Specific and redundant roles of TEAD transcription factors in C2C12 cell and primary myoblast differentiation
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE82193 | GEO | 2017/02/17
SECONDARY ACCESSION(S): PRJNA324273
REPOSITORIES: GEO
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Project description:Defining the function of TEAD transcription factors in myogenic differentiation has proved elusive due to overlapping expression and functional redundancy. Here, we show that siRNA silencing of either Tead1, Tead2 or Tead4 did not effect differentiation of primary myoblasts (PMs) while their simultaneous knockdown strongly impaired differentiation. In contrast in C2C12 cells, silencing of Tead1 or Tead4 impaired differentiation showing a differential requirement for these factors in PMs and C2C12 cells that involved both differential regulation of their expression and intracellular localisation. Through integration of Tead1 and Tead4 ChIP-seq with chromatin modifications, we identify active enhancers associated with genes activated during C2C12 cell differentiation that are bound by combinations of Tead4, Myod1 or Myog and show a signature of frequently co-occuring motifs. We show that distinct but overlapping sets of genes are deregulated by Tead silencing in C2C12 cells and PMs therefore describing for the first time in a comprehensive manner the specific and redundant regulatory roles of Tead factors in myogenic differentiation. We also performed ChIP-seq from mouse muscle in vivo identifying a set of highly transcribed muscle cell-identity genes and revealing that Tead4 binds a distinct repertoire of sites in C2C12 cells and muscle.
2017-02-17 | GSE82190 | GEO
Project description:Defining the function of TEAD transcription factors in myogenic differentiation has proved elusive due to overlapping expression and functional redundancy. Here, we show that siRNA silencing of either Tead1, Tead2 or Tead4 did not effect differentiation of primary myoblasts (PMs) while their simultaneous knockdown strongly impaired differentiation. In contrast in C2C12 cells, silencing of Tead1 or Tead4 impaired differentiation showing a differential requirement for these factors in PMs and C2C12 cells that involved both differential regulation of their expression and intracellular localisation. Through integration of Tead1 and Tead4 ChIP-seq with chromatin modifications, we identify active enhancers associated with genes activated during C2C12 cell differentiation that are bound by combinations of Tead4, Myod1 or Myog and show a signature of frequently co-occuring motifs. We show that distinct but overlapping sets of genes are deregulated by Tead silencing in C2C12 cells and PMs therefore describing for the first time in a comprehensive manner the specific and redundant regulatory roles of Tead factors in myogenic differentiation. We also performed ChIP-seq from mouse muscle in vivo identifying a set of highly transcribed muscle cell-identity genes and revealing that Tead4 binds a distinct repertoire of sites in C2C12 cells and muscle.
2017-02-17 | GSE82192 | GEO
Project description:Specific and redundant roles of TEAD transcription factors in C2C12 cell and primary myoblast differentiation
| PRJNA324273 | ENA
Project description:The TEAD family of transcription factors (TEAD1-4) bind the MCAT element in the regulatory elements of both growth promoting and myogenic differentiation genes. Defining TEAD transcription factor function in myogenesis has proved elusive due to overlapping expression of family members and their functional redundancy. We show that silencing of either Tead1, Tead2 or Tead4 did not effect primary myoblast (PM) differentiation, but that their simultaneous knockdown strongly impaired differentiation. In contrast, Tead1 or Tead4 silencing impaired C2C12 differentiation showing their different contributions in PMs and C2C12 cells. Chromatin immunoprecipitation identified enhancers associated with myogenic genes bound by combinations of Tead4, Myod1 or Myog. Tead4 regulated distinct gene sets in C2C12 cells and PMs involving both activation of the myogenic program and repression of growth and signaling pathways. ChIP-seq from mature mouse muscle fibres in vivo identified a set of highly transcribed muscle cell-identity genes and sites bound by Tead1 and Tead4. Although inactivation of Tead4 in mature muscle fibres caused no obvious phenotype under normal conditions, notexin-induced muscle regeneration was delayed in Tead4 mutants suggesting an important role in myogenic differentiation in vivo. By combining knockdown in cell models in vitro with Tead4 inactivation in muscle in vivo, we provide the first comprehensive description of the specific and redundant roles of Tead factors in myogenic differentiation.
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Project description:Skeletal muscle cell differentiation is a multistage process extensively studied over the years. Even if great improvements have been achieved in defining biological process underlying myogenesis, many molecular mechanisms need still to be clarified.To further highlight this process, we studied cells at undifferentiated, intermediate and highly differentiated stages, and we analyzed, for each condition, morphological and proteomic changes. We also identified the proteins that showed statistical significant changes by a ESI-Q-TOF mass spectrometer. This work provides further evidence of the involvement of particular proteins in skeletal muscle development. Furthermore, the high level of expression of many heat shock proteins, suggests a relationship between differentiation and cellular stress. Intriguingly, the discovery of myogenesis-correlated proteins, known to play a role in apoptosis, suggests a link between differentiation and this type of cell death.
