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Comparative in silico analysis identifies bona fide MyoD binding sites within the Myocyte stress 1 gene promoter.
BMC Molecular Biology ( IF 4.619 ) Pub Date : 2008-05-19 , DOI: 10.1186/1471-2199-9-50
Samir Ounzain 1 , Caroline S Dacwag , Nilesh J Samani , Anthony N Imbalzano , Nelson W Chong
Affiliation  

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.

中文翻译:

计算机比较分析确定了肌细胞应激 1 基因启动子内真正的 MyoD 结合位点。

背景 肌细胞应激 1 (MS1) 是一种横纹肌肌动蛋白结合蛋白,是进化古心肌相关转录因子 (MRTF)/血清反应因子 (SRF) 转录途径的肌肉特异性活性所必需的。迄今为止,对控制 MS1 骨骼肌特异性表达的分子机制知之甚少。此类机制可能在调节 SRF 活动以及因此肌肉决定、分化和再生方面发挥重要作用。在这项研究中,我们采用了计算机比较分析以及实验性启动子表征来描述这些机制。结果 分化的 C2C12 肌肉细胞中 MS1 表达的分析表明 ms1 mRNA 的时间分化依赖性上调。计算机比较序列分析在 5' 上游序列的近端 1.5 kbp 内鉴定了两个保守的推定肌源性调节域。用 ms1 启动子/荧光素酶报告基因和生肌调节因子 (MRF) 过表达质粒共转染 C2C12 成肌细胞,揭示了 ms1 启动子对 MyoD 的特异性敏感性。随后的诱变和 EMSA 分析表明,在已确定的进化保守区域(ECR、α 和β)内的两个不同 E-Box(E1 和 E2)中,MyoD 具有特异性靶向。染色质免疫沉淀 (ChIP) 分析表明,位于 ECR alpha 和 beta 内的 E-Boxes 上 MyoD 的协同结合与 ms1 mRNA 的时间诱导相关。结论 这些发现表明 ms1 mRNA 中组织特异性和分化依赖性上调是由 MyoD 在 ms1 5' 上游序列内不同进化保守 E-Box 的时间结合介导的。我们相信,通过激活 ms1,这是第一项证明 MyoD 活性与 SRF 转录信号传导之间存在直接联系的研究,对理解肌肉决定、分化和再生具有明确的意义。
更新日期:2019-11-01
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