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Novel RNA-binding activity of MYF5 enhances Ccnd1/Cyclin D1 mRNA translation during myogenesis

ABSTRACT: Skeletal muscle contains long multinucleated and contractile structures known as muscle fibers, which arise from the fusion of myoblasts into nucleated myotubes during myogenesis. The myogenic regulatory factor (MRF) MYF5 is the earliest to be expressed during myogenesis and functions as a transcription factor in muscle progenitor cells (satellite cells) and myocytes. In mouse C2C12 myocytes, MYF5 is implicated in the initial steps of myoblast differentiation into myotubes. Ribonucleoprotein immunoprecipitation (RIP) analysis showed that MYF5 bound a subset of myoblast mRNAs; prominent among them was Ccnd1 mRNA, which encodes the key cell cycle regulator CCND1 (Cyclin D1). Biotin-RNA pulldown, UV-crosslinking, and gel shift experiments indicated that MYF5 was capable of binding the 3' untranslated region (UTR) and the coding region (CR) of Ccnd1 mRNA. MYF5 silencing in proliferating growing myoblasts revealed that and MYF5 promoted CCND1 translation, and it also modestly increased transcription of Ccnd1 mRNA. Importantly, silencing MYF5 reduced myoblast growth as well as differentiation of myoblasts into myotubes, while overexpressing MYF5 in C2C12 cells upregulated CCND1 expression. We propose that MYF5 enhances early myogenesis in part by coordinately elevating Ccnd1 transcription and Ccnd1 mRNA translation.

ORGANISM(S): Mus musculus

PROVIDER: GSE73704 | GEO | 2016/08/08

SECONDARY ACCESSION(S): PRJNA297693

REPOSITORIES: GEO

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Novel RNA-binding activity of MYF5 enhances Ccnd1/Cyclin D1 mRNA translation during myogenesis

Project description:Skeletal muscle contains long multinucleated and contractile structures known as muscle fibers, which arise from the fusion of myoblasts into nucleated myotubes during myogenesis. The myogenic regulatory factor (MRF) MYF5 is the earliest to be expressed during myogenesis and functions as a transcription factor in muscle progenitor cells (satellite cells) and myocytes. In mouse C2C12 myocytes, MYF5 is implicated in the initial steps of myoblast differentiation into myotubes. Ribonucleoprotein immunoprecipitation (RIP) analysis showed that MYF5 bound a subset of myoblast mRNAs; prominent among them was Ccnd1 mRNA, which encodes the key cell cycle regulator CCND1 (Cyclin D1). Biotin-RNA pulldown, UV-crosslinking, and gel shift experiments indicated that MYF5 was capable of binding the 3' untranslated region (UTR) and the coding region (CR) of Ccnd1 mRNA. MYF5 silencing in proliferating growing myoblasts revealed that and MYF5 promoted CCND1 translation, and it also modestly increased transcription of Ccnd1 mRNA. Importantly, silencing MYF5 reduced myoblast growth as well as differentiation of myoblasts into myotubes, while overexpressing MYF5 in C2C12 cells upregulated CCND1 expression. We propose that MYF5 enhances early myogenesis in part by coordinately elevating Ccnd1 transcription and Ccnd1 mRNA translation. Four replicates were utilized from either Control (IgG) or MYF5-immunoprecipitated RNA samples from C2C12 cells growing in either growth medium (GM) or differentiation medium (DM) for a total of sixteen samples.

2016-08-08 | E-GEOD-73704 | biostudies-arrayexpress

Genomic mapping of Six1 binding sites during skeletal myoblast differentiation

Project description:In this study, the C2C12 cell line, a model used to study myogenesis and regeneration, was allowed to differentiate from myoblast precursor cells to myotubes. Cells were harvested at 3 different timepoints to perform ChIP-on-Chip of Six1, which is a key muscle regulator. We identified global loci bound by Six1 during skeletal myoblast differentiation. C2C12 Myoblasts were allowed to differentiate into myotubes. Cells at three timepoints were harvested for ChIP-on-Chip, including myoblasts stage, 24h after differentiation and myotubes (96h after differentiation). Myotubes were detached from the undifferentiated myoblast reserve cells using diluted trypsin. 3 independent biological replicates were used for each time point experiment. A microarray set counts 3 arrays (Custom Arrays A, B and C) for a total of approximately 2.9 million probes.

