Targeted Methylation and Gene Silencing of VEGF-A in Human Cells by Using a Designed Dnmt3a-Dnmt3L Single-Chain Fusion Protein with Increased DNA Methylation Activity
Targeted Methylation and Gene Silencing of VEGF-A in Human Cells by Using a Designed Dnmt3a-Dnmt3L Single-Chain Fusion Protein with Increased DNA Methylation Activity
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DOI：
10.1016/j.jmb.2012.11.038
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发表时间：
2013-02-08
影响因子：
5.6
通讯作者：
Jeltsch, Albert
中科院分区：
文献类型：
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作者：
Siddique, Abu Nasar;Nunna, Suneetha;Jeltsch, Albert
Dnmt 3a从头DNA甲基转移酶（Dnmt 3a-C）的C-末端结构域与Dnmt 3L的C-末端结构域形成复合物，这刺激其催化活性。我们产生并表征了这两个域的单链（SC）融合蛋白，其接头长度在16和30个氨基酸残基之间。纯化的sc蛋白在体外表现出比Dnmt 3a-C高约10倍的DNA甲基化活性，并且在细菌细胞中也更有活性。将Dnmt 3a-3L sc酶与靶向血管内皮细胞生长因子A（VEGF-A）启动子的人工锌指蛋白融合后，我们证明了在人类细胞中成功靶向VEGF-A启动子的DNA甲基化，并观察到可以实现基因启动子中12个CpG位点的几乎完全甲基化。Dnmt 3a-3L sc酶的靶向甲基化比Dnmt 3a-C高约两倍，表明Dnmt 3a-3L sc变体作为嵌合DNA甲基转移酶中的催化模块比Dnmt 3a-C更有效。用Dnmt 3a-3L sc变体靶向甲基化VEGF-A启动子导致VEGF-A表达的强烈沉默，表明内源性启动子的人工DNA甲基化是实现人类细胞中相应基因沉默的有力策略。(C)2012爱思唯尔有限公司保留所有权利。
The C-terminal domain of the Dnmt3a de novo DNA methyltransferase (Dnmt3a-C) forms a complex with the C-terminal domain of Dnmt3L, which stimulates its catalytic activity. We generated and characterized single-chain (sc) fusion proteins of both these domains with linker lengths between 16 and 30 amino acid residues. The purified sc proteins showed about 10-fold higher DNA methylation activities than Dnmt3a-C in vitro and were more active in bacterial cells as well. After fusing the Dnmt3a-3L sc enzyme to an artificial zinc-finger protein targeting the vascular endothelial cell growth factor A (VEGF-A) promoter, we demonstrate successful targeting of DNA methylation to the VEGF-A promoter in human cells and observed that almost complete methylation of 12 CpG sites in the gene promoter could be achieved. Targeted methylation by the Dnmt3a-3L sc enzymes was about twofold higher than that of Dnmt3a-C, indicating that Dnmt3a-3L sc variants are more efficient as catalytic modules in chimeric DNA methyltransfeases than Dnmt3a-C. Targeted methylation of the VEGF-A promoter with the Dnmt3a-3L sc variant led to a strong silencing of VEGF-A expression, indicating that the artificial DNA methylation of an endogenous promoter is a powerful strategy to achieve silencing of the corresponding gene in human cells. (C) 2012 Elsevier Ltd. All rights reserved.