期刊
GENES & DEVELOPMENT
卷 31, 期 20, 页码 2085-2098出版社
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1101/gad.297796.117
关键词
SASP; senescence; iPSCs; single-cell RNA-seq; shRNA screens
资金
- Medical Research Council (MRC) Centennary Award
- Biotechnology and Biological Sciences Research Council East of Scotland BioScience Doctoral Training Partnership
- MRC-British Heart Foundation Cardiovascular Stem Cell Research Strategic Development Grant [G0901467]
- Biotechnology and Biological Sciences Research Council [BBS/E/B/000C0425, 1373934] Funding Source: researchfish
- Medical Research Council [MR/K017047/1, MC_U120085810, G0901467, MR/N008715/1] Funding Source: researchfish
- BBSRC [BBS/E/B/000C0425] Funding Source: UKRI
- MRC [MR/K017047/1, MR/N008715/1, MC_U120085810, G0901467] Funding Source: UKRI
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 p16(INK4a), p21(CIP1), and p15(INK4b), 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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