰老。另一方面，mTOR的抑制会减弱衰老相关分泌表型（SASP），而SASP本身有利于重编程。这些相反的作用有助于解释mTOR对重编程的复杂影响。总的来说，我们的研究强调了将功能筛选与scRNA-seq相结合的优势，以加速发现控制复杂表型的途径。
Aarts et al. developed an innovative approach that integrates single-cell RNA sequencing with a shRNA screen in primary human fibroblasts expressing OCT4, SOX2, KLF4, and cMYC to investigate the mechanism of action of the identified candidates. This approach unveiled regulation of senescence as a novel way by which mechanistic target of rapamycin (mTOR) influences reprogramming. Expression of the transcription factors OCT4, SOX2, KLF4, and cMYC (OSKM) reprograms somatic cells into induced pluripotent stem cells (iPSCs). Reprogramming is a slow and inefficient process, suggesting the presence of safeguarding mechanisms that counteract cell fate conversion. One such mechanism is senescence. To identify modulators of reprogramming-induced senescence, we performed a genome-wide shRNA screen in primary human fibroblasts expressing OSKM. In the screen, we identified novel mediators of OSKM-induced senescence and validated previously implicated genes such as CDKN1A. We developed an innovative approach that integrates single-cell RNA sequencing (scRNA-seq) with the shRNA screen to investigate the mechanism of action of the identified candidates. Our data unveiled regulation of senescence as a novel way by which mechanistic target of rapamycin (mTOR) influences reprogramming. On one hand, mTOR inhibition blunts the induction of cyclin-dependent kinase (CDK) inhibitors (CDKIs), including p16INK4a, p21CIP1, and p15INK4b, preventing OSKM-induced senescence. On the other hand, inhibition of mTOR blunts the senescence-associated secretory phenotype (SASP), which itself favors reprogramming. These contrasting actions contribute to explain the complex effect that mTOR has on reprogramming. Overall, our study highlights the advantage of combining functional screens with scRNA-seq to accelerate the discovery of pathways controlling complex phenotypes.
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影响因子：
78.5
作者：
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通讯作者：
Serrano, Manuel
影响因子：
64.5
作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
van Deursen JM
DOI：
10.1146/annurev-pathol-121808-102144
发表时间：
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期刊：
Annual review of pathology
影响因子：
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通讯作者：
Campisi J
影响因子：
64.5
作者：
Dixit, Atray;Pamas, Oren;Li, Biyu;Chen, Jenny;Fulco, Charles P.;Jerby-Amon, Livnat;Marjanovic, Nemanja D.;Dionne, Danielle;Burks, Tyler;Raychowdhury, Raktima;Adamson, Britt;Norman, Thomas M.;Lander, Eric S.;Weissman, Jonathan S.;Friedman, Nir;Regev, Aviv
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