Directed evolution of reprogramming factors by cell selection and sequencing
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA422111 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR6369290.fastq.gz | Fastqsanger.gz | |||
| SRR6369300.fastq.gz | Fastqsanger.gz | |||
| SRR6369310.fastq.gz | Fastqsanger.gz | |||
| SRR6369320.fastq.gz | Fastqsanger.gz | |||
| SRR6369330.fastq.gz | Fastqsanger.gz |
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Project description:Directed biomolecular evolution is widely used to tailor and enhance proteins but has hitherto not been applied in the reprogramming of mammalian cells. Here we describe a novel method to identify artificially enhanced and evolved reprogramming factors by pooled screens with randomised protein libraries, cell selection based on phenotypic readouts and genotyping by amplicon sequencing. We benchmark this approach by identifying artificially enhanced Sox2 and Sox17 factors in pluripotency reprogramming.
2018-07-12 | GSE107987 | GEO
Project description:Directed biomolecular evolution is widely used to tailor and enhance enzymes, fluorescent proteins, and antibodies but has hitherto not been applied in the reprogramming of mammalian cells. Here, we describe a method termed directed evolution of reprogramming factors by cell selection and sequencing (DERBY-seq) to identify artificially enhanced and evolved reprogramming transcription factors. DERBY-seq entails pooled screens with libraries of positionally randomised genes, cell selection based on phenotypic readouts, and genotyping by amplicon sequencing for candidate identification. We benchmark this approach using pluripotency reprogramming with libraries based on the reprogramming factor SOX2 and the reprogramming incompetent endodermal factor SOX17. We identified several SOX2 variants outperforming the wild-type protein in three- and four-factor cocktails. The most effective variants were discovered from the SOX17 library, demonstrating that this factor can be converted into a highly potent inducer of pluripotency with a range of diverse modifications. We propose DERBY-seq as a broad-based approach to discover reprogramming factors for any donor/target cell combination applicable to direct lineage reprogramming in vitro and in vivo.
| S-EPMC6092915 | biostudies-literature
Project description:Two genetic selection systems that couple metabolite hydroxylation or methylation of small molecules to growth of Escherichia coli are presented in this study. One system targets pterin-dependent hydroxylation (tBPt) while another focuses on S-adenosylmethionine-dependent methylation (SAM). Using adaptive laboratory evolution with growth selection, these two systems are demonstrated to not only achieve in vivo directed evolution of enzymes involved in human hormone biosynthesis but also reveal non-intuitive host factors that elude existing synthetic biology approaches. Raw sequencing data for the relevant strains generated in this study are presented here.
2019-01-30 | E-MTAB-6779 | biostudies-arrayexpress
Project description:The evolution of human bipedalism exposed the knee to unique biomechanical challenges, requiring changes in knee anatomy giving rise to the modern-day configuration. In order to better understand the relationship between derived knee morphology and the genetic factors associated with osteoarthritis risk, we performed epigenetic profiling of murine forelimb/hindlimb growth plates to identify regulatory elements shaping formation of specific knee structures, identifying signals of ancient positive selection upon which more recent genetic drift overlaps risk-associated loci. Our functional analyses of an osteoarthritis-risk variant within a reproducibly-associated locus establishes a novel model for studying this degenerative disease.
2020-04-16 | GSE122877 | GEO
Project description:Based on inheritance of acquired characteristics, Lamarckian theory of evolution explains the evolution of biological systems through epigenetics. In a previous study, we have shown how microbial evolution has resulted in a persistent reduction in expression after repeatedly selecting for the lowest PGAL1-YFP-expressing cells. Applying the ATAC-seq assay on samples collected from this 28-days evolution experiment, here we show how genome-wide chromatin compaction change during evolution under selection pressure. We found that the chromatin compaction was altered not only on GAL network genes directly impacted by the selection pressure, showing an example of non-genetic memory, but also at the whole genome level. The GAL network genes experienced chromatin compaction accompanying the reduction in PGAL1-YFP reporter expression; strikingly, the fraction of global genes with differentially compacted chromatin states accounted for about a quarter of the total genome. Moreover, some of the ATAC-seq peaks followed well-defined temporal dynamics. Comparing the peak’s intensity in consecutive days, we found most of the differential compaction to occur between days 0 and 3 when the selection pressure was first applied, and between days 7 and 10 when the pressure was lifted. Among the gene sets enriched for the differential compaction events, some had increased chromatin availability once selection pressure was applied and decreased availability after the pressure was lifted (or vice versa). These results intriguingly show that, despite the lack of targeted selection, transcriptional availability of a large fraction of the genome change in a very diverse manner during evolution and these changes can occur in a relatively short number of generations.
