Oncogenic Fusion Protein EWS/FLI1 Down-regulates Gene Expression by Both Transcriptional and Posttranscriptional Mechanisms
Oncogenic Fusion Protein EWS/FLI1 Down-regulates Gene Expression by Both Transcriptional and Posttranscriptional Mechanisms
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DOI：
10.1074/jbc.m111.225433
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发表时间：
2011-07-01
影响因子：
4.8
通讯作者：
Denny, Christopher T.
中科院分区：
文献类型：
--
作者：
France, Kelly A.;Anderson, Jennifer L.;Denny, Christopher T.
尤因家族肿瘤的特征是RNA结合蛋白EWS与五种ETS转录因子之一（最常见的是FLI1）发生易位。由该易位产生的融合蛋白被认为是一种异常的转录因子，导致基因表达的改变和细胞转化。在这项研究中，我们探究了EWS/FLI1用于改变基因表达的具体过程。利用异源的NIH 3T3细胞和人尤因家族肿瘤细胞系，我们通过对前体mRNA的定量分析表明，EWS/FLI1在转录合成水平上抑制了先前已验证的直接靶基因的表达。染色质免疫沉淀（ChIP）实验显示，在小鼠和人类模型系统中，EWS/FLI1都减少了下调基因启动子处的RNA聚合酶II（Pol II）的量。然而，在下调的靶基因中，同源mRNA和前体mRNA的调节存在显著差异，这表明这些基因也可能在转录后水平受到调控。证实这一点的是，我们发现EWS/FLI1降低了胰岛素样生长因子结合蛋白3（一种在人肿瘤来源的尤因肉瘤细胞系中下调的直接靶基因）的转录半衰期。此外，我们通过再表达实验表明，完整的EWS/FLI1介导的转录抑制需要完整的EWS和ETS结构域。这些数据共同表明，EWS/FLI1可以通过调节转录合成和降解来决定稳态靶基因的表达。
Ewing family tumors are characterized by a translocation between the RNA binding protein EWS and one of five ETS transcription factors, most commonly FLI1. The fusion protein produced by the translocation has been thought to act as an aberrant transcription factor, leading to changes in gene expression and cellular transformation. In this study, we investigated the specific processes EWS/FLI1 utilizes to alter gene expression. Using both heterologous NIH 3T3 and human Ewing Family Tumor cell lines, we have demonstrated by quantitative pre-mRNA analysis that EWS/FLI1 repressed the expression of previously validated direct target genes at the level of transcript synthesis. ChIP experiments showed that EWS/FLI1 decreases the amount of Pol II at the promoter of down-regulated genes in both murine and human model systems. However, in down-regulated target genes, there was a significant disparity between the modulation of cognate mRNA and pre-mRNAs, suggesting that these genes could also be regulated at a posttranscriptional level. Confirming this, we found that EWS/FLI1 decreased the transcript half-life of insulin-like growth factor binding protein 3, a down-regulated direct target gene in human tumor-derived Ewing's sarcoma cell lines. Additionally, we have shown through reexpression experiments that full EWS/FLI1-mediated transcriptional repression requires intact EWS and ETS domains. Together these data demonstrate that EWS/FLI1 can dictate steady-state target gene expression by modulating both transcript synthesis and degradation.