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

DNA的共价修饰，如胞嘧啶甲基化，可以诱导遗传性基因沉默。如果表观遗传修饰可以特异性靶向，那么转录治疗的新方法应该会产生。为了解决这一挑战，我们构建了甲基转移酶，通过适应序列激活的组装策略，它只在期望的位点起作用。这是首次在体内成功应用序列激活酶重组方法。在本研究中，为了确定分裂蛋白结构域在DNA结合和甲基化中的功能，我们分别表达和纯化了分裂蛋白结构域。利用这些结构域，进行DNA结合分析。结果表明，这些结构域与特定的DNA靶标协同结合。这些结构域之间的相互作用是在分裂结构域的目标序列上组装的直接证据。互补蛋白分析已被证明在哺乳动物细胞中蛋白质相互作用的解剖有用。然而，很少有直接的方法来评估分裂结构域相互作用的动力学。此外，为了扩展基因组DNA上的可靶向DNA序列，构建了几个锌指结构域。这些结构域与内源性靶标结合。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。从而优化了甲基转移酶密码子的使用。甲基转移酶的表达显著增加。本研究结果将进一步拓展对分裂蛋白结构域设计的认识。

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.