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Dissection of the cellular function of the ZBED6 transcription factor in mouse myoblast cells, ZBED6-KO Lysate

ABSTRACT: The transcription factor ZBED6 acts as a repressor of Igf2 and affects directly or indirectly the transcriptional regulation of thousands of genes. Here, we use gene editing in mouse C2C12 myoblasts and show that ZBED6 regulates Igf2 exclusively through its binding site 5′-GGCTCG-3′ in intron 1 of Igf2. Deletion of this motif (Igf2ΔGGCT) or complete ablation of Zbed6 leads to ~20-fold up-regulation of IGF2 protein. Quantitative proteomics revealed an activation of Ras signaling pathway in both Zbed6-/- and Igf2ΔGGCT myoblasts, and a significant enrichment of mitochondrial membrane proteins among proteins showing altered expression in Zbed6-/- myoblasts. Both Zbed6-/- and Igf2ΔGGCT myoblasts showed a faster growth rate and developed myotube hypertrophy. These cells exhibited an increased O2 consumption rate, due to IGF2 up-regulation. Transcriptome analysis revealed ~30% overlap between differentially expressed genes in Zbed6-/- and Igf2ΔGGCT myotubes, with an enrichment of up-regulated genes involved in muscle development. In contrast, ZBED6-overexpression in myoblasts led to cell apoptosis, cell cycle arrest, reduced mitochondrial activities and ceased myoblast differentiation. The similarities in growth and differentiation phenotypes observed in Zbed6-/- and Igf2ΔGGCT myoblasts demonstrates that ZBED6 affects mitochondrial activity and myogenesis largely through its regulation of IGF2 expression. This study suggests that the interaction between ZBED6-Igf2 may be a therapeutic target for human diseases where anabolism is impaired.

INSTRUMENT(S): Orbitrap Fusion ETD

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Permanent Cell Line Cell, C2c12 Cell

SUBMITTER: Mårten Larsson

LAB HEAD: Leif Andersson

PROVIDER: PXD012757 | Pride | 2020-07-07

REPOSITORIES: Pride

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Dissection of the cellular function of the ZBED6 transcription factor in mouse myoblast cells IGF2–GGCT, Medium

Project description:The transcription factor ZBED6 acts as a repressor of Igf2 and affects directly or indirectly the transcriptional regulation of thousands of genes. Here, we use gene editing in mouse C2C12 myoblasts and show that ZBED6 regulates Igf2 exclusively through its binding site 5′-GGCTCG-3′ in intron 1 of Igf2. Deletion of this motif (Igf2ΔGGCT) or complete ablation of Zbed6 leads to ~20-fold up-regulation of IGF2 protein. Quantitative proteomics revealed an activation of Ras signaling pathway in both Zbed6-/- and Igf2ΔGGCT myoblasts, and a significant enrichment of mitochondrial membrane proteins among proteins showing altered expression in Zbed6-/- myoblasts. Both Zbed6-/- and Igf2ΔGGCT myoblasts showed a faster growth rate and developed myotube hypertrophy. These cells exhibited an increased O2 consumption rate, due to IGF2 up-regulation. Transcriptome analysis revealed ~30% overlap between differentially expressed genes in Zbed6-/- and Igf2ΔGGCT myotubes, with an enrichment of up-regulated genes involved in muscle development. In contrast, ZBED6-overexpression in myoblasts led to cell apoptosis, cell cycle arrest, reduced mitochondrial activities and ceased myoblast differentiation. The similarities in growth and differentiation phenotypes observed in Zbed6-/- and Igf2ΔGGCT myoblasts demonstrates that ZBED6 affects mitochondrial activity and myogenesis largely through its regulation of IGF2 expression. This study suggests that the interaction between ZBED6-Igf2 may be a therapeutic target for human diseases where anabolism is impaired.

2020-07-07 | PXD012758 | Pride

Dissection of the cellular function of the ZBED6 transcription factor in mouse myoblast cells, ZBED6-KO Medium

2020-07-07 | PXD012769 | Pride

Dissection of the cellular function of the ZBED6 transcription factor in mouse myoblast cells, IGF2-GGCT Lysate

Project description:The transcription factor ZBED6 acts as a repressor of Igf2 and affects directly or indirectly the transcriptional regulation of thousands of genes. Here, we use gene editing in mouse C2C12 myoblasts and show that ZBED6 regulates Igf2 exclusively through its binding site 5′-GGCTCG-3′ in intron 1 of Igf2. Deletion of this motif (Igf2ΔGGCT) or complete ablation of Zbed6 leads to ~20-fold up-regulation of IGF2 protein. Quantitative proteomics revealed an activation of Ras signaling pathway in both Zbed6-/- and Igf2ΔGGCT myoblasts, and a significant enrichment of mitochondrial membrane proteins among proteins showing altered expression in Zbed6-/- myoblasts. Both Zbed6-/- and Igf2ΔGGCT myoblasts showed a faster growth rate and developed myotube hypertrophy. These cells exhibited an increased O2 consumption rate, due to IGF2 up-regulation. Transcriptome analysis revealed ~30% overlap between differentially expressed genes in Zbed6-/- and Igf2ΔGGCT myotubes, with an enrichment of up-regulated genes involved in muscle development. In contrast, ZBED6-overexpression in myoblasts led to cell apoptosis, cell cycle arrest, reduced mitochondrial activities and ceased myoblast differentiation. The similarities in growth and differentiation phenotypes observed in Zbed6-/- and Igf2ΔGGCT myoblasts demonstrates that ZBED6 affects mitochondrial activity and myogenesis largely through its regulation of IGF2 expression. This study suggeststhat the interaction between ZBED6-Igf2 may be a therapeutic target for human diseases where anabolism is impaired.

