Multiple ETS family proteins regulate PF4 gene expression by binding to the same ETS binding site.
Multiple ETS family proteins regulate PF4 gene expression by binding to the same ETS binding site.
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DOI：
10.1371/journal.pone.0024837
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Doi T
中科院分区：
文献类型：
--
作者：
Okada Y;Nobori H;Shimizu M;Watanabe M;Yonekura M;Nakai T;Kamikawa Y;Wakimura A;Funahashi N;Naruse H;Watanabe A;Yamasaki D;Fukada S;Yasui K;Matsumoto K;Sato T;Kitajima K;Nakano T;Aird WC;Doi T
在以往的巨核细胞特异性基因表达机制的研究中，在每个巨核细胞特异性基因启动子中发现了几个ETS基序。虽然这些研究表明，几个ETS家族蛋白调节巨核细胞特异性基因的表达，只有少数ETS家族蛋白已被确定。血小板因子4（plateletfactor 4，PF 4）是巨核细胞特异性基因，其启动子包含多个ETS基序。我们先前已经证明ETS-1与PF 4启动子中的ETS基序结合。然而，其他ETS基序的功能仍不清楚。本研究的目的是研究PF 4启动子中一个新的功能性ETS基序，并鉴定与该基序结合的蛋白质。在电泳迁移率变动试验和染色质免疫沉淀试验中，FLI-1、ELF-1和GABP结合到-51 ETS位点。FLI-1、ELF-1和GABP的表达激活了HepG 2细胞中的PF 4启动子。-51 ETS位点的突变减弱了FLI-1、ELF-1和GABA介导的启动子反式激活。siRNA分析表明FLI-1、ELF-1和GABP调节HEL细胞中PF 4基因的表达。在这三种蛋白质中，只有FLI-1与加塔-1协同激活启动子。此外，在巨核细胞分化过程中，只有FLI-1表达增加。最后，通过使用体外ES细胞分化系统的新型报告基因测定，证实了-51 ETS位点在生理性巨核细胞分化过程中对PF 4启动子激活的重要性。总之，这些数据表明，FLI-1，ELF-1，和GABP调节PF 4基因表达通过-51 ETS网站在巨核细胞和暗示PF 4基因表达的分化阶段特异性调节多种ETS因子。
In previous studies on the mechanism underlying megakaryocyte-specific gene expression, several ETS motifs were found in each megakaryocyte-specific gene promoter. Although these studies suggested that several ETS family proteins regulate megakaryocyte-specific gene expression, only a few ETS family proteins have been identified. Platelet factor 4 (PF4) is a megakaryocyte-specific gene and its promoter includes multiple ETS motifs. We had previously shown that ETS-1 binds to an ETS motif in the PF4 promoter. However, the functions of the other ETS motifs are still unclear. The goal of this study was to investigate a novel functional ETS motif in the PF4 promoter and identify proteins binding to the motif. In electrophoretic mobility shift assays and a chromatin immunoprecipitation assay, FLI-1, ELF-1, and GABP bound to the −51 ETS site. Expression of FLI-1, ELF-1, and GABP activated the PF4 promoter in HepG2 cells. Mutation of a −51 ETS site attenuated FLI-1-, ELF-1-, and GABP-mediated transactivation of the promoter. siRNA analysis demonstrated that FLI-1, ELF-1, and GABP regulate PF4 gene expression in HEL cells. Among these three proteins, only FLI-1 synergistically activated the promoter with GATA-1. In addition, only FLI-1 expression was increased during megakaryocytic differentiation. Finally, the importance of the −51 ETS site for the activation of the PF4 promoter during physiological megakaryocytic differentiation was confirmed by a novel reporter gene assay using in vitro ES cell differentiation system. Together, these data suggest that FLI-1, ELF-1, and GABP regulate PF4 gene expression through the −51 ETS site in megakaryocytes and implicate the differentiation stage-specific regulation of PF4 gene expression by multiple ETS factors.
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