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Project description:Enhanced intracellular Ca2+ signaling by stromal interaction molecule 1 (STIM1)-mediated store-operated Ca2+ entry (SOCE) is required for skeletal muscle differentiation. However, the contribution of STIM2, STIM1's analogue protein, on muscle cell differentiation has not been clearly elucidated. The present study aimed to explore the contribution of STIM2-mediated SOCE on C2C12 myoblast differentiation.Changes in STIM2 expression level (reverse transcription-polymerase chain reaction and Western blotting) and SOCE activity ([Ca2+]i measurement) were measured during 3 days of in vitro differentiation of C2C12 skeletal myoblast. Transcriptional regulation of STIM2 by nuclear factor of activated T cells, cytoplasmic (NFATc) overexpression was observed, and the effect of STIM2 knockdown on NFAT transcriptional activity (luciferase assay) and myoblast differentiation was quantified.Increase of STIM2 protein level and enhanced SOCE activity were observed in differentiating myoblasts. Treatment with a SOCE blocker (2-APB) inhibited the differentiation. Overexpression of NFATc1 increased STIM2 expression and SOCE activity. Knockdown of STIM2 decreased NFAT transcriptional activity, SOCE activity, and differentiation of C2C12 myoblast.It is suggested that STIM2-activated SOCE controls C2C12 myoblast differentiation.
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Project description:Muscle is a contractile tissue responsible for maintaining posture and the movement of all parts of the body. Prolonged oxidizative stress can lead to the damage of cells, tissues, and organs. In this study, we investigated the possibility of oxidative stress in the process of myoblast differentiation of C2C12 cells. First, the myoblast differentiation model of C2C12 cells was constructed and verified by Giemsa staining. The expression of hypoxia inducible factor1-alpha (HIF1-α), hypoxia inducible factor1-beta (HIF1-β), Von Hippel-Lindau (VHL), lysyl oxidase (Lox), EGL-9 family hypoxia-inducible factor 1 (EGLN1), proline 4-hydroxylase alpha 1 (P4HA1) and heme oxygenase-1 (HOMX1) in the process of myoblast differentiation was verified by in vitro experiments and Gene Expression Omnibus (GEO) bioinformatic analysis. We found that with the increased expression of myogenic factor 5 (MYF5), myogenic differentiation 1 (MYOD1), and Desmin, myotube fusion became more obvious during the process of C2C12 cell differentiation. Both experimental and GEO analysis indicated that the expression of HIF1-α, HIF1-β, VHL, LOX, EGLN1 and P4HA1 increased, and the expression of HOMX1 decreased during myogenic differentiation. Therefore, we suggest that the myoblast differentiation of C2C12 cells may be related to oxidative stress. Their possible relationship was proposed, though further studies are needed.
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Project description:The ubiquitin-proteasome system is a major protein degradation pathway in the cell. Proteasomes produce several peptides that are rapidly degraded to free amino acids by intracellular aminopeptidases. Our previous studies reported that proteolysis via proteasomes and aminopeptidases is required for myoblast proliferation and differentiation. However, the role of intracellular aminopeptidases in myoblast proliferation and differentiation had not been clarified. In this study, we investigated the effects of puromycin-sensitive aminopeptidase (PSA) on C2C12 myoblast proliferation and differentiation by knocking down PSA. Aminopeptidase enzymatic activity was reduced in PSA-knockdown myoblasts. Knockdown of PSA induced impaired cell cycle progression in C2C12 myoblasts and accumulation of cells at the G2/M phase. Additionally, after the induction of myogenic differentiation in PSA-knockdown myoblasts, multinucleated circular-shaped myotubes with impaired cell polarity were frequently identified. Cell division cycle 42 (CDC42) knockdown in myoblasts resulted in a loss of cell polarity and the formation of multinucleated circular-shaped myotubes, which were similar to PSA-knockdown myoblasts. These data suggest that PSA is required for the proliferation of myoblasts in the growth phase and for the determination of cell polarity and elongation of myotubes in the differentiation phase.
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Project description:ERK6, a mitogen-activated protein (MAP) kinase-related serine/threonine kinase, is highly expressed in human skeletal muscle and appears to function as a signal transducer during differentiation of myoblasts to myotubes. In transfected 293 cells, activation of the 45-kDa enzyme results in tyrosine-phosphorylated 46- and 56-kDa forms, which phosphorylate myelin basic protein. Overexpression of wild-type ERK6 or the inactive mutant Y185F has no effect on fibroblast and myoblast proliferation, but it enhances or inhibits C2C12 cell differentiation to myotubes, respectively. Our findings suggest ERK6 to be a tissue-specific, differentiation signal-transducing factor that is connected to phosphotyrosine-mediated signaling pathways distinct from those activating other members of the MAP kinase family such as LRK1 and ERK2.
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Project description:The loss of muscle mass in alcoholic myopathy may reflect alcohol inhibition of myogenic cell differentiation into myotubes. Here, using a high content imaging system we show that ethanol inhibits C2C12 myoblast differentiation by reducing myogenic fusion, creating smaller and less complex myotubes compared with controls. Ethanol administration during C2C12 differentiation reduced MyoD and myogenin expression, and microarray analysis identified ethanol activation of the Notch signaling pathway target genes Hes1 and Hey1. A reporter plasmid regulated by the Hes1 proximal promoter was activated by alcohol treatment in C2C12 cells. Treatment of differentiating C2C12 cells with a gamma secretase inhibitor (GSI) abrogated induction of Hes1. On a morphological level GSI treatment completely rescued myogenic fusion defects and partially restored other myotube parameters in response to alcohol. We conclude that alcohol inhibits C2C12 myoblast differentiation and the inhibition of myogenic fusion is mediated by Notch pathway activation.
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