Coupling shRNA screens with single-cell RNA-seq identifies a dual role for mTOR in reprogramming-induced senescence

Marieke Aarts, Athena Georgilis, Meryam Beniazza, Patrizia Beolchi, Ana Banito, Thomas Carroll, Marizela Kulisic, Daniel F Kaemena, Gopuraja Dharmalingam, Nadine Martin, Wolf Reik, Johannes Zuber, Keisuke Kaji, Tamir Chandra, Jesús Gil

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)2085-2098
Number of pages14
JournalGenes and Development
Volume31
Issue number20
DOIs
Publication statusPublished - 14 Nov 2017

Keywords

  • Journal Article

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