Abstract
BackgroundThe Y-box protein MSY3/Csda represses myogenin transcription in skeletal muscle by binding a highly conserved cis-acting DNA element located just upstream of the myogenin minimal promoter (myogHCE). It is not known how this MSY3 activity is controlled in skeletal muscle. In this study, we provide multiple lines of evidence showing that the post-translational phosphorylation of MSY3 by Akt kinase modulates the MSY3 repression of myogenin.MethodsSkeletal muscle and myogenic C2C12 cells were used to study the effects of MSY3 phosphorylation in vivo and in vitro on its sub-cellular localization and activity, by blocking the IGF1/PI3K/Akt pathway, by Akt depletion and over-expression, and by mutating potential MSY3 phosphorylation sites.ResultsWe observed that, as skeletal muscle progressed from perinatal to postnatal and adult developmental stages, MSY3 protein became gradually dephosphorylated and accumulated in the nucleus. This correlated well with the reduction of phosphorylated active Akt. In C2C12 myogenic cells, blocking the IGF1/PI3K/Akt pathway using LY294002 inhibitor reduced MSY3 phosphorylation levels resulting in its accumulation in the nuclei. Knocking down Akt expression increased the amount of dephosphorylated MSY3 and reduced myogenin expression and muscle differentiation. MSY3 phosphorylation by Akt in vitro impaired its binding at the MyogHCE element, while blocking Akt increased MSY3 binding activity. While Akt over-expression rescued myogenin expression in MSY3 overexpressing myogenic cells, ablation of the Akt substrate, (Ser126 located in the MSY3 cold shock domain) promoted MSY3 accumulation in the nucleus and abolished this rescue. Furthermore, forced expression of Akt in adult skeletal muscle induced MSY3 phosphorylation and myogenin derepression.ConclusionsThese results support the hypothesis that MSY3 phosphorylation by Akt interferes with MSY3 repression of myogenin circuit activity during muscle development. This study highlights a previously undescribed Akt-mediated signaling pathway involved in the repression of myogenin expression in myogenic cells and in mature muscle. Given the significance of myogenin regulation in adult muscle, the Akt/MSY3/myogenin regulatory circuit is a potential therapeutic target to counteract muscle degenerative disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0043-9) contains supplementary material, which is available to authorized users.
Highlights
The Y-box protein MSY3/cold shock domain A (Csda) represses myogenin transcription in skeletal muscle by binding a highly conserved cis-acting DNA element located just upstream of the myogenin minimal promoter
MyogHCE deletion analyzed using reporter transgenic mice, led to a persistence of myogenin expression along the muscle fiber during the period of muscle maturation [16]. These results show that both MSY3 and the myogHCE play a crucial role in myogenin down-regulation that occurs during the refinement of synaptogenesis, when acetylcholine receptor (AChR) expression becomes restricted at synaptical sites in the muscle fiber [30, 31]
MSY3 is progressively dephosphorylated as skeletal muscle matures We previously demonstrated that MSY3 protein expression progressively increased as muscle matures, from late stages of fetal development (15 dpc) to the mature stages of muscle growth in postnatal life (2 months old, 2MM)
Summary
The Y-box protein MSY3/Csda represses myogenin transcription in skeletal muscle by binding a highly conserved cis-acting DNA element located just upstream of the myogenin minimal promoter (myogHCE). It is not known how this MSY3 activity is controlled in skeletal muscle. In fibroblasts MSY3 represses VEGF expression by preferentially binding single-stranded sense DNA of the hypoxia responsive region (HRR) of the VEGF promoter [24]; it regulates proliferation and density in epithelial cells, by shuttling between the tight junction and the nucleus, where it regulates the expression of cell cycle progression genes [25,26,27]. These results show that both MSY3 and the myogHCE play a crucial role in myogenin down-regulation that occurs during the refinement of synaptogenesis, when AChR expression becomes restricted at synaptical sites in the muscle fiber [30, 31]
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