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Multiphasic and dynamic changes in alternative splicing during induction of pluripotency are coordinated by numerous RNA binding proteins

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

ORGANISM(S): Mus musculus

SUBMITTER: Yi Xing

PROVIDER: E-GEOD-76233 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Multiphasic and dynamic changes in alternative splicing during induction of pluripotency are coordinated by numerous RNA binding proteins [iPS]

Project description:Alternative splicing (AS) plays a critical role in cell fate transitions, development and disease. Recent studies have shown that AS also influences pluripotency and somatic cell reprogramming. We profiled transcriptome-wide AS changes that occur during reprogramming of fibroblasts to pluripotency. This analysis revealed distinct phases of AS during reprogramming, including a splicing program that is unique to transgene-independent iPS cells. Changes in the expression of alternative splicing factors Zcchc24, Esrp1, Mbnl1/2 and Rbm47 were demonstrated to be key contributors to phase-specific AS. RNA binding motif enrichment analysis near alternatively spliced exons provided further insight into the combinatorial regulation of AS during reprogramming by different RNA binding proteins. Ectopic expression of Esrp1 enhanced reprogramming, in part by modulating the AS of the epithelial specific transcription factor Grhl1.These data represent a comprehensive temporal analysis of the dynamic regulation of AS during the acquisition of pluripotency. Mouse embryo fibroblasts were isolated from an E12.5 embryo that was homozygous for a TET-OP-OKSM polycistronic transgene targeted to the collagen 1A locus and homozygous for rtTA targeted to the Rosa26 locus. Therefore the expression of the â??4 factors/Yamanaka factorsâ?? that induce pluripotency can be induced with doxycycline treatment. Using this model we isolated RNA in triplicate over a timecourse of 0, 4, 7, 10, 15, 20, days of dox treatment as well as 3 transgene independent clones. Ssea1 MACS purification (Miltenyi Biotec) was used at each time point except zero.

2016-03-15 | E-GEOD-70022 | biostudies-arrayexpress

Multiphasic and dynamic changes in alternative splicing during induction of pluripotency are coordinated by numerous RNA binding proteins [ESRP DKO]

Project description:Alternative splicing (AS) plays a critical role in cell fate transitions, development and disease. Recent studies have shown that AS also influences pluripotency and somatic cell reprogramming. We profiled transcriptome-wide AS changes that occur during reprogramming of fibroblasts to pluripotency. This analysis revealed distinct phases of AS during reprogramming, including a splicing program that is unique to transgene-independent iPS cells. Changes in the expression of alternative splicing factors Zcchc24, Esrp1, Mbnl1/2 and Rbm47 were demonstrated to be key contributors to phase-specific AS. RNA binding motif enrichment analysis near alternatively spliced exons provided further insight into the combinatorial regulation of AS during reprogramming by different RNA binding proteins. Ectopic expression of Esrp1 enhanced reprogramming, in part by modulating the AS of the epithelial specific transcription factor Grhl1.These data represent a comprehensive temporal analysis of the dynamic regulation of AS during the acquisition of pluripotency. ES cells from 3 independent E3.5 blastocysts from either Control (Esrp1 WT/WT; Esrp2 -/-) or Esrp DKO (Esrp1 floxed/floxed; Esrp2 -/-) were transfected with pLVX-EGFP-Cre, puro selected and RNA was isolated 6 days later.

2016-03-15 | E-GEOD-76222 | biostudies-arrayexpress

Determination of a comprehensive alternative splicing regulatory network and the combinatorial regulation by key factors during Epithelial-to-Mesenchymal Transition [EMT.time course]

Project description:The epithelial to mesenchymal transition (EMT) is an essential biological process during embryonic development and has also been implicated in cancer metastasis. Previous studies have characterized transcriptional regulation and key transcription factors that impact EMT. However, the role of alternative splicing (AS) regulation in EMT has only recently emerged and remains relatively uncharacterized. Here we used a robust in vitro EMT model to dynamically and comprehensively characterize splicing switches during EMT in a temporal manner. We generated a H358 clone stably expressing a doxycycline (Dox)-inducible cDNA encoding a Zeb1-mCherry fusion protein. Over a 7-day time course following Dox treatment, cells have undergone EMT. We harvested total RNA and protein at each day of the EMT time course and a no Dox-treated control in biological triplicates. We made cDNA libraries for each replicate and subjected them to RNA-seq.

2016-04-05 | E-GEOD-75487 | biostudies-arrayexpress

Determination of a comprehensive alternative splicing regulatory network and the combinatorial regulation by key factors during Epithelial-to-Mesenchymal Transition [ESRP KD]

Project description:Epithelial specific splicing regulatory protein 1 and 2 (ESRP1 and ESRP2) are important regulators of alternative splicing during EMT. To study the alternative splicing events regulated by ESRP1/2 at a genome wide scale, we used lentiviral shRNAs to knockdown ESRP1/2 in H358 cells and performed RNA-seq in biological triplicates. We used lentiviral based shRNAs targeting ESRP1 and ESRP2 to knockdown both regulators in human H358 cells. We harvested total RNA and protein from ESRP1/2 knockdown and control knockdown in biological triplicates. We made cDNA libraries for each replicate and subjected them to RNA-seq.

2016-04-05 | E-GEOD-75489 | biostudies-arrayexpress

Determination of a comprehensive alternative splicing regulatory network and the combinatorial regulation by key factors during Epithelial-to-Mesenchymal Transition [RBM47 KD]

Project description:RBM47 is a RNA binding protein that is known to regulate alternative splicing. To study the genome wide regulatory role of RBM47 in alternative splicing and determine the potential function of RBM47 during EMT, we used lentiviral shRNAs to knockdown RBM47 in H358 cells and performed RNA-seq in biological triplicates. We used lentiviral based shRNAs to knockdown RBM47 in human H358 cells. We harvested total RNA and protein from RBM47 knockdown and control knockdown in biological triplicates. We made cDNA libraries for each replicate and subjected them to RNA-seq.

