Abstract
BackgroundThe expression of myogenic regulatory factors (MRFs) consisting of MyoD, Myf5, myogenin (MyoG) and MRF4 characterizes various phases of skeletal muscle development including myoblast proliferation, cell-cycle exit, cell fusion and the maturation of myotubes to form myofibers. Although it is well known that the function of MyoG cannot be compensated for other MRFs, the molecular mechanism by which MyoG controls muscle cell differentiation is still unclear. Therefore, in this study, RNA-Seq technology was applied to profile changes in gene expression in response to MyoG knock-down (MyoGkd) in primary bovine muscle satellite cells (MSCs).ResultsAbout 61–64% of the reads of over 42 million total reads were mapped to more than 13,000 genes in the reference bovine genome. RNA-Seq analysis identified 8,469 unique genes that were differentially expressed in MyoGkd. Among these genes, 230 were up-regulated and 224 were down-regulated by at least four-fold. DAVID Functional Annotation Cluster (FAC) and pathway analysis of all up- and down-regulated genes identified overrepresentation for cell cycle and division, DNA replication, mitosis, organelle lumen, nucleoplasm and cytosol, phosphate metabolic process, phosphoprotein phosphatase activity, cytoskeleton and cell morphogenesis, signifying the functional implication of these processes and pathways during skeletal muscle development. The RNA-Seq data was validated by real time RT-PCR analysis for eight out of ten genes as well as five marker genes investigated.ConclusionsThis study is the first RNA-Seq based gene expression analysis of MyoGkd undertaken in primary bovine MSCs. Computational analysis of the differentially expressed genes has identified the significance of genes such as SAP30-like (SAP30L), Protein lyl-1 (LYL1), various matrix metalloproteinases, and several glycogenes in myogenesis. The results of the present study widen our knowledge of the molecular basis of skeletal muscle development and reveal the vital regulatory role of MyoG in retaining muscle cell differentiation.
Highlights
Skeletal muscle formation is a multi-step process that requires proliferation of myocytes, expression of muscle-specific myogenic regulatory factors (MRFs) including MyoD, Myf5, myogenin (MyoG) and MRF4, cell cycle withdrawal, myotube formation by the fusion of mononucleated cells and maturation of myotubes into myofibers [1], [2], [3], [4], [5]
MRFs are basic helix-loop-helix transcription factors [6] that cooperate with several transcription factors of the myocytes enhancer factor-2 (MEF2) family [7] to regulate myogenesis. bHLH proteins heterodimerize with E-proteins [8], enabling binding to the E-Box consensus sequence (CANNTG) present in the regulatory regions of muscle specific genes [9], [10]
The expression level of MyoG, which is known to play a role in myogenic differentiation [33], was determined by realtime RT-PCR at different time points of primary bovine cells differentiation
Summary
Skeletal muscle formation is a multi-step process that requires proliferation of myocytes, expression of muscle-specific myogenic regulatory factors (MRFs) including MyoD, Myf, myogenin (MyoG) and MRF4 (or Myf6), cell cycle withdrawal, myotube formation by the fusion of mononucleated cells and maturation of myotubes into myofibers [1], [2], [3], [4], [5]. BHLH proteins heterodimerize with E-proteins [8], enabling binding to the E-Box consensus sequence (CANNTG) present in the regulatory regions of muscle specific genes [9], [10] Among these MRFs, MyoD is highly expressed during the mid-G1 phase and between the S and M phases of the cell cycle, but absent during the G0 phase [11], whereas Myf is highly expressed during the G0 phase and decreases during the G1 phase [12]. The expression of myogenic regulatory factors (MRFs) consisting of MyoD, Myf, myogenin (MyoG) and MRF4 characterizes various phases of skeletal muscle development including myoblast proliferation, cell-cycle exit, cell fusion and the maturation of myotubes to form myofibers. In this study, RNA-Seq technology was applied to profile changes in gene expression in response to MyoG knockdown (MyoGkd) in primary bovine muscle satellite cells (MSCs)
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