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Genes Dev. 2017 Oct 15;31(20):2085-2098. doi: 10.1101/gad.297796.117. Epub 2017 Nov 14.

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

Author information

1
Medical Research Council (MRC) London Institute of Medical Sciences (LMS), London W12 0NN, United Kingdom.
2
Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, London W12 0NN, United Kingdom.
3
MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh EH16 4UU, United Kingdom.
4
WaferGen Biosystems, L-1526 Luxembourg, Luxembourg.
5
Epigenetics Programme, The Babraham Institute, Cambridge CB22 3AT, United Kingdom.
6
Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna 1030, Austria.
7
MRC Human Genetics Unit, University of Edinburgh, Edinburgh EH4 2XU, United Kingdom.

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.

KEYWORDS:

SASP; iPSCs; senescence; shRNA screens; single-cell RNA-seq

PMID:
29138277
PMCID:
PMC5733499
DOI:
10.1101/gad.297796.117
[Indexed for MEDLINE]
Free PMC Article

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