A +220 GATA motif mediates basal but not endotoxin-repressible expression of the von Willebrand factor promoter in Hprt-targeted transgenic mice.
A +220 GATA motif mediates basal but not endotoxin-repressible expression of the von Willebrand factor promoter in Hprt-targeted transgenic mice.
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DOI：
10.1111/j.1538-7836.2009.03501.x
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发表时间：
2009-08
期刊：
影响因子：
--
通讯作者：
Aird WC
中科院分区：
文献类型：
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作者：
Liu J;Kanki Y;Okada Y;Jin E;Yano K;Shih SC;Minami T;Aird WC
血管性血友病因子（vonWillebrand factor，VWF）基因是内皮细胞时空异质性的标志物。在+220处的加塔基序与体外基础VWF表达有关。其他研究表明，GATA 3和VWF在炎症介质的应答中转录下调。我们的目标是确定+220加塔基序在体内介导VWF启动子表达的重要性，并阐明加塔元件是否在VWF表达的空间和/或时间调节中起作用。在人脐静脉内皮细胞（HUVEC）中进行ChIP和电泳迁移率变动测定。将含有3.6kb的人VWF启动子的报告基因构建体转染到培养的内皮细胞中或靶向小鼠的Hprt基因座，所述启动子具有和不具有+220加塔元件突变。Hprt靶向小鼠经历内毒素血症。在蛋白质-DNA结合测定中，+220加塔基序结合加塔-2、-3和-6。加塔位点的突变导致HUVEC中基础启动子活性降低。当靶向小鼠的Hprt基因座时，加塔突变导致表达LacZ的血管床中的启动子活性显著成比例地降低。脂多糖（LPS）的全身给药导致VWF mRNA表达和启动子活性的广泛减少。LPS介导的VWF启动子抑制不受加塔突变的影响。VWF启动子的-2182和第一个内含子末端之间的区域包含LPS介导的基因抑制的信息。+220加塔基序对VWF基因的基础表达很重要，但对LPS抑制的表达不重要。
The von Willebrand factor (VWF) gene is a marker for spatial and temporal heterogeneity of the endothelium. A GATA motif at +220 has been implicated in basal VWF expression in vitro. Other studies have shown that GATA3 and VWF are transcriptionally downregulated in response to inflammatory mediators. Our goal was to determine the importance of the +220 GATA motif in mediating expression of VWF promoter in vivo, and to elucidate whether the GATA element plays a role in spatial and/or temporal regulation of VWF expression. ChIP and electrophoretic mobility shift assays were carried out in human umbilical vein endothelial cells (HUVEC). Reporter gene constructs containing 3.6 kb of the human VWF promoter with and without amutation of the +220 GATA element were transfected into cultured endothelial cells or targeted to the Hprt locus of mice. The Hprt-targeted mice were subjected to endotoxemia. In protein-DNA binding assays, the +220 GATA motif bound GATA-2, -3 and -6. Mutation of the GATA site resulted in reduced basal promoter activity in HUVEC. When targeted to the Hprt locus of mice, the GATA mutation resulted in a significant, proportionate reduction of promoter activity in LacZ expressing vascular beds. Systemic administration of lipopolysaccharide (LPS) resulted in a widespread reduction in VWF mRNA expression and promoter activity. LPS-mediated repression of the VWF promoter was unaffected by the GATA mutation. A region of the VWF promoter between −2182 and the end of the first intron contains information for LPS-mediated gene repression. The +220 GATA motif is important for basal, but not LPS-repressible expression of the VWF gene.