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Project description:Ectopic expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM) can reprogram somatic cells into induced pluripotent stem cells (iPSCs). These iPSCs are highly similar to embryonic stem cells and can be used for regenerative medicine, drug screening and disease modelling. Despite recent advances, reprogramming is a slow and inefficient process. This suggests that there are several safeguarding mechanisms to counteract cell fate conversion. Cellular senescence is one of these barriers, which is mediated through activation of the tumour suppressors p53/p21CIP1, p15INK4b and p16INK4a. In this study, we have screened for shRNAs blunting reprogramming-induced senescence. We integrated single-cell RNA sequencing (scRNA-Seq) with shRNA screening to investigate the mechanism of action of the identified candidates.
2018-02-09 | GSE95020 | GEO
Project description: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.
| S-EPMC5733499 | biostudies-literature
Project description:Coupling shRNA screens with single-cell RNA-Seq identifies mechanisms regulating senescence during reprogramming
| PRJNA375744 | ENA
Project description:BackgroundHigh throughput screens with RNA interference technology enable loss-of-function analyses of gene activities in mammalian cells. While the construction of genome-scale shRNA libraries has been successful, results of large-scale screening of those libraries can be difficult to analyze because of the relatively high noise levels and the fact that not all shRNAs in a library are equally effective in silencing gene expression.ResultsWe have screened a library consisting of 43,828 shRNAs directed against 8,500 human genes for functions that are necessary in cell detachment induced by a constitutively activated c-Abl tyrosine kinase. To deal with the issues of noise and uncertainty of knockdown efficiencies, we employed an analytical strategy that combines quantitative data analysis with biological knowledge, i.e. Gene Ontology and pathway information, to increase the power of the RNAi screening technique. Using this strategy we found 16 candidate genes to be involved in Abl-induced disruption of cell adhesion, and verified that the knockdown of IL6ST is associated with enhanced cell attachment.ConclusionOur results suggest that the power of genome-wide quantitative shRNA screens can be significantly increased when analyzed using a systems biology-based approach to identify functional gene networks.
| S-EPMC2441606 | biostudies-literature
Project description:The generation of induced pluripotent stem cells (iPSCs) from differentiated cells following forced expression of OCT4, KLF4, SOX2, and C-MYC (OKSM) is slow and inefficient, suggesting that transcription factors have to overcome somatic barriers that resist cell fate change. Here, we performed an unbiased serial shRNA enrichment screen to identify potent repressors of somatic cell reprogramming into iPSCs. This effort uncovered the protein modifier SUMO2 as one of the strongest roadblocks to iPSC formation. Depletion of SUMO2 both enhances and accelerates reprogramming, yielding transgene-independent, chimera-competent iPSCs after as little as 38 hr of OKSM expression. We further show that the SUMO2 pathway acts independently of exogenous C-MYC expression and in parallel with small-molecule enhancers of reprogramming. Importantly, suppression of SUMO2 also promotes the generation of human iPSCs. Together, our results reveal sumoylation as a crucial post-transcriptional mechanism that resists the acquisition of pluripotency from fibroblasts using defined factors.
| S-EPMC4939549 | biostudies-literature
Project description:RNAi screening using pooled shRNA libraries is a valuable tool for identifying genetic regulators of biological processes. However, for a successful pooled shRNA screen, it is imperative to thoroughly optimize experimental conditions to obtain reproducible data. Here we performed viability screens with a library of ?10,000 shRNAs at two different fold representations (100- and 500-fold at transduction) and report the reproducibility of shRNA abundance changes between screening replicates determined by microarray and next generation sequencing analyses. We show that the technical reproducibility between PCR replicates from a pooled screen can be drastically improved by ensuring that PCR amplification steps are kept within the exponential phase and by using an amount of genomic DNA input in the reaction that maintains the average template copies per shRNA used during library transduction. Using these optimized PCR conditions, we then show that higher reproducibility of biological replicates is obtained by both microarray and next generation sequencing when screening with higher average shRNA fold representation. shRNAs that change abundance reproducibly in biological replicates (primary hits) are identified from screens performed with both 100- and 500-fold shRNA representation, however a higher percentage of primary hit overlap between screening replicates is obtained from 500-fold shRNA representation screens. While strong hits with larger changes in relative abundance were generally identified in both screens, hits with smaller changes were identified only in the screens performed with the higher shRNA fold representation at transduction.
