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Enhanced CLIP uncovers IMP protein-RNA targets in human pluripotent stem cells important for cell adhesion and survival

ABSTRACT: This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

ORGANISM(S): Homo sapiens

SUBMITTER: Gene Yeo

PROVIDER: E-GEOD-78509 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Enhanced CLIP uncovers IMP protein-RNA targets in human pluripotent stem cells important for cell adhesion and survival [eCLIP-Seq]

Project description:Human pluripotent stem cells (hPSCs) require precise control of post-transcriptional RNA networks to maintain proliferation and survival. Using a recently developed enhanced UV crosslinking and immunoprecipitation (eCLIP) approach, we identify RNA targets of the IMP/IGF2BP family of RNA-binding proteins in hPSCs. At the broad region- and binding site-level IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3'UTR-enriched targets. RNA Bind-N-Seq applied to recombinant full-length IMP1 and IMP2 reveals CA-rich motifs that are enriched in eCLIP-defined binding sites. We observe that IMP1 loss in hPSCs recapitulates IMP1 phenotypes, including a reduction in cell adhesion and an increase in cell death. For cell adhesion, in hPSCs we find IMP1 maintains levels of integrin mRNA, specifically regulating RNA stability of ITGB5. Additionally, we show IMP1 can be linked to hPSC survival via direct target BCL2. Thus, transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles. eCLIP-seq was performed in biological replicate for IGF2BP1/IMP1 and IGF2BP2/IMP2, as well as one replicate each for IGF2BP3/IMP3, RBFOX2, and an IgG control. Each sample has a size-matched input control for analysis

2016-04-07 | E-GEOD-78507 | biostudies-arrayexpress

Enhanced CLIP uncovers IMP protein-RNA targets in human pluripotent stem cells important for cell adhesion and survival [RNA-Seq]

2016-04-07 | E-GEOD-78508 | biostudies-arrayexpress

Enhanced CLIP (eCLIP) enables robust and scalable transcriptome-wide discovery and characterization of RNA binding protein binding sites

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

2016-03-28 | E-GEOD-77634 | biostudies-arrayexpress

Primary cilium-autophagy-Nrf2 (PAN) axis links the cell cycle to neuroectoderm fate in human embryonic stem cells

Project description:Under defined differentiation conditions human embryonic stem cells (hESCs) can be directed toward a mesendodermal (ME) or neuroectoderm (NE) fate, the first decision during hESC differentiation. Coupled with G1 lengthening a divergent ciliation pattern emerged within the first 24 hours of induced lineage specification and these changes heralded a neuroectoderm decision before any neural precursor markers were expressed. By day 2, increased ciliation in NE precursors induced autophagy that resulted in the inactivation of Nrf2. Nrf2 binds directly to upstream regions of the OCT4 and NANOG genes to promote their expression and represses NE derivation. Nrf2 suppression was sufficient to rescue poorly neurogenic iPSC lines. Only after these events have been initiated do neural precursor markers get expressed at day 4. Thus we have identified a primary cilium-autophagy-Nrf2 (PAN) axis coupled to cell cycle progression that directs hESCs toward NE. Transcriptome analysis of hESC-derived neuroectoderm and mesendoderm cells

2016-03-24 | E-GEOD-69982 | biostudies-arrayexpress

Transcriptome of iPSC-derived Neural Cells with Heterozygous Knockout in CHD8

Project description:CHD8, encoding Chromodomain helicase DNA binding protein 8, is a top autism spectrum disorders (ASDs) risk gene. To better understanding the molecular links between CHD8 functions and ASD, we have applied the CRISPR/Cas9 technology to knockout one copy of CHD8 in induced pluripotent stem cells (iPSCs) to mimic the loss of function status that would exist in the developing human embryo prior to neuronal differentiation. Transcriptome profiling (RNA-seq) in neural progenitors and early differentiating neurons revealed that CHD8 hemizygosity (CHD8+/-) affected the expression of several thousands of genes, enriched for functions of neural development, Î²-catenin/Wnt signaling, extracellular matrix, and skeletal system development. Moreover, CHD8 regulates multiple genes implicated in ASD, schizophrenia and genes associated with brain volume. iPSCs derived from a healthy subject were transduced with CRISPR/Cas9 vectors with single guide RNA sequences to target the N-terminal of CHD8 protein to generate truncated mutation seach of the two target sequences. Two clones, one with a 2-bp (KO1) and the other with a 10-bp (KO2) heterozygous deletion were found.The CHD8+/- iPSC lines were used to generate NPCs and early differentiating neurons for RNA-seq analysis, together with samples prepared from the parental clones, for a total of 8 samples (two biological replicates of wild-type (WT) and CHD8+/- at two neurodevelopmental stages).