2010-07-05 | E-GEOD-20150 | biostudies-arrayexpress

Quantitative Myogenesis C2C12 Proteome with Dimethyl-Labelling

Project description:The Z-disc is a protein-rich structure critically important for myofibril development and integrity. In order to monitor the quantiative changes in C2C12 myoblast during myogenesis, a quantitative dimethyl-labelling approach was performed with d0 myoblasts, d5 myotubes and electrical puls stimulated d5 myotubes.

2017-01-02 | PXD004960 | Pride

ADAMTSL2 regulates Wnt signaling in myogenesis

Project description:ADAMTSL2 mutations cause geleophysic dysplasia, which is characterized by short stature, pseudomuscular build, joint stiffness and tight skin. To elucidate the role of ADAMTSL2 in skeletal muscle myogenesis, we used C2C12 myoblasts, which differentiate and form myotubes when serum is reduced as a model system for myogenesis. Adamtsl2 was depleted by stably expressing shRNA targeting Adamtsl2 mRNA. Upon shRNA-mediated depletion of Adamtsl2, C2C12 myoblasts did not differentiate and did not form myosin heavy chain-positive myotubes in cell culture.

2021-10-16 | GSE185894 | GEO

Comprehensive analysis of the proteome, phosphoproteome and N-linked sialylated glycoproteome of C2C12 myoblasts

Project description:C2C12 myoblast is a model that has been used extensively to study the process of skeletal muscle differentiation. Proteomics has advanced our understanding of skeletal muscle biology and the process of myogenesis. However, there is still no deep coverage of C2C12 myoblast proteome, which is important for the understanding of key drivers for the differentiation of skeletal muscle cells. Here, we conducted a multi-dimensional proteome profiling with TiSH strategy to get a comprehensive analysis of proteome, phosphoproteome and N-linked sialylated glycoproteome of C2C12 myoblasts. A total of 8313 protein groups were identified in C2C12 myoblasts, including 7827 protein groups from non-modified peptides, 3803 phosphoproteins and 977 formerly N-linked sialylated glycoproteins.

2020-11-27 | PXD011334 | Pride

C2C12 murine myoblast cell line and 48 h 10 uM BIO treated C2C12 cellulate

Project description:In urodele amphibians, limb regeneration involves the dedifferentiation of muscle myotubes into single cells that may acquire pluripotent potential. We have employed small molecules (myoseverin and BIO) to attempt to reproduce this behavior in mammalian muscle culture. C2C12 myotubes derived from the C2C12 myoblast cell line were induced to undergo cellularization by myoseverin treatment, which destabilizes tubulin filaments. The GSK-3 inhibitor, BIO, was then used to induce dedifferentiation. Induce neuron formation; the cells were incubated with 250 nM reversine for 48 h, and neural induction media (DMEM/F12 supplemented with N2 (Invitrogen)) and 1.5 uM all-trans retinoic acid for 7 days. C2C12 murine myoblast cell line and 48 h 10 uM BIO treated C2C12 cellulate (derived by 20 M myoseverin treatment for 48 h) C2C12 myoblasts were differentiated into myotubes with 2%horse serum in DMEM for 8 days (from 2-4 d, 10 uM AraC treatment was also used to kill any remaining myoblasts). Next, myotubes were cellularized by 20 uM myoseverin treatment for 48 h. 24 h after myoseverin treatment, myotubes were treated with 10 uM BIO for 2d.

