University of Leicester
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10.1186_1471-2199-9-50.pdf (621.1 kB)

Comparative in silico analysis identifies bona fide MyoD binding sites within the Myocyte stress 1 gene promoter.

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journal contribution
posted on 2012-10-24, 09:03 authored by Samir Ounzain, C.S. Dacwag, Nilesh J. Samani, A.N. Imbalzano, Nelson W. Chong
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, 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. 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.

History

Citation

BMC Molecular Biology, 2008, 9:50

Version

  • VoR (Version of Record)

Published in

BMC Molecular Biology

Publisher

BioMed Central Ltd

eissn

1471-2199

Copyright date

2008

Available date

2012-10-24

Publisher version

http://www.biomedcentral.com/1471-2199/9/50

Language

eng