Uroplakin lb gene transcription in urothelial tumor cells is regulated by CpG methylation
Uroplakin lb gene transcription in urothelial tumor cells is regulated by CpG methylation
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DOI：
10.1593/neo.05364
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发表时间：
2005-12-01
期刊：
影响因子：
4.8
通讯作者：
Jackson, P
中科院分区：
文献类型：
--
作者：
Cowled, P;Kanter, I;Jackson, P
Uroplakin lb 是尿路上皮细胞表面的结构蛋白。在许多移行细胞癌中，尿斑蛋白 1b mRNA 水平显着降低或缺失，但其分子机制仍未确定。之前，我们发现尿斑蛋白 1b 表达的丧失与近端启动子内 Sp1/NF kappa B 结合基序的 CpG 甲基化相关。在本研究中，我们表明报告基因活性被该启动子区域中三个 CpG 对的甲基化完全阻断。使用纯化蛋白质或核提取物进行的凝胶位移分析表明，Sp1 和 NF kappa B 与包含三个 CpG 对中的两个的基序结合。有趣的是，在凝胶迁移分析中，这两个 CpG 位点的甲基化并没有阻止蛋白质与启动子的结合。此外，这两个 CpG 的突变不会影响报告基因活性，但跨越每个 CpG 的 6 bp 片段的突变会部分抑制报告基因活性，表明这些位点具有功能。在使用表达单个蛋白质的质粒的转染实验中，证实了调节报告基因活性需要 Sp1 和 NF kappa B。我们的数据表明，特定 CpG 位点的甲基化可以通过阻断 Sp1 和 NF kappa B 的结合，至少部分地沉默尿斑蛋白 lb 启动子，尽管可能还涉及其他因素。
Uroplakin lb is a structural protein on the surface of urothelial cells. Levels of uroplakin lb mRNA are dramatically reduced or absent in many transitional cell carcinomas, but the molecular mechanisms responsible remain undetermined. Previously, we showed that loss of uroplakin lb expression correlated with CpG methylation of Sp1/NF kappa B-binding motifs within the proximal promoter. In this study, we show that reporter activity was completely blocked by the methylation of three CpG pairs in this promoter region. Gel shift analysis using purified proteins or nuclear extracts showed that Sp1 and NF kappa B bound to motifs encompassing two of the three CpG pairs. Interestingly, the methylation of these two CpG sites did not prevent the binding of proteins to the promoter in gel shift analyses. Additionally, mutation of these two CpGs did not affect reporter activity, but mutation of 6-bp fragment spanning each CpG partially inhibited reporter activity, suggesting that these sites were functional. A requirement for both Sp1 and NF kappa B in regulating reporter activity was confirmed in transfection experiments using plasmids expressing individual proteins. Our data suggest that the methylation of specific CpG sites can silence the uroplakin lb promoter, at least in part, by blocking the binding of Sp1 and NF kappa B, although other factors may be involved.