2011-12-08 | E-GEOD-34238 | biostudies-arrayexpress

Expression profiling FSHD vs. control myoblasts and myotubes

Project description:The gene expression pathways leading to muscle pathology in facioscapulohumeral dystrophy (FSHD) remain to be elucidated. This muscular dystrophy is caused by a contraction of an array of tandem 3.3-kb repeats (D4Z4) at 4q35.2. We compared expression of control and FSHD myoblasts and myotubes (three preparations each) on exon microarrays (Affymetrix Human Exon 1.0 ST) and validated FSHD-specific differences for representative genes by qRT-PCR on additional myoblast cell strains. The FSHD and control myoblasts used for these experiments were shown to grow and differentiate into myotubes equally efficiently as control myoblasts. There were no significant FSHD-control differences in RNA levels for MYOD1 and MYOG at the myoblast and myotube stages and for MYF5 and MYF6 at the myoblast stage. In contrast, 295 other genes were dysregulated at least 2-fold in FSHD vs. control myoblasts (p <0.01, adjusted for multiple comparisons). Remarkably, only 10% of the FSHD-associated gene dysregulation at the myoblast stage was downregulation. At the myotube stage, about ten times as many genes exhibited FSHD-associated downregulated as at the myoblast stage and twice as many genes displayed FSHD-associated upregulation. The FSHD-related changes in RNA levels appear to be due to posttranscriptional as well as transcriptional alterations. Among the prominently dysregulated pathways were signaling and oxidative stress pathways. By comparing expression profiles of control myoblasts and myotubes to each other and to 19 non-muscle cell types profiled identically, our study also revealed many new myogenesis associations for genes not previously annotated as muscle-specific. Keywords: Disease state analysis and time course for differentiation

2011-09-29 | GSE26145 | GEO

Genomic mapping of Six1 binding sites during skeletal myoblast differentiation

2010-07-05 | GSE20150 | GEO

Phosphoproteomics of C2C12 differentiation

Project description:Phosphoproteomic analysis of myogenesis in C2C12 myoblasts differentiating into myotubes. Four time points were analysed (0min, 30min, 24h and 5d) with four biological replicates.

2024-01-26 | PXD023413 | Pride

Expression Profiling of Early Myogenesis - Illumina Dataset

Project description:Myogenesis is a tightly controlled process involving the transcriptional activation and repression of thousands of genes. Although many components of the transcriptional network are known for the later phases of myogenesis, relatively little work has described the transcriptional landscape within the first 24 hours, when myoblasts commit to differentiate. Through dense temporal sampling of differentiating C2C12 myoblasts, we identify 266 transcriptional regulators (TRs) whose expression is altered within the first 12 hours of myogenesis. A high-content shRNA screen of 76 TRs involving 427 stable lines identified 48 genes whose knockdown significantly inhibits differentiation of C2C12 myoblasts. These include known regulators of myogenesis (Myod1, Myog and Myf5), as well as 26 regulators not previously associated with the process. Of the TRs differentially expressed within the first 24 hours, two-thirds inhibited differentiation when knocked down. Surprisingly, a similar proportion (67%) of shRNAs targeting TRs whose expression did not change during differentiation also inhibited myogenesis, suggesting that both stably and differentially expressed TRs are essential for this complex differentiation program. This implies that microarray-based approaches that concentrate functional validation studies on differentially-expressed genes will fail to identify many genes that are critically implicated in complex biological processes. C2C12 myoblasts were differentiated into myotubes and sampled at various timepoints for gene expression measurement on Illumina Mouse WG-6v1.1 arrays. Cells grown in separate plates were harvested at 12 different timepoints: t_0h, t_1h, t_1.5h, t_2h, t_3h, t_6h, t_9h, t_12h, t_24h, t_48h, t_96h, t_144h. All harvests were performed in triplicate using growths from successive passages.

2012-03-21 | E-GEOD-17038 | biostudies-arrayexpress

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