Expression data from individual MEF2A isoform knockdown in C2C12 myotubes
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ABSTRACT: Differentiation of muscle tissue is regulated by a complex network of transcription factors. The MEF2 family of transcription factors are important players in muscle development and differentiation. We knocked down expression of MEF2 isoforms in a cell culture model of skeletal muscle differentiation and assessed global expression pattern changes between MEF2 knockdowns, and with a negative control.
Project description:Differentiation of muscle tissue is regulated by a complex network of transcription factors. The MEF2 family of transcription factors are important players in muscle development and differentiation. We knocked down expression of MEF2 isoforms in a cell culture model of skeletal muscle differentiation and assessed global expression pattern changes between MEF2 knockdowns, and with a negative control. C2C12 myoblasts were transduced with shRNA viral vectors to knockdown expression of individual MEF2 proteins. Cells were then induced to differentiate and were harvested 3 days post-differentiation for Affymetrix global gene expression analysis. Each array was pooled RNA from two separate transductions, and each treatment group was analyzed in triplicate.
Project description:Regulation of homeostasis and development of cardiac muscle tissues is controlled by a core set of transcription factors. The MEF2 family plays a critical role in these processes. We knocked down expression of MEF2 isoforms in a primary cell culture model of cardiomyocytes and assessed global expression pattern changes between MEF2 knockdowns, and with a negative control.
Project description:During the last decade, skeletal muscle-secreted proteins have been identified with important roles in intercellular communications. To investigate whether muscle-derived exosomes participate in this molecular dialog, we determined and compared the protein contents of the exosome-like vesicles (ELVs) released from C2C12 murine myoblasts during proliferation (ELV-MB), and after differentiation into myotubes (ELV-MT). Using a proteomic approach combined with electron microscopy, western-blot and bioinformatic analyses, we compared the protein repertoires within ELV-MB and ELV-MT. RAW files were processed using MaxQuant [28] version 1.3.0.3. Spectra were searched against the Uniprot database (August 2012 version, Mus musculus taxonomy 10090, 86644 sequences, Bos taurus taxonomy 9913, 34280 sequences and Equus caballus taxonomy 9796, 24299 sequences) and the frequently observed contaminants database (notably containing protein sequences from serum proteins) embedded in MaxQuant. Trypsin was chosen as the enzyme and 2 missed cleavages were allowed. Precursor mass error tolerances were set respectively at 20 ppm and 6 ppm for first and main searches. Fragment mass error tolerance was set to 0.5 Da. Peptide modifications allowed during the search were: trioxidation (C, fixed), acetyl (N-ter, variable), dioxidation (M, variable), oxidation (M, variable) and deamidation (NQ, variable). Minimum peptide length was set to 7 amino acids. Minimum number of peptides, razor+unique peptides and unique peptides were set respectively to 2, 2 and1. Maximum false discovery rates - calculated by employing a reverse database strategy - were set to 0.01 at peptide and protein levels.
Project description:We report that differentiation of the C2C12 myoblast line on patterened hydrogels results in the increased expression of muscle sarcomere genes and more closely models expression changes that happen in in vivo muscle differentiation than do C2C12 myoblasts cultured on unpatterened substrates.
Project description:In this study, we have identified MEF2A-sensitive genes in atrial and ventricular chambers of the adult heart. MEF2A is a member of the myocyte enhancer factor 2 (MEF2) family of transcription factors. MEF2 proteins are expressed in skeletal and cardiac muscle tissues and are conserved across many mammalian species, but the gene programs regulated by MEF2A in adult cardiac chambers are largely unknown. We compared gene expression profiles between WT and Mef2a knockout atria and ventricles from adult mice, and the results identified distinct and overlapping sets of genes sensitive to the loss of MEF2A in the adult heart.
Project description:Nrf2 (NF-E2-related-factor-2) contributes to the maintenance of glucose homeostasis in vivo. Nrf2 suppresses blood glucose levels by protecting pancreatic β-cells from oxidative stress and improving peripheral tissue glucose utilization. To elucidate the molecular mechanisms by which Nrf2 contributes to the maintenance of glucose homeostasis, we generated skeletal muscle (SkM)-specific Keap1-knockout (Keap1MuKO) mice that express abundant Nrf2 in SkM and then examined Nrf2-target gene expression in this tissue. In Keap1MuKO mice, blood glucose levels were significantly downregulated, and the levels of glycogen branching enzyme (Gbe1) mRNA, along with those of glycogen branching enzyme (GBE) protein, were significantly upregulated in mouse SkM. Consistent with this result, chemical Nrf2-inducers promoted Gbe1 mRNA expression in both mouse SkM and C2C12 myotubes. Chromatin-immunoprecipitation analysis demonstrated that Nrf2 binds the Gbe1 upstream promoter regions. In Keap1MuKO mice, muscle glycogen content was strongly reduced, and forced GBE expression in C2C12 myotubes promoted glucose uptake. Therefore, our results demonstrate that Nrf2-induction in SkM increases GBE expression and reduces muscle glycogen content, resulting in improved glucose tolerance.
Project description:We identified genes expressed in mouse skeletal muscle, during the process of muscle regeneration after injury, which are dysregulated in the absence of Mef2a expression. MEF2A is a member of the evolutionarily conserved MEF2 transcription factor family which has known roles in cardiac muscle development and function, but is not well studied in skeletal muscle. We performed a comparison of gene expression profiles in wild type and MEF2A knockout tibialis anterior muscle, seven days post-injury with cardiotoxin. The results indicated that a variety of genes expressed during muscle regeneration, predominantly microRNAs in the Gtl2-Dio3 locus, are dysregulated by the loss of MEF2A expression.
Project description:Nrf2 (NF-E2-related-factor-2) contributes to the maintenance of glucose homeostasis in vivo. Nrf2 suppresses blood glucose levels by protecting pancreatic β-cells from oxidative stress and improving peripheral tissue glucose utilization. To elucidate the molecular mechanisms by which Nrf2 contributes to the maintenance of glucose homeostasis, we generated skeletal muscle (SkM)-specific Keap1-knockout (Keap1MuKO) mice that express abundant Nrf2 in SkM and then examined Nrf2-target gene expression in this tissue. In Keap1MuKO mice, blood glucose levels were significantly downregulated, and the levels of glycogen branching enzyme (Gbe1) mRNA, along with those of glycogen branching enzyme (GBE) protein, were significantly upregulated in mouse SkM. Consistent with this result, chemical Nrf2-inducers promoted Gbe1 mRNA expression in both mouse SkM and C2C12 myotubes. Chromatin-immunoprecipitation analysis demonstrated that Nrf2 binds the Gbe1 upstream promoter regions. In Keap1MuKO mice, muscle glycogen content was strongly reduced, and forced GBE expression in C2C12 myotubes promoted glucose uptake. Therefore, our results demonstrate that Nrf2-induction in SkM increases GBE expression and reduces muscle glycogen content, resulting in improved glucose tolerance. Chromatin occupancy of Nrf2 under CDDO-Im-treated condition were generated by deep sequencing, in dupliplicate
Project description:The novel lncRNA ChRO1 (Chromatin ReOrganization associated lncRNA 1) which we discovered seems to be involved in myogenesis by association with heterochromatin reorganization during muscle differentiation. We wanted to identify which genes are influenced by ChRO1 depletion in muscle differentiation. Analysis of the function of ChRO1 during myogenesis in gene expression level. The hypothesis in present study was that ChRO1 promotes myogenesis. Results provide important information that ChRO1 is involved in expression of various myogenic genes.