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Conserved functional antagonism between CELF and MBNL proteins regulates stem cell-specific alternative splicing and regeneration in planarians (dataset 2)

ABSTRACT: Here, we analyze the RNA-binding preferences of the planarian smed-mbnl-like-2, smed-mbnl-1 (isoform X1), smed-bruli, smed-mbnl-1 (isoform Xins), and smed-mbnl-like-1 protein using RNAcompete.

ORGANISM(S): synthetic construct

PROVIDER: GSE75554 | GEO | 2016/08/01

SECONDARY ACCESSION(S): PRJNA304513

REPOSITORIES: GEO

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Conserved functional antagonism between CELF and MBNL proteins regulates stem cell-specific alternative splicing and regeneration in planarians

Project description:Here, we analyze the RNA-binding preferences of the planarian smed-mbnl-like-2, smed-mbnl-1 (isoform X1), smed-bruli, smed-mbnl-1 (isoform Xins), and smed-mbnl-like-1 protein using RNAcompete. In the RNAcompete assay, a purified GST-tagged protein is incubated with an excess of RNA pool and bound RNA from individual pulldown experiments are directly labeled and hybridized to a custom Agilent 244K microarray.

2016-08-01 | E-GEOD-75554 | biostudies-arrayexpress

Conserved functional antagonism between CELF and MBNL proteins regulates stem cell-specific alternative splicing and regeneration in planarians

Project description:Understanding gene regulation in stem cells is key to understanding stem cell biology.Contrary to transcriptional regulation the function of alternative splicing (AS) in stem cells is poorly understood. Here we study function and mechanisms of AS in a powerful in vivo model for stem cell biology, the planarian S. mediterranea. Knockdown of AS factors, computational analyses of massive RNA-sequencing data, phenotyping, and biochemical binding assays revealed a stem cell specific AS program comprising hundreds of alternative exons, intron retention events, and microexons. The conserved AS factors CELF/MBNL are main regulators of this program. We show that they antagonize each other by directly promoting or repressing stem cell specific AS events. Knockdown of CELF/MBNL leads to defective regeneration by affecting self-renewal and differentiation, respectively. Thus, AS is of key importance for stem cell regulation in vivo and antagonistic regulation by CELF/MBNL is likely an ancestral feature of animal stem cells. Sequencing of polyA-selected RNA from Schmidtea mediterranea individuals after control, smed-bruno-like(RNAi) and mbnl(RNAi) after 20,25,30 days after RNAi

2016-08-01 | E-GEOD-75296 | biostudies-arrayexpress

Conserved functional antagonism between CELF and MBNL proteins regulates stem cell-specific alternative splicing and regeneration in planarians

Project description:Understanding gene regulation in stem cells is key to understanding stem cell biology. Contrary to transcriptional regulation the function of alternative splicing (AS) in stem cells is poorly understood. Here we study function and mechanisms of AS in a powerful in vivo model for stem cell biology, the planarian S. mediterranea. Knockdown of AS factors, computational analyses of massive RNA-sequencing data, phenotyping, and biochemical binding assays revealed a stem cell specific AS program comprising hundreds of alternative exons, intron retention events, and microexons. The conserved AS factors CELF/MBNL are main regulators of this program. We show that they antagonize each other by directly promoting or repressing stem cell specific AS events. Knockdown of CELF/MBNL leads to defective regeneration by affecting self-renewal and differentiation, respectively. Thus, AS is of key importance for stem cell regulation in vivo and antagonistic regulation by CELF/MBNL is likely an ancestral feature of animal stem cells. mRNA profiles of various samples of planarian cells, including control and knockdowns of key splicing factors in whole worms, and FACS-purified fractions

2016-08-01 | E-GEOD-75594 | biostudies-arrayexpress

Conserved functional antagonism between CELF and MBNL proteins regulates stem cell-specific alternative splicing and regeneration in planarians (dataset 3)

Project description:Understanding gene regulation in stem cells is key to understanding stem cell biology. Contrary to transcriptional regulation the function of alternative splicing (AS) in stem cells is poorly understood. Here we study function and mechanisms of AS in a powerful in vivo model for stem cell biology, the planarian S. mediterranea. Knockdown of AS factors, computational analyses of massive RNA-sequencing data, phenotyping, and biochemical binding assays revealed a stem cell specific AS program comprising hundreds of alternative exons, intron retention events, and microexons. The conserved AS factors CELF/MBNL are main regulators of this program. We show that they antagonize each other by directly promoting or repressing stem cell specific AS events. Knockdown of CELF/MBNL leads to defective regeneration by affecting self-renewal and differentiation, respectively. Thus, AS is of key importance for stem cell regulation in vivo and antagonistic regulation by CELF/MBNL is likely an ancestral feature of animal stem cells.

2016-08-01 | GSE75594 | GEO

Conserved functional antagonism between CELF and MBNL proteins regulates stem cell-specific alternative splicing and regeneration in planarians (dataset 1)

2016-08-01 | GSE75296 | GEO

H3K36me3 is significantly more enriched at Set1-affected loci than at MLL1/2-affected loci in planarian stem cells

Project description:Histone H3 lysine 4 trimethylation (H3K4me3) is known to correlate with both active and poised genomic loci, yet many questions remain regarding its functional roles in vivo. We identify functional genomic targets of two H3K4 methyltransferases, Set1 and MLL1/2, in both the stem cells (X1) and differentiated tissue (Xins) of the planarian flatworm Schmidtea mediterranea. We show that, despite their common substrate, these enzymes target distinct genomic loci in vivo, which are distinguishable by the footprint each enzyme leaves on the chromatin template, i.e., the breadth of the H3K4me3 peak. Whereas Set1 targets are broadly associated with the maintenance of the stem cell population, MLL1/2 targets are specifically enriched for genes involved in ciliogenesis. These data not only confirm that chromatin regulation is fundamental to planarian stem cell function, but also provide evidence for post-embryonic functional specificity of H3K4me3 methyltransferases in vivo. To test this, we looked at the H3K4me3 chromatin profile in WW, X1 and Xins cell types upon knockdown of set1, mll1/2, as well as WT. Examine binding of H3K36me3 in WT in Planarian. The experiment performed yielded a total of 2 samples. These samples validate the main ChIP samples.

