A three-kilobase fragment of the human Robo4 promoter directs cell type-specific expression in endothelium
A three-kilobase fragment of the human Robo4 promoter directs cell type-specific expression in endothelium
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DOI：
10.1161/01.res.0000269779.10644.dc
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发表时间：
2007-06-22
影响因子：
20.1
通讯作者：
Aird, William C.
中科院分区：
文献类型：
--
作者：
Okada, Yoshiaki;Yano, Kiichiro;Aird, William C.
Robo 4是roundabout家族的成员，仅在内皮细胞中表达，并与内皮细胞迁移和血管生成有关。在这里，我们报告的人Robo 4启动子的克隆和表征。3-kb 5 '侧翼区指导体外内皮细胞特异性表达。缺失和突变分析揭示了位于-2528和-2941的两个12-bp回文DNA序列、位于-42和-153的两个SP1共有基序以及位于-119的ETS共有基序的功能重要性。在电泳迁移率变动测定使用supershifting抗体，SP1基序结合SP1蛋白，而ETS网站结合异源二聚体成员的ETS家族，GA结合蛋白（GABP）。这些DNA-蛋白质相互作用通过染色质免疫沉淀试验证实。用针对GABP和SP1的小干扰RNA转染原代人内皮细胞导致内源性Robo 4 mRNA表达显著降低（约50%）。将3 kb Robo 4启动子与LacZ偶联，并通过同源重组将所得盒引入小鼠的Hprt基因座。在成人器官的血管系统（特别是微血管）、肿瘤异种移植物和胚胎中观察到报告基因活性，其中它与内皮细胞特异性标志物CD 31共定位。成人组织和肿瘤中的LacZ mRNA水平与内源性Robo 4、CD 31和血管内皮钙粘蛋白的mRNA水平相关。此外，报告基因表达的模式与LacZ敲入内源性Robo 4基因座的小鼠中观察到的模式相似。总之，这些数据表明，3- kb的上游启动子的人Robo 4包含信息的细胞类型特异性表达在完整的内皮细胞。
Robo4, a member of the roundabout family, is expressed exclusively in endothelial cells and has been implicated in endothelial cell migration and angiogenesis. Here we report the cloning and characterization of the human Robo4 promoter. The 3-kb 5'-flanking region directs endothelial cell-specific expression in vitro. Deletion and mutation analyses revealed the functional importance of two 12-bp palindromic DNA sequences at -2528 and -2941, 2 SP1 consensus motifs at -42 and -153, and an ETS consensus motif at -119. In electrophoretic mobility shift assays using supershifting antibodies, the SP1 motifs bound SP1 protein, whereas the ETS site bound a heterodimeric member of the ETS family, GA binding protein (GABP). These DNA-protein interactions were confirmed by chromatin immunoprecipitation assays. Transfection of primary human endothelial cells with small interfering RNA against GABP and SP1 resulted in a significant (approximate to 50%) reduction in endogenous Robo4 mRNA expression. The 3-kb Robo4 promoter was coupled to LacZ, and the resulting cassette was introduced into the Hprt locus of mice by homologous recombination. Reporter gene activity was observed in the vasculature of adult organs (particularly in microvessels), tumor xenografts, and embryos, where it colocalized with the endothelial cell-specific marker CD31. LacZ mRNA levels in adult tissues and tumors correlated with mRNA levels for endogenous Robo4, CD31, and vascular endothelial cadherin. Moreover, the pattern of reporter gene expression was similar to that observed in mice in which LacZ was knocked into the endogenous Robo4 locus. Together, these data suggest that 3- kb upstream promoter of human Robo4 contains information for cell type-specific expression in the intact endothelium.