Physical and functional interactions between the human DNMT3L protein and members of the de novo methyltransferase family
Physical and functional interactions between the human DNMT3L protein and members of the de novo methyltransferase family
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DOI：
10.1002/jcb.20447
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发表时间：
2005-08-01
影响因子：
4
通讯作者：
Chédin, F
中科院分区：
文献类型：
--
作者：
Chen, ZX;Mann, JR;Chédin, F
新生甲基转移酶样蛋白DNMT3L是生殖细胞中印迹基因甲基化所必需的。虽然酶无活性，但人类DNMT3L被证明是小鼠Dnmt3a从头甲基化的一般刺激因子。已经在小鼠和人类中描述了几种具有发育阶段和组织特异性表达模式的DNMT3A和DNMT3B亚型，从而对DNMT3L刺激的生理伴侣的身份提出了质疑。在这里，我们使用了一种基于片段体的体内甲基转移酶试验，系统地分析了人类DNMT3A和DNMT3B的五种亚型的活性和受人类DNMT3L的刺激。我们的研究结果表明，人类DNMT3A、DNMT3A2、DNMT3B1和DNMT3B2具有催化能力，而DNMT3B3在我们的实验中没有活性。我们还报道了所有四种活性同工异构体的活性在与DNMT3L共表达时显着增加，尽管程度不同。这是首次全面描述人类DNMT3A和DNMT3B亚型的体内活性及其与DNMT3L的功能相互作用。为了进一步阐明DNMT3L刺激DNA甲基化的机制，我们详细绘制了介导人类DNMT3L与人类DNMT3A和DNMT3B相互作用的结构域。我们的研究结果表明，DNMT3L的c端是与DNMT3A和DNMT3B相互作用所需的唯一区域，并且相互作用通过DNMT3A和DNMT3B的c端催化结构域进行。讨论了这些发现对新生甲基转移酶和基因组印迹的调控的意义。
The de novo methyltransferase-like protein, DNMT3L, is required for methylation of imprinted genes in germ cells. Although enzymatically inactive, human DNMT3L was shown to act as a general stimulatory factor for de novo methylation by murine Dnmt3a. Several isoforms of DNMT3A and DNMT3B with development-stage and tissue-specific expression patterns have been described in mouse and human, thus bringing into question the identity of the physiological partner(s) for stimulation by DNMT3L. Here, we used an episome-based in vivo methyltransferase assay to systematically analyze five isoforms of human DNMT3A and DNMT3B for activity and stimulation by human DNMT3L. Our results show that human DNMT3A, DNMT3A2, DNMT3B1, and DNMT3B2 are catalytically competent, while DNMT3B3 is inactive in our assay. We also report that the activity of all four active isoforms is significantly increased upon co-expression with DNMT3L, albeit to varying extents. This is the first comprehensive description of the in vivo activities of the poorly characterized human DNMT3A and DNMT3B isoforms and of their functional interactions with DNMT3L. To further elucidate the mechanism by which DNMT3L stimulates DNA methylation, we have mapped in detail the domains that mediate interaction of human DNMT3L with human DNMT3A and DNMT3B. Our results show that the C-terminus of DNMT3L is the only region required for interaction with DNMT3A and DNMT3B and that interaction takes place through the C-terminal catalytic domain of DNMT3A and DNMT3B. The implications of these findings for the regulation of de novo methyltransferases and genomic imprinting are discussed.