title: Comparative in silico analysis identifies bona fide MyoD binding sites within the Myocyte stress 1 gene promoter.
abstract: BACKGROUND
  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. 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.
  
  
  RESULTS
  Analysis 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, alpha and beta). Chromatin immunoprecipitation (ChIP) analysis indicates that co-ordinated binding of MyoD at E-Boxes located within ECRs alpha and beta correlates with the temporal induction in ms1 mRNA.
  
  
  CONCLUSIONS
  These 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.
authors.affiliation: Cardiology Group, Department of Cardiovascular Sciences, University of Leicester, Clinical Sciences Wing, Glenfield General Hospital, Leicester, LE3 9QP, UK. so41@le.ac.uk
authors.forename: Samir
authors.initials: S
authors.lastname: Ounzain
authors.forename: Caroline S
authors.initials: CS
authors.lastname: Dacwag
authors.forename: Nilesh J
authors.initials: NJ
authors.lastname: Samani
authors.forename: Anthony N
authors.initials: AN
authors.lastname: Imbalzano
authors.forename: Nelson W
authors.initials: NW
authors.lastname: Chong
mesh_terms: D000818:Animals; D001665:Binding Sites; D019295:Computational Biology; D053263:Gene Regulatory Networks; D006801:Humans; D051379:Mice; D024510:Muscle Development; D009124:Muscle Proteins / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D017570:MyoD Protein / Q000378:metabolism*; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic
chemical_list: D009124:Muscle Proteins; D017570:MyoD Protein