Identification of a DNA methylation-dependent activator sequence in the pseudoxanthoma elasticum gene, ABCC6
Identification of a DNA methylation-dependent activator sequence in the pseudoxanthoma elasticum gene, ABCC6
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DOI：
10.1074/jbc.m501139200
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发表时间：
2005-05-13
影响因子：
4.8
通讯作者：
Váradi, A
中科院分区：
文献类型：
--
作者：
Arányi, T;Ratajewski, M;Váradi, A
ABCC 6编码MRP 6，MRP 6是ABC蛋白家族的成员，具有未知的生理作用。人类ABCC 6及其两个假基因共享99%相同的DNA序列。ABCC 6的功能缺失突变与弹性假黄瘤（PXE）的发生有关，这是一种影响弹性组织的隐性遗传性疾病。在编码区发现了各种致病突变;然而，真正的PXE患者的ABCC 6编码区的突变检出率仅为80%。这表明调控区的多态性或突变可能有助于疾病的发展。在这里，我们报告的ABCC 6基因启动子的第一个特征。5'调控区的系统发育计算机分析揭示了两个嵌入CpG岛的进化保守序列元件的存在。对ABCC 6和假基因的DNA甲基化的研究确定了近端启动子中CpG岛的甲基化与细胞系中ABCC 6表达水平之间的相关性。激活子和阻遏子序列均通过报告基因测定在近端启动子中被发现。最有效的激活子序列是非表达细胞中内源基因上受DNA甲基化保护的保守元件之一。最后，该序列的体外甲基化抑制荧光素酶启动子构建体的转录活性。总之，这些结果确定了ABCC 6启动子中的DNA甲基化依赖性激活子序列。
ABCC6 encodes MRP6, a member of the ABC protein family with an unknown physiological role. The human ABCC6 and its two pseudogenes share 99% identical DNA sequence. Loss-of-function mutations of ABCC6 are associated with the development of pseudoxanthoma elasticum (PXE), a recessive hereditary disorder affecting the elastic tissues. Various disease-causing mutations were found in the coding region; however, the mutation detection rate in the ABCC6 coding region of bona fide PXE patients is only similar to 80%. This suggests that polymorphisms or mutations in the regulatory regions may contribute to the development of the disease. Here, we report the first characterization of the ABCC6 gene promoter. Phylogenetic in silico analysis of the 5' regulatory regions revealed the presence of two evolutionarily conserved sequence elements embedded in CpG islands. The study of DNA methylation of ABCC6 and the pseudogenes identified a correlation between the methylation of the CpG island in the proximal promoter and the ABCC6 expression level in cell lines. Both activator and repressor sequences were uncovered in the proximal promoter by reporter gene assays. The most potent activator sequence was one of the conserved elements protected by DNA methylation on the endogenous gene in non-expressing cells. Finally, in vitro methylation of this sequence inhibits the transcriptional activity of the luciferase promoter constructs. Altogether these results identify a DNA methylation-dependent activator sequence in the ABCC6 promoter.