Development of orthogonal combinations of artificial DNA methyltrasferases and their substrates for study of heritable patterns of DNA methylation.

开发人工 DNA 甲基转移酶及其底物的正交组合，用于研究 DNA 甲基化的遗传模式。

基本信息

批准号：
23710251
负责人：
NOMURA Wataru
金额：
$ 3万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Young Scientists (B)
财政年份：
2011
资助国家：
日本
起止时间：
2011 至 2012
项目状态：
已结题

项目摘要

Covalent modification of DNA, such as cytosine methylation, can induce heritable gene silencing. If epigenetic modifications can be specifically targeted, new approaches to transcriptional therapy should result. To address this challenge we have constructed methyltransferases that would act only at a desired site by adapting the sequence-enabled assembly strategy. This is the first successful application of the sequence-enable enzyme reassembly approach in vivo. In this study, to determine the functions of split protein domains in DNA binding and methylation, the split domains were expressed and purified separately. Utilizing these domains, DNA binding analyses were performed. The results indicate cooperative binding of the domains to the specific DNA targets. This interaction between the domains shows a direct evidence of assembly on the target sequence of split domains. The complementary protein assays have been shown their usefulness in dissection of protein interaction in mammalian cells. However, a few of direct approach to evaluate kinetics of interaction of split domains have been performed. Moreover, to expand the targetable DNA sequences on genomic DNA, several zinc finger domains were constructed. These domains showed DNA binding on endogenous targets. To perform efficient DNA methylation in mammalian cells, the expression of methyltransferase would be a key factor. Thus, the codon usage of methyltransferase was optimized. The expression of methyltransferase was greatly increased. The present results would expand the knowledge in the design of split protein domains.

DNA的共价修饰，例如胞嘧啶甲基化，可以诱导可遗传的基因沉默。如果可以专门针对表观遗传修饰，则应导致转录疗法的新方法。为了应对这一挑战，我们已经构建了甲基转移酶，这些甲基转移酶只能通过调整启用序列的组装策略来在所需的站点上起作用。这是体内序列启用酶重新组装方法的首次成功应用。在这项研究中，为了确定DNA结合和甲基化中分裂蛋白结构域的功能，分别表达了分裂结构域并分别纯化。利用这些域，进行了DNA结合分析。结果表明域与特定DNA靶标的合作结合。域之间的这种相互作用显示了分裂域目标序列上组装的直接证据。互补的蛋白质测定已显示出它们在哺乳动物细胞中蛋白质相互作用的解剖方面的有用性。但是，已经进行了一些评估分裂域相互作用动力学的直接方法。此外，为了扩展基因组DNA上的靶向DNA序列，构建了几个锌指域。这些结构域显示DNA在内源性靶标上的结合。为了在哺乳动物细胞中进行有效的DNA甲基化，甲基转移酶的表达将是一个关键因素。因此，优化了甲基转移酶的密码子使用。甲基转移酶的表达大大增加。目前的结果将扩大分裂蛋白质结构域设计的知识。