Comparative in silico analysis identifies bona fide MyoD binding sites within the Myocyte stress 1 gene promoter
UMass Chan Affiliations
Department of Cell BiologyDocument Type
Journal ArticlePublication Date
2008-05-21Keywords
AnimalsBinding Sites
*Computational Biology
Gene Regulatory Networks
Humans
Mice
*Muscle Development
Muscle Proteins
Mutagenesis, Site-Directed
MyoD Protein
NIH 3T3 Cells
*Promoter Regions, Genetic
Cell Biology
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Show full item recordAbstract
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. CONCLUSION: 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.Source
BMC Mol Biol. 2008 May 19;9:50. doi:10.1186/1471-2199-9-50. Link to article on publisher's siteDOI
10.1186/1471-2199-9-50Permanent Link to this Item
http://hdl.handle.net/20.500.14038/34863PubMed ID
18489770Related Resources
Link to Article in PubMedRights
© 2008 Ounzain et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.ae974a485f413a2113503eed53cd6c53
10.1186/1471-2199-9-50