Targeted DNA demethylation of the Arabidopsis genome using the human TET1 catalytic domain.
Targeted DNA demethylation of the Arabidopsis genome using the human TET1 catalytic domain.
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DOI：
10.1073/pnas.1716945115
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发表时间：
2018-02-27
影响因子：
11.1
通讯作者：
Jacobsen SE
中科院分区：
文献类型：
--
作者：
Gallego-Bartolomé J;Gardiner J;Liu W;Papikian A;Ghoshal B;Kuo HY;Zhao JM;Segal DJ;Jacobsen SE
DNA甲基化是一种表观遗传修饰，参与基因沉默。对这种修饰的研究通常依赖于使用影响甲基化维持的突变体或化学物质。这些方法导致甲基化的全局变化，并且使得研究甲基化对特定位点的基因表达或染色质的影响变得困难。在这项研究中，我们开发了针对植物DNA去甲基化的工具。我们报告了有效的靶向去甲基化和对全局甲基化模式的最小影响，并表明在一种情况下，靶向去甲基化是可遗传的。这些工具可用于解决有关DNA甲基化生物学的基本问题，以及开发新的生物技术策略来修改基因表达和创建新的植物性状表观等位基因。DNA甲基化是一种重要的表观遗传修饰，参与基因调控和转座因子沉默。DNA甲基化的变化可以是可遗传的，因此可以导致稳定的表观等位基因的形成。植物中稳定表观等位基因的一个充分表征的例子是fwa，其由FLOWWWA AGENINGEN（FWA）基因启动子中DNA胞嘧啶甲基化（5 mC）的缺失组成，引起FWA的上调和可遗传的晚花表型。在这里，我们证明了人类去甲基化酶TEN-ELEVEN TRANSLOCATION 1（TET 1cd）的催化结构域与设计用于靶向FWA启动子的人工锌指（ZF）之间的融合可以引起高效的靶向去甲基化、FWA上调和可遗传的晚花表型。另外设计的ZF-TET 1cd融合靶向CACTA 1转座子的甲基化区域也引起靶向去甲基化和表达变化。最后，我们使用TET 1cd和修改的SunTag系统开发了基于CRISPR/dCas 9的靶向去甲基化系统。与ZF-TET 1cd融合体类似，SunTag-TET 1cd系统在针对FWA或CACTA 1基因座时能够靶向去甲基化并激活基因表达。我们的研究提供了在基因组中特定位点靶向去除5 mC的工具，具有高特异性和最小的脱靶效应。这些工具提供了为感兴趣的性状开发新的表观等位基因的机会，并重新激活先前沉默的基因、转基因或转座子的表达。
DNA methylation is an epigenetic modification involved in gene silencing. Studies of this modification usually rely on the use of mutants or chemicals that affect methylation maintenance. Those approaches cause global changes in methylation and make difficult the study of the impact of methylation on gene expression or chromatin at specific loci. In this study, we develop tools to target DNA demethylation in plants. We report efficient on-target demethylation and minimal effects on global methylation patterns, and show that in one case, targeted demethylation is heritable. These tools can be used to approach basic questions about DNA methylation biology, as well as to develop new biotechnology strategies to modify gene expression and create new plant trait epialleles. DNA methylation is an important epigenetic modification involved in gene regulation and transposable element silencing. Changes in DNA methylation can be heritable and, thus, can lead to the formation of stable epialleles. A well-characterized example of a stable epiallele in plants is fwa, which consists of the loss of DNA cytosine methylation (5mC) in the promoter of the FLOWERING WAGENINGEN (FWA) gene, causing up-regulation of FWA and a heritable late-flowering phenotype. Here we demonstrate that a fusion between the catalytic domain of the human demethylase TEN-ELEVEN TRANSLOCATION1 (TET1cd) and an artificial zinc finger (ZF) designed to target the FWA promoter can cause highly efficient targeted demethylation, FWA up-regulation, and a heritable late-flowering phenotype. Additional ZF–TET1cd fusions designed to target methylated regions of the CACTA1 transposon also caused targeted demethylation and changes in expression. Finally, we have developed a CRISPR/dCas9-based targeted demethylation system using the TET1cd and a modified SunTag system. Similar to the ZF–TET1cd fusions, the SunTag–TET1cd system is able to target demethylation and activate gene expression when directed to the FWA or CACTA1 loci. Our study provides tools for targeted removal of 5mC at specific loci in the genome with high specificity and minimal off-target effects. These tools provide the opportunity to develop new epialleles for traits of interest, and to reactivate expression of previously silenced genes, transgenes, or transposons.
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影响因子：
10.5
作者：
Bartee, L;Malagnac, F;Bender, J
通讯作者：
Bender, J
影响因子：
56.9
作者：
Jacobsen, SE;Meyerowitz, EM
通讯作者：
Meyerowitz, EM
DOI：
10.1093/bioinformatics/btu638
发表时间：
2015-01-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Anders S;Pyl PT;Huber W
通讯作者：
Huber W
DOI：
10.4236/abb.2016.75023
发表时间：
2016-01-01
期刊：
Advances in Bioscience and Biotechnology
影响因子：
--
作者：
Hollwey, E.;Watson, M.;Meyer, P.
通讯作者：
Meyer, P.
影响因子：
14.9
作者：
Chen H;Kazemier HG;de Groote ML;Ruiters MH;Xu GL;Rots MG
通讯作者：
Rots MG