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Project description:Background: Direct cardiac reprogramming is currently being investigated for the generation of cells with a true cardiomyocyte (CM) phenotype. Based on the original approach of cardiac transcription factor-induced reprogramming of fibroblasts into CM-like cells, various modifications of that strategy have been developed. However, they uniformly suffer from poor reprogramming efficacy and a lack of translational tools for target cell expansion and purification. Therefore, our group has developed a unique approach to generate proliferative cells with a pre-CM phenotype that can be expanded in vitro to yield substantial cell doses. Methods: Cardiac fibroblasts were reprogrammed toward CM fate using lentiviral transduction of cardiac transcriptions factors (GATA4, MEF2C, TBX5, and MYOCD). The resulting cellular phenotype was analyzed by RNA sequencing and immunocytology. Live target cells were purified based on intracellular CM marker expression using molecular beacon technology and fluorescence-activated cell sorting. CM commitment was assessed using 5-azacytidine–based differentiation assays and the therapeutic effect was evaluated in a mouse model of acute myocardial infarction using echocardiography and histology. The cellular secretome was analyzed using mass spectrometry. Results: We found that proliferative CM precursor-like cells were part of the phenotype spectrum arising during direct reprogramming of fibroblasts toward CMs. These induced CM precursors (iCMPs) expressed CPC- and CM-specific proteins and were selectable via hairpin-shaped oligonucleotide hybridization probes targeting Myh6/7-mRNA–expressing cells. After purification, iCMPs were capable of extensive expansion, with preserved phenotype when under ascorbic acid supplementation, and gave rise to CM-like cells with organized sarcomeres in differentiation assays. When transplanted into infarcted mouse hearts, iCMPs prevented CM loss, attenuated fibrotic scarring, and preserved ventricular function, which can in part be attributed to their substantial secretion of factors with documented beneficial effect on cardiac repair. Conclusions: Fibroblast reprogramming combined with molecular beacon-based cell selection yields an iCMP-like cell population with cardioprotective potential. Further studies are needed to elucidate mechanism-of-action and translational potential.
2023-06-24 | PXD042806 | Pride
Project description:Oct4, Sox2, Klf4, and cMyc (OSKM) reprogram somatic cells to pluripotency. To gain a mechanistic understanding of their function, we mapped OSKM-binding, stage-specific transcription-factors (TFs), and chromatin-states in discrete reprogramming stages and performed loss- and gain-of-function experiments. We found that early in reprogramming OSK extensively bind somatic-enhancers and initiate their decommissioning by recruiting Hdac1. Concurrently, OSK engage other sites, including specific pluripotency-enhancers, and induce the relocation of somatic TFs to these sites and away from somatic-enhancers, extending somatic-enhancer decommissioning genome-wide. Pluripotency-enhancer selection early in reprogramming occurs predominantly at sites with high OSK-motif densities and requires collaborative binding by OSK. Most pluripotency-enhancers are selected later and occupied by OS and stage-specific-TFs like Esrrb. Overexpression of stage-specific-TFs influences reprogramming efficiency by changing OSK-occupancy, somatic-enhancer decommissioning, and pluripotency-enhancer selection. We propose that collaborative interactions among OSK and with stage-specific-TFs direct both somatic-enhancer decommissioning and pluripotency-enhancer selection, which drives the enhancer reorganization underlying reprogramming
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Project description:Oct4, Sox2, Klf4, and cMyc (OSKM) reprogram somatic cells to pluripotency. To gain a mechanistic understanding of their function, we mapped OSKM-binding, stage-specific transcription-factors (TFs), and chromatin-states in discrete reprogramming stages and performed loss- and gain-of-function experiments. We found that early in reprogramming OSK extensively bind somatic-enhancers and initiate their decommissioning by recruiting Hdac1. Concurrently, OSK engage other sites, including specific pluripotency-enhancers, and induce the relocation of somatic TFs to these sites and away from somatic-enhancers, extending somatic-enhancer decommissioning genome-wide. Pluripotency-enhancer selection early in reprogramming occurs predominantly at sites with high OSK-motif densities and requires collaborative binding by OSK. Most pluripotency-enhancers are selected later and occupied by OS and stage-specific-TFs like Esrrb. Overexpression of stage-specific-TFs influences reprogramming efficiency by changing OSK-occupancy, somatic-enhancer decommissioning, and pluripotency-enhancer selection. We propose that collaborative interactions among OSK and with stage-specific-TFs direct both somatic-enhancer decommissioning and pluripotency-enhancer selection, which drives the enhancer reorganization underlying reprogramming
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Project description:Oct4, Sox2, Klf4, and cMyc (OSKM) reprogram somatic cells to pluripotency. To gain a mechanistic understanding of their function, we mapped OSKM-binding, stage-specific transcription-factors (TFs), and chromatin-states in discrete reprogramming stages and performed loss- and gain-of-function experiments. We found that early in reprogramming OSK extensively bind somatic-enhancers and initiate their decommissioning by recruiting Hdac1. Concurrently, OSK engage other sites, including specific pluripotency-enhancers, and induce the relocation of somatic TFs to these sites and away from somatic-enhancers, extending somatic-enhancer decommissioning genome-wide. Pluripotency-enhancer selection early in reprogramming occurs predominantly at sites with high OSK-motif densities and requires collaborative binding by OSK. Most pluripotency-enhancers are selected later and occupied by OS and stage-specific-TFs like Esrrb. Overexpression of stage-specific-TFs influences reprogramming efficiency by changing OSK-occupancy, somatic-enhancer decommissioning, and pluripotency-enhancer selection. We propose that collaborative interactions among OSK and with stage-specific-TFs direct both somatic-enhancer decommissioning and pluripotency-enhancer selection, which drives the enhancer reorganization underlying reprogramming
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