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

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

Identification of genes involved in reprogramming-induced senescence

Senescence is a state of irreversible growth arrest first described in human fibroblasts when they undergo serial passage in vitro. Senescence can also occur in response to different stresses, such as replicative exhaustion, DNA damage or overexpression of oncogenes. The growth arrest associated with senescence is implemented by the activation of the p53/p21Cip1 and p16INK4a/Rb pathways. The expression in somatic cells of a combination of pluripotency-associated transcription factors, Oct4, Sox2, Klf4 and cMyc (OSKM) has been shown to generate embryonic stem cell-like cells termed induced pluripotent stem cells (iPSCs). iPSCs have brought new expectations to the study of stem cell biology, drugs development and regenerative medicine. However, the overall low efficiency of the reprogramming and its slow kinetics suggest that there might be barriers limiting the derivation of iPSCs. The expression of the reprogramming factors in normal somatic cells has been shown to activate the p53 and p16INK4a pathways, triggering a senescence response that limits the cells available to be reprogrammed, in what has been called reprogramming induced-senescence (RIS). Our aim is to identify modulators of RIS. We hypothesized that this could help not just to improve the efficiency of generation of iPSCs but could also help to clarify the link between tumour suppressor pathways, pluripotency and de-differentiation. To this end, we carried out a screen to identify shRNAs whose expression bypass the growth arrest imposed by the four reprogramming factors in IMR90 human primary fibroblasts. We screened a genome-wide shRNA library and took advantage of next generation sequencing (NGS) to detect the relative enrichment of shRNAs. We shortlisted 68 genes to validate their ability to bypass the OSKM-induced arrest. Of those, 8 genes consistently rescued the growth arrest in independent experiments. We performed a preliminary characterisation of IFIH1 and IGSF4D. The long-term goal would be to expand the characterisation of these candidates, assess whether they might also have a role in oncogene and replicative induced senescence and investigate whether they affect the efficiency of reprogramming to iPSCs. In addition, conducting a secondary screen with the top candidates shortlisted from the primary screen could contribute to the identification of additional genes that affect reprogramming and senescence.Open Acces

MicroRNA Regulation of Cbx7 Mediates a Switch of Polycomb Orthologs during ESC Differentiation

The Polycomb Group (PcG) of chromatin modifiers regulates pluripotency and differentiation. Mammalian genomes encode multiple homologs of the Polycomb repressive complex 1 (PRC1) components, including five orthologs of the Drosophila Polycomb protein (Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8). We have identified Cbx7 as the primary Polycomb ortholog of PRC1 complexes in embryonic stem cells (ESCs). The expression of Cbx7 is downregulated during ESC differentiation, preceding the upregulation of Cbx2, Cbx4, and Cbx8, which are directly repressed by Cbx7. Ectopic expression of Cbx7 inhibits differentiation and X chromosome inactivation and enhances ESC self-renewal. Conversely, Cbx7 knockdown induces differentiation and derepresses lineage-specific markers. In a functional screen, we identified the miR-125 and miR-181 families as regulators of Cbx7 that are induced during ESC differentiation. Ectopic expression of these miRNAs accelerates ESC differentiation via regulation of Cbx7. These observations establish a critical role for Cbx7 and its regulatory miRNAs in determining pluripotency