Sp1-targeted inhibition of gene transcription by WP631 in transfected lymphocytes
Sp1-targeted inhibition of gene transcription by WP631 in transfected lymphocytes
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DOI：
10.1021/bi036185e
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发表时间：
2004-06-15
期刊：
影响因子：
2.9
通讯作者：
Portugal, J
中科院分区：
文献类型：
--
作者：
Mansilla, S;Priebe, W;Portugal, J
Sp1转录因子与DNA的结合被认为是小配体干扰基因转录的潜在靶标。我们试图将体内（细胞培养）sp1与DNA结合的直接抑制与调节细胞周期进程的通路网络的间接影响区分开来，后者可能在不直接干扰Sp1-DNA相互作用的情况下减少转录。我们测试了Sp3蛋白是否可以抑制Sp1激活的转录并干扰药物- dna相互作用，Sp3蛋白的推定结合序列与Sp1位点重叠。一个由wtGLUT1（野生型葡萄糖转运蛋白1）基因启动子或突变的mut2GLUT1启动子组成的模型系统，与CAT（氯霉素乙酰转移酶）报告基因相连接，用于分析瞬时转染Jurkat T淋巴细胞中Sp1和Sp3转录因子过表达的影响。Bisanthracycline WP631是一种有效的sp1激活转录的体外抑制剂，研究了其在转染Jurkat T淋巴细胞中特异性抑制转录的能力。mut2GLUT1启动子被用来进一步区分WP631对Sp1-DNA复合物的干扰和sp3诱导的抑制，因为在该启动子中sp3结合位点被取消，取而代之的是WP631的高亲和力结合位点。
The binding of Sp1 transcription factor to DNA is considered a potential target for small ligands designed to interfere with gene transcription. We attempted to distinguish the direct inhibition of the Sp1-binding to DNA in vivo (cell culture) from more indirect effects due to the network of pathways that modulate cell cycle progression, which may decrease transcription without direct interference with Sp1-DNA interactions. We tested whether the Sp3 protein, whose putative binding sequence overlaps the Sp1 site, can inhibit Sp1-activated transcription and interfere with drug-DNA interactions. A well-characterized model system consisting of a wtGLUT1 (wild-type glucose transporter 1) gene promoter, or a mutated mut2GLUT1 promoter, linked to a CAT (chloramphenicol acetyltransferase) reporter gene, was used to analyze the effects of overexpressed Sp1 and Sp3 transcription factors in transiently transfected Jurkat T lymphocytes. Bisanthracycline WP631, a potent inhibitor of Sp1-activated transcription in vitro, was assayed for its ability to specifically inhibit transcription in transfected Jurkat T lymphocytes. The mut2GLUT1 promoter was used to further discriminate between the WP631 interference with Sp1-DNA complexes and Sp3-induced inhibition, since the Sp3-binding site is canceled in this promoter and replaced by a high-affinity binding site for WP631.