Ewing's sarcoma fusion protein, EWS/Fli-1 and Fli-1 protein induce PLD2 but not PLD1 gene expression by binding to an ETS domain of 5′ promoter
Ewing's sarcoma fusion protein, EWS/Fli-1 and Fli-1 protein induce PLD2 but not PLD1 gene expression by binding to an ETS domain of 5′ promoter
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DOI：
10.1038/sj.onc.1209973
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发表时间：
2007-03-01
期刊：
影响因子：
8
通讯作者：
Murate, T.
中科院分区：
文献类型：
--
作者：
Kikuchi, R.;Murakami, M.;Murate, T.
有报道EWS/Fli-1的短干扰RNA（siRNA）下调尤文肉瘤（EWS）细胞系磷脂酶D（PLD）2，提示PLD 2是异常转录因子EWS/Fli-1的靶点。在此，我们进一步研究了EWS/Fli-1和Fli-1在另一个EWS细胞系中以及在EWS/Fli-1-或Fli-1-转染的细胞系中对PLD 2基因表达的调节。EWS/Fli-1-或Fli-1-过表达的细胞显示出更高的PLD 2但不是PLD 1蛋白表达和增强的细胞增殖相比，模拟转染。用1-丁醇或PLD 2的siRNA处理这些细胞抑制细胞生长，表明PLD在细胞生长促进中的关键作用。在EWS/Fli-1或Fli-1-转染子中，PLD 2 mRNA水平也增加，但PLD 1 mRNA水平没有增加。在确定转录起始点后，我们克隆了PLD 1和PLD 2的50个启动子，并分析了启动子活性。结果表明，EWS/Fli-1和Fli- 1通过与PLD 2启动子的成红细胞转化特异性结构域（距转录起始位点约126至约120 bp）结合来增加PLD 2基因表达，该结构域是最小和最强的区域。电泳迁移率变动分析表明，DNA结合结构域和反式激活结构域是增强PLD 2基因表达所必需的。
It was reported that short interfering RNA (siRNA) of EWS/Fli-1 downregulated phospholipase D (PLD)2 in Ewing's sarcoma (EWS) cell line, suggesting that PLD2 is the target of aberrant transcription factor, EWS/Fli-1. Here, we further investigated the regulation of PLD2 gene expression by EWS/Fli-1 and Fli-1 in another EWS cell line, and also in EWS/Fli-1- or Fli-1-transfected cell line. EWS/Fli-1- or Fli-1-overexpressed cells showed higher PLD2 but not PLD1 protein expression and enhanced cell proliferation as compared to mock transfectant. The treatment of these cells with 1-butanol or siRNA of PLD2 inhibited cell growth, suggesting the pivotal role of PLD in cell growth promotion. PLD2 but not PLD1 mRNA level was also increased in EWS/Fli-1 or Fli-1-transfectants. After determining the transcription initiation points, we cloned the 50 promoter of both PLD1 and PLD2 and analysed promoter activities. Results showed that EWS/Fli-1 and Fli- 1 increase PLD2 gene expression by binding to an erythroblast transformation-specific domain (-126 to -120 bp from the transcription initiation site) of PLD2 promoter, which is the minimal and most powerful region. Electrophoresis mobility shift assay using truncated proteins showed that both DNA-binding domain and trans-activating domain were necessary for the enhanced gene expression of PLD2.