Methylated DNA-binding proteins from arabidopsis
Methylated DNA-binding proteins from arabidopsis
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DOI：
10.1104/pp.103.026708
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发表时间：
2003-12-01
期刊：
影响因子：
7.4
通讯作者：
Sano, H
中科院分区：
文献类型：
--
作者：
Ito, M;Koike, A;Sano, H
5-甲基胞嘧啶(m(5)C)在表观遗传控制中起关键作用，通常被含有甲基-CpG结合结构域(MBD)的蛋白质识别。数据库筛选已经从拟南芥中鉴定出至少12个可能的甲基CpG结合蛋白；我们已经为其中7个分离到了相应的cDNA。绿色荧光蛋白融合蛋白分析显示，尽管大小和氨基酸序列不同，但所有7种蛋白质都专一地迁移到细胞核中，这表明这与染色质结构有关。然而，用细菌表达的蛋白质和CPGS中含有m(5)C的合成寡核苷酸进行的DNA结合分析表明，只有一个能与之特异结合，命名为AtMBD5。进一步的分析表明，AtMBD5有效地与CpNpN(N为A、T或C)中的m(5)C结合，但不能与CpNpG序列中的m(5)C结合，这两个序列都经常存在于植物DNA中。其他6种蛋白要么显示非特异性DNA结合，要么不能识别m(5)C。RNA印迹杂交和免疫印迹分析表明，AtMBD5基本上存在于所有组织中。利用真核启动子驱动的绿色荧光蛋白，AtMBD5在雌蕊和根尖中得到了有效表达。由于CpG中的m(5)Cs和CpNpN中的m(5)Cs分别被认为在基因表达和基因沉默中起作用，因此本研究结果提示AtMBD5可能在活跃的增殖细胞中对基因的调节和/或自卫具有不同的功能。
The 5-methylcytosines (m(5)C) play a critical role in epigenetic control, often being recognized by proteins containing a methyl-CpG-binding domain (MBD). Database screening has identified at least 12 putative methyl-CpG-binding proteins from Arabidopsis; we have isolated corresponding cDNAs for seven of them. Despite variation in size and amino acid sequence, all seven proteins exclusively migrate into the nucleus as revealed by green fluorescent protein fusion protein assay, suggesting a relationship with chromatin structure. However, DNA-binding assays using bacterially expressed proteins and synthetic oligonucleotides containing m(5)C in CpGs showed only one to specifically bind, designated AtMBD5. Further analysis showed that AtMBD5 efficiently binds to m(5)C in CpNpN (N is A, T, or C) but not in CpNpG sequences, both frequently found in plant DNA. The other six proteins showed either nonspecific DNA binding or no ability to recognize m(5)C. RNA-blot hybridization and immunoblot analysis indicated AtMBD5 to be present essentially in all tissues. Using green fluorescent protein driven by the authentic promoter, AtMBD5 was found to be actively expressed in pistils and root tips. Because m(5)Cs in CpG and CpNpN are considered to function in gene expression and gene silencing, respectively, the present results suggest that AtMBD5 may have distinct functions in regulation and/or self defense of genes in actively proliferating cells.