Methylation in hMLH1 promoter interferes with its binding to transcription factor CBF and inhibits gene expression
Methylation in hMLH1 promoter interferes with its binding to transcription factor CBF and inhibits gene expression
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DOI：
10.1038/sj.onc.1204891
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发表时间：
2001-10-25
期刊：
影响因子：
8
通讯作者：
Kim, YS
中科院分区：
文献类型：
--
作者：
Deng, GR;Chen, A;Kim, YS
微卫星不稳定性（MSI）是由错配修复基因（例如hMLH1、hMSH2）功能障碍引起的。 hMLH1 表达缺失和 hMLH1 启动子中 CpG 位点甲基化经常出现在伴有 MSI 的散发性结直肠癌中。在本研究中，通过使用含有不同长度的hMLH1启动子和荧光素酶报告基因的构建体进行瞬时转染测定，我们定位了hMLH1启动子的近端区域，该区域在调节该基因中起主要作用。事实上，无论 hMLH1 表达水平如何，所有宿主细胞系中的荧光素酶活性都很高，这表明转录机制即使在非表达细胞中也是完整的。当hMLH1启动子在转染前体外甲基化时，转染子中的荧光素酶活性显着降低。这一观察结果表明甲基化导致 hMLH1 启动子活性的抑制。通过电泳迁移率变动分析（EMSA），我们在该区域鉴定了一个CCAAT盒，它特异性结合转录因子CBF。 CCAAT盒的突变不仅抑制了其与CBF因子的结合，而且降低了其驱动荧光素酶基因表达的能力。通过用表达显性失活 CBF-B 突变体的质粒抑制启动子活性，进一步证实了 CBF 在激活转录中的作用。 CCAAT 盒上游两个碱基对的 CpG 位点的甲基化抑制了 CBF 与 CCAAT 盒的结合。我们得出结论，相邻 CpG 位点的甲基化会抑制 CBF 转录与相应 CCAAT 盒的结合，并且是结肠癌细胞中 hMLH1 基因沉默的原因之一。
Microsatellite instability (MSI) is caused by the dysfunction of mismatch repair genes, such as hMLH1, hMSH2. Loss of hMLH1 expression and methylation of CpG sites in hMLH1 promoter are frequently present in sporadic colorectal cancer with MSI. In this study, by transient transfection assay with constructs containing different lengths of hMLH1 promoter and a luciferase reporter gene, we located a proximal region of hMLH1 promoter, which plays a main role in regulating the gene. The fact that luciferase activities were high in all host cell lines regardless of their hMLH1 expression levels indicates that the transcription machinery is intact even in non-expressing cells. When hMLH1 promoter was in vitro methylated before transfection, the luciferase activities in the transfectants were significantly reduced. This observation indicates that methylation causes the inhibition of hMLH1 promoter activity. By electrophoretic mobility shift assay (EMSA), we identified a CCAAT box in this region, which specifically bound transcription factor CBF. Mutations in CCAAT box not only inhibited its binding to CBF factor, but also reduced its ability to drive the expression of luciferase gene. The role of CBF in activating transcription was further substantiated by inhibition of promoter activity with a plasmid expressing a dominant negative CBF-B mutant. Methylation at a CpG site two base pairs upstream of the CCAAT box inhibited the binding of CBF to CCAAT box. We conclude that methylation of an adjacent CpG site inhibits binding of the CBF transcription to the corresponding CCAAT box, and is one of the causes of hMLH1 gene silencing in colon cancer cells.