2015-12-17 | E-GEOD-74419 | biostudies-arrayexpress

RNA Seq analysis of sorted Schmidtea mediterranea X1 and Xins cells to identify factors specific to neoblasts

Project description:Adult stem cells are tissue-specific cells with the capacity to self-renew and differentiate to continually replace cells lost to normal physiological turnover or injury. Neoblasts, the planarian stem cells, are widely distributed throughout the body mesenchyme, driving constitutive renewal of tissues during homeostasis and endowing planarians with the remarkable capacity to regenerate wholly from tiny tissue fragments. Neoblasts are the only dividing cells in planarians and are believed to be collectively comprised of both a heterogeneous population of pluripotent cells with broad differentiation potential and also lineage-committed progenitor cells that give rise to specific tissues. Recent technology has allowed one to isolate stem cells so we used a well-established method to isolate planarian stem cells by Hoechst blue staining and flow cytometry. To understand the molecular mechanisms underlying neoblast differentiation, we performed an RNA-Seq analysis of X1 and Xins cells looking at the differentially expressed genes between the two populations. Examine gene expression profiles of adult flatwormâ??s X1 and Xins cell types. The experiment was performed in quadruplicate yielding a total of 8 samples.

2015-12-17 | E-GEOD-73027 | biostudies-arrayexpress

Set1 and MLL1/2 target distinct sets of functionally different genomic loci in vivo: An in depth ChIP-seq analysis of H3K4me3 targets in vivo and upon Set1 and mll1 knockdown

Project description:Histone H3 lysine 4 trimethylation (H3K4me3) is known to correlate with both active and poised genomic loci, yet many questions remain regarding its functional roles in vivo. We identify functional genomic targets of two H3K4 methyltransferases, Set1 and MLL1/2, in both the stem cells (X1) and differentiated tissue (Xins) of the planarian flatworm Schmidtea mediterranea. We show that, despite their common substrate, these enzymes target distinct genomic loci in vivo, which are distinguishable by the footprint each enzyme leaves on the chromatin template, i.e., the breadth of the H3K4me3 peak. Whereas Set1 targets are broadly associated with the maintenance of the stem cell population, MLL1/2 targets are specifically enriched for genes involved in ciliogenesis. These data not only confirm that chromatin regulation is fundamental to planarian stem cell function, but also provide evidence for post-embryonic functional specificity of H3K4me3 methyltransferases in vivo. To test this, we looked at the H3K4me3 chromatin profile in WW, X1 and Xins cell types upon knockdown of set1, mll1/2, as well as WT. Examine differential binding of H3K4me3 in different experimental conditions. The experiment performed yielded a total of 58 samples.

2015-12-17 | E-GEOD-74153 | biostudies-arrayexpress

Conserved functional antagonism of CELF and MBNL proteins controls stem cell-specific alternative splicing in planarians.

Project description:In contrast to transcriptional regulation, the function of alternative splicing (AS) in stem cells is poorly understood. In mammals, MBNL proteins negatively regulate an exon program specific of embryonic stem cells; however, little is known about the in vivo significance of this regulation. We studied AS in a powerful in vivo model for stem cell biology, the planarian Schmidtea mediterranea. We discover a conserved AS program comprising hundreds of alternative exons, microexons and introns that is differentially regulated in planarian stem cells, and comprehensively identify its regulators. We show that functional antagonism between CELF and MBNL factors directly controls stem cell-specific AS in planarians, placing the origin of this regulatory mechanism at the base of Bilaterians. Knockdown of CELF or MBNL factors lead to abnormal regenerative capacities by affecting self-renewal and differentiation sets of genes, respectively. These results highlight the importance of AS interactions in stem cell regulation across metazoans.

| S-EPMC4978528 | biostudies-literature

Transcriptome analysis reveals strain-specific and conserved stemness genes in Schmidtea mediterranea

Project description:The transcriptome of the planarian Schmidtea mediterranea is not well characterized. We have used RNA-Seq to characterize the transcriptome in both sexual and asexual strains of S. mediterranea from both untreated and irradiated animals. Moreover, we have performed RNA-Seq on RNA purified from FACS sorted neoblasts and differentiated cells. Together these studies expand our understanding of the planarian transcriptome and have identified strain-specific, neoblast-specific, and conserved transcripts. RNA-Seq was performed on RNA isolated from untreated and irradiated S. mediterranea animals from both the sexual and asexual strains, on FACS purified X1 neoblasts, X2 neoblasts, and Xins differentiated cells. One of the raw data files for GSM847465 is missing. The fasta file is provided at http://genome.vcell.uchc.edu/GenomeData02/Graveley_Lab_Public_Data/Planarian/S.mediterranea_SexNIRmRNA3.fa.gz

2012-04-24 | E-GEOD-34326 | biostudies-arrayexpress

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