Identification of the promoter of human transcription factor Sp3 and evidence of the role of factors Sp1 and Sp3 in the expression of Sp3 protein.
Identification of the promoter of human transcription factor Sp3 and evidence of the role of factors Sp1 and Sp3 in the expression of Sp3 protein.
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DOI：
10.1016/j.gene.2005.02.007
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发表时间：
2005-05
期刊：
影响因子：
3.5
通讯作者：
Z. Lou;V. Maher;J. Mccormick
中科院分区：
文献类型：
--
作者：
Z. Lou;V. Maher;J. Mccormick
在一项过表达转录因子 Sp1 在人类纤维肉瘤细胞系形成肿瘤中的作用的研究中 [Cancer Res., 65 (2005) 1007]，我们发现 Sp1 特异性核酶的表达不仅降低了 Sp1 蛋白的水平，而且还降低了 Sp3 蛋白的水平，并且当纤维肉瘤细胞系中这两种转录因子的蛋白水平降低到接近正常人中的水平时成纤维细胞，细胞系不能再形成肿瘤。如果 Sp1 基因和 Sp3 基因的启动子都具有 Sp1/Sp3 转录因子结合位点，并且这些位点对细胞中 Sp1 和 Sp3 蛋白的表达水平至关重要，则 Sp1 特异性核酶可以降低 Sp1 蛋白和 Sp3 蛋白的表达水平。 Sp1最小启动子已被鉴定，它有两个Sp1/Sp3位点[J.生物。化学。 276（2001）22126]。为了表征 Sp3 启动子，我们分离了 Sp3 基因 5' 侧翼区域的 2.1 kb，其中包含 Sp1/Sp3 结合位点，并使用表达报告基因测定表明其具有启动子活性。然后，我们系统地减小了 5' 侧翼区域的大小，并确定 nt-339 至 nt-39 片段（其中在 nt-181 处包含一个 Sp1/Sp3 结合位点，在 nt-168 处包含另一个结合位点）保留了与 2.1kb 区域相同的启动子活性。电泳迁移率变动分析表明 Sp3 蛋白和 Sp1 蛋白均与这两个位点结合。通过突变这些结合位点中的一个或两个，我们使用报告基因测定表明每个位点都是完整启动子活性所必需的。然后，我们设计了一种Sp3特异性核酶，在Sp1蛋白和Sp3蛋白高水平表达的人纤维肉瘤细胞系中表达它，结果发现，这两种蛋白的表达水平确实显着降低。
In a study of the role of transcription factor Sp1 in the formation of tumors by human fibrosarcoma cell lines that overexpress it [Cancer Res., 65 (2005) 1007], we found that expression of an Sp1-specific ribozyme, not only reduced the level of Sp1 protein, but also that of Sp3 protein, and that when the protein levels of these two transcription factors in the fibrosarcoma cell lines were reduced to near that found in normal human fibroblasts, the cell lines could no longer form tumors. An Sp1-specific ribozyme could reduce the level of expression of both Sp1 protein and Sp3 protein if the promoter of the Sp1 gene and that of the Sp3 gene both have Sp1/Sp3 transcription factor binding sites and if such sites are critically responsible for the level of expression of both Sp1 and Sp3 protein in the cells. The Sp1 minimal promoter has been identified and it has two Sp1/Sp3 sites [J. Biol. Chem. 276 (2001) 22126]. To characterize the Sp3 promoter, we isolated 2.1 kb of the 5′-flanking region of the Sp3 gene, which contains Sp1/Sp3 binding sites, and using an expression reporter assay, showed that it has promoter activity. We then systematically reduced the size of the 5′ flanking region, and determined that the nt-339 to nt-39 fragment, which contains an Sp1/Sp3 binding site at nt-181 and another at nt-168, retained the same promoter activity as the 2.1kb region. Electrophoretic mobility shift assays indicated that both Sp3 protein and Sp1protein bind to these two sites. By mutating either or both of these binding sites, we showed using the reporter assay that each site is required for full promoter activity. We then designed an Sp3-specific ribozyme, expressed it in a human fibrosarcoma cell line in which Sp1 protein and Sp3 protein are expressed at high levels, and found that, indeed, the level of expression of both proteins was significantly reduced.