2015-11-05 | E-GEOD-71594 | biostudies-arrayexpress

A Human Pluripotent Stem Cell Surface N-Glycoprotein resource including New Markers, Extracellular Epitopes, and Drug Targets

Project description:Detailed knowledge of cell surface proteins for isolating well-defined populations of human pluripotent stem cells (hPSCs) would significantly enhance their characterization and translational potential. Normal H9 human embryonic stem cells and the KB3 human induced pluripotent stem cell lines were analyzed by Cell Surface Capture Technology, and in parallel transcript profiles from five independent samples (i.e., Replicas 1-5 for each) were performed to facilitate protein and transcriptomic comparisons. The study compared gene expression profiles of pluripotent stem cells with Cell Surface Capture technology generated N-glycoprotein surfaceome analyses of the same cell types.

2014-03-12 | E-GEOD-55805 | biostudies-arrayexpress

QuantSeq and RNAseq libraries to study developmental alternative polyadenylation

Project description:QuantSeq-Rev method to generate highly strand-specific next-generation sequencing (NGS) libraries enabling transcript quantification and identification of the 3'end of polyadenylated RNAs

2019-02-25 | E-MTAB-5090 | biostudies-arrayexpress

Transcription profiling by array of ESC derived hepatocytes treated with either vehicle or high energy substrates to model non alcoholic fatty liver disease

Project description:Female H9 ESCs were maintained on matrigel and differentiated into hepatocyte like cells as previously described (Szkolnicka D, Farnworth SL, Lucendo-Villarin B, Hay DC. Curr Protoc Stem Cell Biol. 2014 Aug 1;30:1G.5.1-12. doi: 10.1002/9780470151808.sc01g05s30) . Sodium L-Lactate (L7022, Sigma, Gillingham, UK) 10mM, Sodium Pyruvate 1mM (Sigma, Dorset, UK) and Octanoic Acid (C2875, Sigma, Gillingham, UK) 2mM for 48 hours before harvesting.

2018-01-30 | E-MTAB-6227 | biostudies-arrayexpress

Transcriptome analysis of pancreatic progenitors differentiated from H9 human embryonic stem cells and induced pluripotent stem cells derived from a Mitchell-Riley Syndrome patient

Project description:Skin biopsies were obtained from a patient with Mitchell-Riley syndrome caused by a homozygous frame-shift mutation (c.1129C>T) in the RFX6 gene that leads to a premature stop codon (p.Arg377X). The patient suffered severe pancreatic agenesis, in common with other Mitchell-Riley syndrome patients. Fibroblasts from the biopsy were reprogrammed to generate a human induced pluripotent stem cell (hiPSC) line (MRS2-6). To assess the effects of the mutant RFX6 allele on pancreas formation and identify direct targets of the transcription factor RFX6, MRS2-6 and H9 control human embryonic stem cells (hESC) were differentiated into pancreatic progenitors. Samples were harvested for RNA isolation and whole transcriptome analysis at days 4 (definitive endoderm), 7 and 8 (gut tube), and 12 (pancreatic progenitors).

2020-03-30 | E-MTAB-6626 | biostudies-arrayexpress

Genome-wide chromatin binding analysis (ChIP-seq) of HA epitope tagged RFX6 protein in human embryonic stem cell derived pancreatic progenitors

Project description:To identify direct transcriptional targets of RFX6, we performed chromatin immunoprecipitation of HA epitope tagged RFX6 followed by massively parallel DNA sequencing (ChIP-seq). Using CRISPR/Cas9 gene editing, the HA epitope was inserted into the 3' end of the RFX6 gene in H9 hESC. Pluripotent cells were then differentiated into PDX1+RFX6+ pancreatic progenitors and endogenous RFX6-HA was immunoprecipitated with an anti-HA antibody. To eliminate background signal caused by non-specific antibody binding, a control experiment using wild-type H9 hESC was performed in parallel.

2020-10-23 | E-MTAB-9335 | biostudies-arrayexpress

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