| S-EPMC3411659 | biostudies-literature
Project description:RNA interference (RNAi) has opened promising avenues to better understand gene function. Though many RNAi screens report on the identification of genes, very few, if any, have been further studied and validated. Data discrepancy is emerging as one of RNAi main pitfalls. We reasoned that a systematic analysis of lethality-based screens, since they score for cell death, would examine the extent of hit discordance at inter-screen level. To this end, we developed a methodology for literature mining and overlap analysis of several screens using both siRNA and shRNA flavors, and obtained 64 gene lists censoring an initial list of 7,430 nominated genes. We further performed a comparative analysis first at a global level followed by hit re-assessment under much more stringent conditions. To our surprise, none of the hits overlapped across the board even for PLK1, which emerged as a strong candidate in siRNA screens; but only marginally in the shRNA ones. Furthermore, EIF5B emerges as the most common hit only in the shRNA screens. A highly unusual and unprecedented result was the observation that 5,269 out of 6,664 nominated genes (~80%) in the shRNA screens were exclusive to the pooled format, raising concerns as to the merits of pooled screens which qualify hits based on relative depletions, possibly due to multiple integrations per cell, data deconvolution or inaccuracies in intracellular processing causing off-target effects. Without golden standards in place, we would encourage the community to pay more attention to RNAi screening data analysis practices, bearing in mind that it is combinatorial in nature and one active siRNA duplex or shRNA hairpin per gene does not suffice credible hit nomination. Finally, we also would like to caution interpretation of pooled shRNA screening outcomes.
| S-EPMC3885821 | biostudies-literature
Project description:Broad-spectrum antiviral drugs targeting host processes could potentially treat a wide range of viruses while reducing the likelihood of emergent resistance. Despite great promise as therapeutics, such drugs remain largely elusive. Here we used parallel genome-wide high-coverage short hairpin RNA (shRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screens to identify the cellular target and mechanism of action of GSK983, a potent broad-spectrum antiviral with unexplained cytotoxicity. We found that GSK983 blocked cell proliferation and dengue virus replication by inhibiting the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH). Guided by mechanistic insights from both genomic screens, we found that exogenous deoxycytidine markedly reduced GSK983 cytotoxicity but not antiviral activity, providing an attractive new approach to improve the therapeutic window of DHODH inhibitors against RNA viruses. Our results highlight the distinct advantages and limitations of each screening method for identifying drug targets, and demonstrate the utility of parallel knockdown and knockout screens for comprehensive probing of drug activity.
| S-EPMC4836973 | biostudies-literature
Project description:Replication stress induced by nucleotide deficiency plays an important role in cancer initiation. Replication stress in primary cells typically activates the cellular senescence tumor-suppression mechanism. Senescence bypass correlates with development of cancer, a disease characterized by metabolic reprogramming. However, the role of metabolic reprogramming in the cellular response to replication stress has been little explored. Here, we report that ataxia telangiectasia mutated (ATM) plays a central role in regulating the cellular response to replication stress by shifting cellular metabolism. ATM inactivation bypasses senescence induced by replication stress triggered by nucleotide deficiency. This was due to restoration of deoxyribonucleotide triphosphate (dNTP) levels through both upregulation of the pentose phosphate pathway via increased glucose-6-phosphate dehydrogenase (G6PD) activity and enhanced glucose and glutamine consumption. These phenotypes were mediated by a coordinated suppression of p53 and upregulation of c-MYC downstream of ATM inactivation. Our data indicate that ATM status couples replication stress and metabolic reprogramming during senescence.
| S-EPMC4431925 | biostudies-literature
Project description:Ammonia is a cytotoxic metabolite with pleiotropic molecular and metabolic effects, including senescence induction. During dysregulated ammonia metabolism, which occurs in chronic diseases, skeletal muscle becomes a major organ for nonhepatocyte ammonia uptake. Muscle ammonia disposal occurs in mitochondria via cataplerosis of critical intermediary metabolite α-ketoglutarate, a senescence-ameliorating molecule. Untargeted and mitochondrially targeted data were analyzed by multiomics approaches. These analyses were validated experimentally to dissect the specific mitochondrial oxidative defects and functional consequences, including senescence. Responses to ammonia lowering in myotubes and in hyperammonemic portacaval anastomosis rat muscle were studied. Whole-cell transcriptomics integrated with whole-cell, mitochondrial, and tissue proteomics showed distinct temporal clusters of responses with enrichment of oxidative dysfunction and senescence-related pathways/proteins during hyperammonemia and after ammonia withdrawal. Functional and metabolic studies showed defects in electron transport chain complexes I, III, and IV; loss of supercomplex assembly; decreased ATP synthesis; increased free radical generation with oxidative modification of proteins/lipids; and senescence-associated molecular phenotype-increased β-galactosidase activity and expression of p16INK, p21, and p53. These perturbations were partially reversed by ammonia lowering. Dysregulated ammonia metabolism caused reversible mitochondrial dysfunction by transcriptional and translational perturbations in multiple pathways with a distinct skeletal muscle senescence-associated molecular phenotype.
| S-EPMC8783680 | biostudies-literature