2020-07-07 | PXD012789 | Pride

miRNA mimic transfected SC and MIPC transcriptome analysis

Project description:In the present study, we analyzed two populations of myogenic cells isolated from mouse skeletal muscle, that is, myoblasts derived from satellite cells (SCs) and muscle interstitial progenitor cells (MIPCs). We found that SDF-1 treatment of myoblasts led to down-regulation of miR10a, miR151, miR425, and miR5100. Therefore, to follow the impact of these miRNAs on myogenic cells, SC-derived myoblasts and MIPCs were transfected with selected miRNA mimics in the concentration of 50 mol/L and changes in their transcriptome were analyzed.

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Cooperative target mRNA destabilization and translation inhibition by miR-58 microRNA family in C. elegans [RNA-Seq]

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2015-06-05 | E-GEOD-60420 | biostudies-arrayexpress

Microarray analysis of gene expression regulation by Vgll3 in mouse satellite cell-derived myoblasts

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2019-06-17 | E-MTAB-7614 | biostudies-arrayexpress

Canonical Wnt signalling regulates nuclear export of Setdb1 during skeletal muscle terminal differentiation [ChIP-seq]

Project description:The histone 3 lysine 9 (H3K9)-specific methyltransferase (KMT) Setdb1 is essential for both stem cell pluripotency and terminal differentiation of different cell types. To shed light on Setdb1 role(s) in these mutually exclusive processes, we used mouse skeletal myoblasts as a model of terminal differentiation. Ex vivo studies on isolated single myofibres showed that Setdb1 is required for muscle adult stem cells expansion following activation and in vitro studies on skeletal myoblasts confirmed that Setdb1 suppresses terminal myoblast differentiation. We used genome-wide analyses to identify Setdb1 direct target genes in myoblasts and observed a release of Setdb1 from the promoter of selected target genes upon myoblast terminal differentiation, concomitant to a nuclear export of Setdb1 to the cytoplasm. We demonstrated that both genomic release and cytoplasmic Setdb1 relocalisation during differentiation were dependent on canonical Wnt signalling. Taken together, our findings uncover a functional link between Setdb1 and canonical Wnt signalling in skeletal muscle cells, which affects the expression of a subset of Setdb1 target genes. We revealed Wnt-dependent subcellular relocalisation of Setdb1 as a novel mechanism regulating Setdb1 functions. ChIP-seq of Setdb1 and H3K9me3 in Myoblast cells (C2C12)

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Mouse Dux is myotoxic and shares partial functional homology with its human paralog DUX4

Project description:We report the RNA-seq experiments performed in human myoblasts transfected with human DUX4 and mouse Dux. Comparison of genes up- and down-regulated by DUX4 and Dux in human myoblasts to identify pathways similiarly regulated by both transcription factors.

2016-08-24 | E-GEOD-85935 | biostudies-arrayexpress

Non-lethal stress-induced discrete proteolysis is caspase-3/7 dependent

Project description:Cells are in constant adaptation to environmental changes to insure their proper functioning. When exposed to stresses, cells activate specific pathways to elicit adaptive modifications. Those changes can be mediated by selective modulation of gene and protein expression as well as by post-translational modifications, such as phosphorylation and proteolytic processing. Protein cleavage, as a controlled and limited post-translational modification, is involved in diverse physiological processes such as the maintenance of protein homeostasis, activation of repair pathways, apoptosis and the regulation of proliferation. Here we assessed by quantitative proteomics the proteolytic landscape in two cell lines subjected to low cisplatin concentrations used as a mild non-lethal stress paradigm. This landscape was compared to the one obtained in the same cells stimulated with cisplatin concentrations inducing apoptosis. These analyses were performed in wild-type cells and in cells lacking the two main executioner caspases: caspase-3 and caspase-7. Ninety proteins were found to be cleaved at one or a few sites (discrete cleavage) in low stress conditions compared to four hundred and forty in apoptotic cells. Many of the cleaved proteins in stressed cells were also found to be cleaved in apoptotic conditions. As expected in apoptotic cells, ~80% of the cleavage events were dependent on caspase 3/caspase 7. Strikingly, upon exposure to non-lethal stresses, no discrete cleavage was detected in cells lacking caspase-3 and caspase-7. This indicates that the proteolytic landscape in stressed viable cells fully depends on the activity of executioner caspases. These results suggest that the so-called executioner caspases fulfill important stress adaptive responses distinct from their role in apoptosis.

2021-07-07 | PXD023488 | Pride

Linc-smad7 promotes myoblast differentiation and muscle regeneration via sponging miR-125b

Project description:Long noncoding RNAs (lncRNAs) are involved in the regulation of skeletal muscle development. In the present study, differentially expressed lncRNAs were identified from RNA-seq data derived from myoblasts and myotubes. We conducted studies to elucidate the function and molecular mechanism of action of Linc-smad7 during skeletal muscle development. Our findings show that Linc-smad7 is upregulated during the early phase of myoblasts differentiation. In in vitro studies, we showed that overexpression of Linc-smad7 promoted the arrest of myoblasts in G1 phase, inhibited DNA replication, and induced myoblast differentiation. Our in vivo studies suggest that Linc-smad7 stimulates skeletal muscle regeneration in cardiotoxin-induced muscle injury. Mechanistically, Linc-smad7 overexpression increased smad7 and IGF2 protein levels. On the contrary, overexpression of miR-125b reduced smad7 and IGF2 protein levels. Results of RNA immunoprecipitation analysis and biotin-labeled miR-125b capture suggest that Linc-smad7 could act as a competing endogenous RNA (ceRNA) for miRNA-125b. Taken together, our findings suggest that the novel noncoding regulator Linc-smad7 regulates skeletal muscle development.

2017-11-01 | GSE106365 | GEO

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