Promoter-specific activation and demethylation by MBD2/demethylase
Promoter-specific activation and demethylation by MBD2/demethylase
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DOI：
10.1074/jbc.c200408200
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发表时间：
2002-09-27
影响因子：
4.8
通讯作者：
Szyf, M
中科院分区：
文献类型：
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作者：
Detich, N;Theberge, J;Szyf, M
MBD 2是甲基-CpG结合蛋白家族中唯一一个被报道既是转录抑制因子又是DNA去甲基化酶（dMTase）的成员。为了理解MBD 2/dMTase的明显矛盾的功能，我们研究了dMTase过表达对瞬时转染到HEK 293细胞中的各种体外甲基化启动子活性的影响。我们发现，MBD 2/dMTase表达载体（His-dMTase）的强制表达以时间和剂量依赖性方式差异激活两种甲基化报告基因，pSV 40-CAT（与氯霉素乙酰转移酶（CAT）报告基因相邻的SV 40无增强子启动子）和pGL 2 T + I4 xTBRE（与荧光素酶报告基因相邻的p21启动子区域）。His-dMTase在96小时后使pSV 40-CAT表达增加3-10倍，而pGL 2 T + I4 xTBRE表达仅在48小时后增加2-3倍，并且在96小时时没有进一步增加。基因激活不是普遍的，因为p19-ARF启动子没有作用。然后，我们评估激活是否可能是由于启动子区域内的去甲基化。使用亚硫酸氢盐作图，我们发现外源性表达的His-dMTase诱导SV 40启动子内的10个CpG位点中的8个去甲基化。使用mC 32 pG寡核苷酸、底物和来自His-dMTase或空pcDNA3.1His载体转染的HEK 293细胞的纯化Q-Sepharose级分的体外CpG脱甲基酶测定也证实了His-dMTase增加细胞中脱甲基酶活性的观察结果。我们建议，一个单一的蛋白质同时具有阻遏物和去甲基化酶的功能已经发展到协调一个程序，需要抑制一些甲基化基因和激活其他。
MBD2 is the only member of a family of methyl-CpG-binding proteins that has been reported to be both a transcriptional repressor and a DNA demethylase (dMTase). To understand the apparently contradictory function of MBD2/dMTase, we studied the effects of dMTase overexpression on the activity of various in vitro methylated promoters transiently transfected into HEK293 cells. We found that forced expression of a MBD2/dMTase expression vector (His-dMTase) differentially activated two methylated reporters, pSV40-CAT (the SV40 enhancerless promoter adjacent to the chloramphenicol acetyltransferase (CAT) reporter gene) and pGL2T+I4xTBRE (a region of the p21 promoter next to the luciferase reporter gene), in a time- and dose-dependent manner. His-dMTase increased pSV40-CAT expression by 3-10-fold after 96 h, while pGL2T+I4xTBRE expression was increased by 2-3-fold after only 48 h and did not further increase at 96 h. Gene activation was not universal because no effect was seen with the p19-ARF promoter. We then assessed whether activation might be due to demethylation within the promoter region. Using bisulfite mapping, we found that exogenous expression of His-dMTase induced demethylation at 8 of the 10 CpG sites within the SV40 promoter. The observation that His-dMTase increases the demethylase activity in the cells was also confirmed using an in vitro CpG demethylase assay with a mC32pG oligonucleotide, substrate and purified Q-Sepharose fractions from HEK293 cells transfected with His-dMTase or empty pcDNA3.1His vector. We propose that a single protein possessing both repressor and demethylase functions has evolved to coordinate a program that requires suppression of some methylated genes and activation of others.