Arányi Tamás, Ratajewski Marcin, Bardóczy Viola, Pulaski Lukasz, Bors András, Tordai Attila, Váradi András
Institute of Enzymology, Hungarian Academy of Sciences, H-1113 Budapest, Hungary.
J Biol Chem. 2005 May 13;280(19):18643-50. doi: 10.1074/jbc.M501139200. Epub 2005 Mar 9.
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 approximately 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.
ABCC6编码MRP6，它是ABC蛋白家族的成员，其生理作用尚不清楚。人类ABCC6及其两个假基因共享99%相同的DNA序列。ABCC6的功能丧失突变与弹性假黄瘤（PXE）的发生有关，PXE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ABCC6编码区的突变检测率仅约为80%。这表明调控区的多态性或突变可能导致该疾病的发生。在此，我们报告了ABCC6基因启动子的首次特征描述。对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ABCC6及其假基因的DNA甲基化研究确定了近端启动子中CpG岛的甲基化与细胞系中ABCC6表达水平之间的相关性。通过报告基因分析在近端启动子中发现了激活剂和抑制剂序列。最有效的激活剂序列是在非表达细胞中受内源性基因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了荧光素酶启动子构建体的转录活性。总之，这些结果确定了ABCC6启动子中一个依赖DNA甲基化的激活剂序列。