2016-04-05 | E-GEOD-75491 | biostudies-arrayexpress

Determination of a comprehensive alternative splicing regulatory network and the combinatorial regulation by key factors during Epithelial-to-Mesenchymal Transition

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

2016-04-05 | E-GEOD-75492 | biostudies-arrayexpress

Dnmt3a haploinsufficiency transforms Flt3-ITD myeloproliferative disease into a rapid, spontaneous, and fully-penetrant acute myeloid leukemia (Bulk RNA-Seq)

Project description:Cytogenetically normal acute myeloid leukemia (CN-AML) represents nearly 50% of human acute myeloid leukemia (AML) cases with a 5-year overall survival of approximately 30%. In CN-AML with poorer prognosis, mutations in the de novo DNA methyltransferase (DNMT3A) and the FMS-like tyrosine kinase 3 (Flt3) commonly co-occur (1-3). We demonstrate that mice with Flt3-internal-tandem duplication (Flt3ITD) and inducible deletion of Dnmt3a spontaneously develop a rapidly-lethal, completely-penetrant, and transplantable AML of normal karyotype. These murine AML retain a single Dnmt3a floxed allele, revealing the oncogenic potential of Dnmt3a haploinsufficiency. FLT3-ITD/DNMT3A-mutant primary human and murine AML demonstrate a similar pattern of global DNA methylation. In the murine model, rescuing DNMT3A expression was accompanied by DNA re-methylation and loss of clonogenic potential, suggesting that Dnmt3a-mutant oncogenic effects are reversible. Differentially methylated genomic regions were associated with changes in the expression of nearby genes. Moreover, dissection of the cellular architecture of the AML model using single-cell RNA-Seq, flow cytometry and colony assays identified clonogenic subpopulations that differentially express genes that are sensitive to the methylation of nearby genomic loci and varied in response to Dnmt3a levels. Thus, Dnmt3a haploinsufficiency transforms Flt3ITD myeloproliferative disease by modulating methylation-sensitive gene expression within a clonogenic AML subpopulation. To identify the gene expression changes associated with Dnmt3a loss of function in human and murine Flt3-ITD and Dnmt3a-mutant AML (Bulk RNA-Seq).

2016-03-27 | E-GEOD-77846 | biostudies-arrayexpress

Combining transcriptome and proteome analysis to identify and characterize the RpoS regulon in Pseudomonas fluorescens

Project description:Background: Microorganisms are the major cause of food spoilage during storage, processing and distribution. Pseudomonas fluorescens is a typical spoilage bacterium that contributes to a large extent to the spoilage process of proteinaceous food. RpoS is considered an important global regulator involved in stress survival and virulence in many pathogens. Our previous work revealed that RpoS contributed to the spoilage activities of P. fluorescens by regulating resistance to different stress conditions, extracellular acylated homoserine lactone (AHL) levels, extracellular protease and total volatile basic nitrogen (TVB-N) production. However, RpoS-dependent genes in P. fluorescens remained undefined. Results: RNA-seq transcriptomics analysis combined with quantitative proteomics analysis basing on multiplexed isobaric tandem mass tag (TMT) labeling was performed for the P. fluorescens wild-type strain UK4 and its derivative carrying a rpoS mutation. A total of 375 differentially expressed genes (DEGs) and 212 differentially expressed proteins (DEPs) were identified in these two backgrounds. The DGEs were further verified by qRT-PCR tests, and the genes directly regulated by RpoS were confirmed by 5’-RACE-PCR sequencing. The combining transcriptome and proteome analysis revealed a role of this regulator in several cellular processes, including polysaccharide metabolism, intracellular secretion and extracellular structures, cell well biogenesis, stress responses, ammonia and biogenic amine production, which may contribute to biofilm formation, stress resistance and spoilage activities of P. fluorescens. Moreover, in this work we indeed observed that RpoS contributed to the production of the macrocolony biofilm’s matrix.

2019-11-12 | PXD010845 | Pride

Multiphasic and Dynamic Changes in Alternative Splicing during Induction of Pluripotency Are Coordinated by Numerous RNA-Binding Proteins.

Project description:Alternative splicing (AS) plays a critical role in cell fate transitions, development, and disease. Recent studies have shown that AS also influences pluripotency and somatic cell reprogramming. We profiled transcriptome-wide AS changes that occur during reprogramming of fibroblasts to pluripotency. This analysis revealed distinct phases of AS, including a splicing program that is unique to transgene-independent induced pluripotent stem cells (iPSCs). Changes in the expression of AS factors Zcchc24, Esrp1, Mbnl1/2, and Rbm47 were demonstrated to contribute to phase-specific AS. RNA-binding motif enrichment analysis near alternatively spliced exons provided further insight into the combinatorial regulation of AS during reprogramming by different RNA-binding proteins. Ectopic expression of Esrp1 enhanced reprogramming, in part by modulating the AS of the epithelial specific transcription factor Grhl1. These data represent a comprehensive temporal analysis of the dynamic regulation of AS during the acquisition of pluripotency.

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Dnmt3a haploinsufficiency transforms Flt3-ITD myeloproliferative disease into a rapid, spontaneous, and fully-penetrant acute myeloid leukemia

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

2016-03-27 | E-GEOD-77849 | biostudies-arrayexpress