Heterodimeric DNA methyltransferases as a platform for creating designer zinc finger methyltransferases for targeted DNA methylation in cells.
Heterodimeric DNA methyltransferases as a platform for creating designer zinc finger methyltransferases for targeted DNA methylation in cells.
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DOI：
10.1093/nar/gkp1126
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发表时间：
2010-03
影响因子：
14.9
通讯作者：
Ostermeier M
中科院分区：
文献类型：
--
作者：
Meister GE;Chandrasegaran S;Ostermeier M
将甲基化靶向特定基因组位点的能力将进一步研究DNA甲基化的生物学作用，并可能提供沉默基因表达和治疗涉及异常低甲基化的疾病的工具。DNA甲基转移酶与锌指的端对端融合已显示使甲基化偏向于所需区域。然而，该策略是固有地受限的，因为甲基转移酶结构域保持活性，而不管锌指结构域是否在其同源位点处结合并且可以甲基化非靶位点。我们展示了一种替代策略，其中DNA甲基转移酶的片段，在其甲基化DNA的能力受损，融合到两个锌指设计结合9 bp的位点侧翼的甲基化靶位点。利用天然异源二聚体DNA甲基转移酶M.EcoHK31I，它甲基化5′-YGGCCR-3′的内部胞嘧啶，我们证明了这种策略可以产生一个甲基转移酶，能够在大肠杆菌中的目标位点显着水平的甲基化，在非目标位点的甲基化检测不到的水平。然而，在锌指甲基转移酶的较高表达水平下可以检测到一些非靶甲基化，这表明需要进一步改进以获得所需的排他性靶特异性。
The ability to target methylation to specific genomic sites would further the study of DNA methylation’s biological role and potentially offer a tool for silencing gene expression and for treating diseases involving abnormal hypomethylation. The end-to-end fusion of DNA methyltransferases to zinc fingers has been shown to bias methylation to desired regions. However, the strategy is inherently limited because the methyltransferase domain remains active regardless of whether the zinc finger domain is bound at its cognate site and can methylate non-target sites. We demonstrate an alternative strategy in which fragments of a DNA methyltransferase, compromised in their ability to methylate DNA, are fused to two zinc fingers designed to bind 9 bp sites flanking a methylation target site. Using the naturally heterodimeric DNA methyltransferase M.EcoHK31I, which methylates the inner cytosine of 5′-YGGCCR-3′, we demonstrate that this strategy can yield a methyltransferase capable of significant levels of methylation at the target site with undetectable levels of methylation at non-target sites in Escherichia coli. However, some non-target methylation could be detected at higher expression levels of the zinc finger methyltransferase indicating that further improvements will be necessary to attain the desired exclusive target specificity.
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影响因子：
4.1
作者：
JACOBS, D;BROWN, NL
通讯作者：
BROWN, NL
DOI：
10.1073/pnas.0609502103
发表时间：
2006-12-26
影响因子：
11.1
作者：
Minczuk, Michal;Papworth, Monika A.;Klug, Aaron
通讯作者：
Klug, Aaron
影响因子：
46.9
作者：
van Steensel, B;Henikoff, S
通讯作者：
Henikoff, S
影响因子：
8
作者：
Deng, GR;Chen, A;Kim, YS
通讯作者：
Kim, YS
影响因子：
3.7
作者：
Fung, Wai-To;Sze, Kong-Hung;Shaw, Pang-Chui
通讯作者：
Shaw, Pang-Chui