An Sp1/Sp3 binding polymorphism confers methylation protection.
An Sp1/Sp3 binding polymorphism confers methylation protection.
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DOI：
10.1371/journal.pgen.1000162
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发表时间：
2008-08-22
期刊：
影响因子：
4.5
通讯作者：
Issa JP
中科院分区：
文献类型：
--
作者：
Boumber YA;Kondo Y;Chen X;Shen L;Guo Y;Tellez C;Estécio MR;Ahmed S;Issa JP
数百个基因在癌症中显示出异常的DNA超甲基化，但人们对这种超甲基化的原因知之甚少。我们发现RIL是癌症中常见的甲基化靶点。在寻找影响RIL高甲基化的因素时，我们在其转录起始点附近发现了一个12bp的多态序列，它产生了一个长的等位基因。对纯合子肿瘤的焦磷酸测序显示短等位基因的甲基化增加了2.1倍(P<；0.001)。对rIL杂合子癌症的亚硫酸氢盐测序显示，短等位基因的甲基化程度是长等位基因的3.1%(P<；0.001)。未甲基化的长和短EBV转化细胞系之间的表达水平比较显示，体内表达没有差异。电泳迁移率改变分析表明，长等位基因的插入区域与Sp1和Sp3转录因子结合，这是短等位基因所没有的结合。瞬时转染rIL等位基因特异性转基因后，早期没有发现额外的Sp1位点对转录的影响。然而，甲基化种子构建体的稳定转染显示，随着从头开始甲基化的最终扩散，短等位基因的转录水平逐渐下降。相反，长等位基因的表达水平随着时间的推移表现出稳定的水平，荧光素酶和∼的甲基化水平通过亚硫酸氢盐测序降低了2-3倍(P<；0.001)，这表明Sp1位点的多态防止了时间依赖的沉默。我们的发现表明，在某些基因中，癌症中的高甲基化是由启动子上的蛋白质-DNA相互作用决定的，并提供了一种新的机制，通过这种机制，遗传多态可以影响表观遗传状态。在癌症中引导DNA高甲基化的因素还知之甚少。我们发现候选抑癌基因RIL是癌症中常见的甲基化靶点。在这里，我们报告了它的转录起始点附近的一个12个碱基的多态序列，它产生了一个长的等位基因。甲基化分析表明，在老年结肠癌、结肠癌和白血病中，短等位基因的甲基化程度是长等位基因的2.13.1倍(P<；0.001)。在EBV转化的细胞系中，短等位基因和长等位基因的表达水平相似。凝胶迁移率改变分析表明，长等位基因的插入区段与Sp1和Sp3转录因子结合。RIL等位基因特异性转基因早期对转录没有影响，但甲基化种子构建体表现出从短等位基因开始逐渐减少转录，最终重新甲基化的扩散。相比之下，荧光素酶测定的长等位基因随着时间的推移表现出稳定的表达，亚硫酸氢盐测序表明∼甲基化水平降低了2-3倍(P<；0.001)，这表明Sp1位点的多态防止了依赖时间的沉默。我们的发现表明，在某些基因中，癌症中的高甲基化是由启动子上的蛋白质-DNA相互作用决定的，并提供了一种新的机制，通过这种机制，遗传多态可以影响表观遗传状态。
Hundreds of genes show aberrant DNA hypermethylation in cancer, yet little is known about the causes of this hypermethylation. We identified RIL as a frequent methylation target in cancer. In search for factors that influence RIL hypermethylation, we found a 12-bp polymorphic sequence around its transcription start site that creates a long allele. Pyrosequencing of homozygous tumors revealed a 2.1-fold higher methylation for the short alleles (P<0.001). Bisulfite sequencing of cancers heterozygous for RIL showed that the short alleles are 3.1-fold more methylated than the long (P<0.001). The comparison of expression levels between unmethylated long and short EBV-transformed cell lines showed no difference in expression in vivo. Electrophorectic mobility shift assay showed that the inserted region of the long allele binds Sp1 and Sp3 transcription factors, a binding that is absent in the short allele. Transient transfection of RIL allele-specific transgenes showed no effects of the additional Sp1 site on transcription early on. However, stable transfection of methylation-seeded constructs showed gradually decreasing transcription levels from the short allele with eventual spreading of de novo methylation. In contrast, the long allele showed stable levels of expression over time as measured by luciferase and ∼2–3-fold lower levels of methylation by bisulfite sequencing (P<0.001), suggesting that the polymorphic Sp1 site protects against time-dependent silencing. Our finding demonstrates that, in some genes, hypermethylation in cancer is dictated by protein-DNA interactions at the promoters and provides a novel mechanism by which genetic polymorphisms can influence an epigenetic state. The factors that guide DNA hypermethylation in cancer are poorly understood. We identified the candidate tumor-suppressor gene, RIL, as a frequent methylation target in cancer. Here, we report on a 12-bp polymorphic sequence around its transcription start site that creates a long allele. Methylation analysis showed that, in aging colon, colon cancer, and leukemias, the short allele had 2.1–3.1-fold higher methylation than the long allele (P<0.001). Short and long alleles had similar expression levels in EBV-transformed cell lines. Electrophorectic mobility shift assay showed that the inserted region of the long allele binds Sp1 and Sp3 transcription factors. Transfection of RIL allele-specific transgenes showed no effects of the additional Sp1 site on transcription early on, but methylation-seeded constructs showed gradually decreasing transcription from the short allele with eventual spreading of de novo methylation. By contrast, the long allele showed stable expression over time as measured by luciferase, and ∼2–3-fold lower levels of methylation by bisulfite sequencing (P<0.001), suggesting that the polymorphic Sp1 site protects against time-dependent silencing. Our finding demonstrates that in some genes, hypermethylation in cancer is dictated by protein-DNA interactions at the promoters and provides a novel mechanism by which genetic polymorphisms can influence an epigenetic state.
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