College of Life Sciences and Biotechnology, Ningbo University, Ningbo, Zhejiang, China.
DNA Cell Biol. 2012 May;31(5):845-55. doi: 10.1089/dna.2011.1263. Epub 2011 Dec 7.
Myostatin (MSTN) is a negative regulator of skeletal muscle growth and development. There are two types of MSTNs in fish, but little is known about their gene regulation. Here, the 5' flanking fragments of 1029 bp from MSTN-1 and 643 bp from MSTN-2 were cloned, sequenced, and analyzed in Larimichthys crocea. Both fragments contained CAAT box and several putative cis-regulatory elements. However, putative TATA box, MyoD, MEF3, SP1, USF, and GH-CSE sites were identified only in the L. crocea MSTN-1 (lcMSTN-1) promoter. Transcriptional activities of four fragments (1013, 841, 514, and 261 bp) truncated from lcMSTN-1 upstream region and two fragments (643 and 296 bp) from lcMSTN-2 upstream region were examined in vitro, using transient transfection in CIK and L6 cells. In CIK cells, the promoter activity correlated positively with the length of truncated fragments in both MSTN-1 and 2. The lcMSTN-2 promoter showed a higher activity than lcMSTN-1 in the corresponding region, which was consistent with MSTN gene expression in vivo. In L6 cells, lcMSTN-2 upstream showed an extremely high luciferase activity. These data indicated that both cloned 5' flanking sequences contained functional promoters, and that transcription regulation of lcMSTN-1 and 2 promoters was significantly different between mammalian and fish cells.
肌肉生长抑制素 (MSTN) 是骨骼肌生长和发育的负调控因子。鱼类中有两种类型的 MSTN，但它们的基因调控知之甚少。在这里，克隆并测序了虹鳟 MSTN-1 的 5' 侧翼片段 1029bp 和 MSTN-2 的 643bp，并在虹鳟中进行了分析。这两个片段都包含 CAAT 盒和几个推测的顺式调控元件。然而，仅在虹鳟 MSTN-1（lcMSTN-1）启动子中鉴定出了 TATA 盒、MyoD、MEF3、SP1、USF 和 GH-CSE 位点。从 lcMSTN-1 上游区域截断的四个片段（1013、841、514 和 261bp）和 lcMSTN-2 上游区域的两个片段（643 和 296bp）的转录活性在体外通过 CIK 和 L6 细胞的瞬时转染进行了研究。在 CIK 细胞中，MSTN-1 和 2 中截断片段的长度与启动子活性呈正相关。与体内 MSTN 基因表达一致，lcMSTN-2 启动子在相应区域的活性高于 lcMSTN-1。在 L6 细胞中，lcMSTN-2 上游表现出极高的荧光素酶活性。这些数据表明，克隆的 5' 侧翼序列均包含功能性启动子，并且 lcMSTN-1 和 2 启动子在哺乳动物和鱼类细胞中的转录调控有显著差异。
