Research on the regulation of vertebrates' genome DNA methylation

高等真核生物基因组发生甲基化修饰。这种甲基化影响的不是编码，而是表达信息。甲基化修饰是由三个DNA甲基转移酶(Dnmt)和一个相关因子Dnmt3L催化的，没有催化活性。本研究旨在阐明DNMT对DNA甲基化模式产生和维持的分子机制，并获得以下结果：DNMT1在双链DNA上连续甲基化半甲基化CpG，有助于维持基因组甲基化模式。出乎意料的是，Dnmt1经常无法甲基化半甲基化的位置。DNMT1的N-末端部分形成独立的结构域结构，并具有识别DNA小丛的DNA结合活性。我们发现Np95是体内维持DNA甲基化模式所必需的新因子。这些性质有助于理解DNA甲基化模式的维持机制。De novo类型的酶DNMT3A几乎不甲基化核小体核心区，但DNMT3b可以显著甲基化。DNMT3A有效地甲基化了核小体的裸露连接区，其活性被连接子组蛋白H1抑制。组蛋白H1可能参与了DNMT3A产生DNA甲基化模式的调控。在发生整体甲基化的雄性原始生殖细胞(PGC)中，DNMT3A的亚型Dnmt3a2和Dnmt3L高表达。Dnmt3L是甲基化的先决条件。我们发现Dnmt3a2在生理盐分条件下不能表现出催化活性，但在Dnmt3L存在下恢复了活性。提示Dnmt3a2的这种依赖于盐的催化活性是PGC中需要Dnmt3L的全局甲基化的原因。Dnmt3的这些特性有助于理解DNA甲基化模式的创建。

Genome in higher eukaryotes suffers methylation modification. This methylation affects not the coding but the expression information. The methylation modification is catalyzed by three DNA methyltransferases (Dnmt) and one related factor Dnmt3L, which has no catalytic activity. In the research project, we aimed to elucidate the molecular mechanism of the Dnmt on the creation and the maintenance of DNA methylation patterns, and obtained the following results.Dnmtl, which contributes to the maintenance of genome methylation patterns, processively methylates hemimethylated CpG on a double-stranded DNA. Unexpectedly, Dnmt1 frequently fails to methylate hemimethylated positions. The N-terminal portion of Dnmt1 forms independent domain structure, and possesses DNA binding activity that recognizes minor grove of DNA. We found Np95 as a new factor that is necessary for the maintenance of DNA methylation patterns in vivo. These properties help in understanding the mechanism of the maintenance of DNA methylation patterns.De novo-type enzyme Dnmt3a hardly methylates nucleosome core region, but that Dnmt3b can significantly methylates. Dnmt3a efficiently methylates the naked linker region of nucleosomes, and the activity is inhibited by linker histone H1. Histone H1 may contribute to the regulation in creating DNA methylation patterns by Dnmt3a. In male primordial germ cells (PGC), in which global methylation occurs, Dnmt3a2, which is an isoform of Dnmt3a, and Dnmt3L are highly expressed. Dnmt3L is prerequisite for the global methylation. We found that Dnmt3a2 cannot show catalytic activity under physiological salt conditions, but restores the activity in the presence of Dnmt3L. It is suggested that this salt-dependent catalytic activity of Dnmt3a2 is the reason why the global methylation in PGC requires Dnmt3L. These properties of Dnmt3 help in understanding the creation of DNA methylation patterns.