Abstract
BackgroundMyocyte stress 1 (MS1) is a striated muscle actin binding protein required for the muscle specific activity of the evolutionary ancient myocardin related transcription factor (MRTF)/serum response factor (SRF) transcriptional pathway. To date, little is known about the molecular mechanisms that govern skeletal muscle specific expression of MS1. Such mechanisms are likely to play a major role in modulating SRF activity and therefore muscle determination, differentiation and regeneration. In this study we employed a comparative in silico analysis coupled with an experimental promoter characterisation to delineate these mechanisms.ResultsAnalysis of MS1 expression in differentiating C2C12 muscle cells demonstrated a temporal differentiation dependent up-regulation in ms1 mRNA. An in silico comparative sequence analysis identified two conserved putative myogenic regulatory domains within the proximal 1.5 kbp of 5' upstream sequence. Co-transfecting C2C12 myoblasts with ms1 promoter/luciferase reporters and myogenic regulatory factor (MRF) over-expression plasmids revealed specific sensitivity of the ms1 promoter to MyoD. Subsequent mutagenesis and EMSA analysis demonstrated specific targeting of MyoD at two distinct E-Boxes (E1 and E2) within identified evolutionary conserved regions (ECRs, α and β). Chromatin immunoprecipitation (ChIP) analysis indicates that co-ordinated binding of MyoD at E-Boxes located within ECRs α and β correlates with the temporal induction in ms1 mRNA.ConclusionThese findings suggest that the tissue specific and differentiation dependent up-regulation in ms1 mRNA is mediated by temporal binding of MyoD at distinct evolutionary conserved E-Boxes within the ms1 5' upstream sequence. We believe, through its activation of ms1, this is the first study to demonstrate a direct link between MyoD activity and SRF transcriptional signalling, with clear implications for the understanding of muscle determination, differentiation and regeneration.
Highlights
Myocyte stress 1 (MS1) is a striated muscle actin binding protein required for the muscle specific activity of the evolutionary ancient myocardin related transcription factor (MRTF)/ serum response factor (SRF) transcriptional pathway
Serum response factor (SRF), a MADS box transcription factor related to the myocyte enhancer factor (MEF), regulates skeletal muscle gene expression through binding of a DNA sequence known as the serum response element (SRE) or CArG box [9,10,11]
Quantitative RT-PCR was conducted using 1 μg of total RNA obtained from subconfluent C2C12 myoblasts (MB) and from C2C12 myotubes differentiated for 3 days (MT)
Summary
Myocyte stress 1 (MS1) is a striated muscle actin binding protein required for the muscle specific activity of the evolutionary ancient myocardin related transcription factor (MRTF)/ serum response factor (SRF) transcriptional pathway. Little is known about the molecular mechanisms that govern skeletal muscle specific expression of MS1. Such mechanisms are likely to play a major role in modulating SRF activity and muscle determination, differentiation and regeneration. A specialised group of transcription factors control this process of myogenic specification and differentiation These factors, designated the myogenic regulatory factors (MRFs), include four basic helix-loop-helix (bHLH) E-Box binding proteins: MyoD, Myf, Myogenin and MRF4 [4]. Serum response factor (SRF), a MADS box transcription factor related to the MEFs, regulates skeletal muscle gene expression through binding of a DNA sequence known as the serum response element (SRE) or CArG box [9,10,11]. Confirming an important role for myogenic SRF activity, a conditional skeletal muscle specific knockout of SRF results in severe skeletal muscle myopathy that results in perinatal lethality [17]
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