Characterization of the human DNA methyltransferase splice variant Dnmt1b
Characterization of the human DNA methyltransferase splice variant Dnmt1b
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DOI：
10.1074/jbc.275.15.10754
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发表时间：
2000-04-14
影响因子：
4.8
通讯作者：
MacLeod, AR
中科院分区：
文献类型：
--
作者：
Bonfils, C;Beaulieu, N;MacLeod, AR
胞嘧啶 DNA 甲基化的组织和基因特异性模式是脊椎动物基因组的特征，DNA 甲基化模式的产生和正确维持对于胚胎发育至关重要，通过靶向破坏 Dnmt1（负责维持 DNA 甲基化的基因）或靶向破坏 Dnmt3a 或 Dnmt3b，参与新形成的甲基化模式生成的基因。最近，鉴定了由 Dnmt1 的选择性剪接产生的新 mRNA Dnmt1b (Hsu, D. W., Lin, M. J., Lee, T. L., Wen, S. C., Chen, X., and Shen, C. K., (1999) Proc. Natl. Acad Sci. U. S. A. 96, 9751-9756)。 Dnmt1b mRNA 的丰度通过半定量逆转录聚合酶链式反应进行估计，并被认为编码主要的 C-5 DNA 甲基转移酶亚型。在此，我们报告了这种新型 DNA 甲基转移酶转录物 Dnmt1b 及其蛋白质产物在人类细胞系和新鲜分离的人外周血单核细胞中的表征。通过定量 RNase 保护分析确定，Dnmt1b 转录物的丰度范围为人类细胞中 Dnmt1 的 6% 至 25%。针对 Dnmt1 5' 和 3' 末端的第二代反义抑制剂抑制细胞中 Dnmt1 和 Dnmt1b 的积累。从杆状病毒表达系统纯化的 Dnmt1b 蛋白被证明是一种功能性 DNA 甲基转移酶，并且 DNA 和 S-腺苷-L-甲硫氨酸的米氏常数与杆状病毒表达的 Dnmt1 相似，然而，针对 Dnmt1b 表位产生的抗体表明，Dnmt1b 蛋白的存在水平约为 2-5%。 因此，Dnmt1 仅代表人类细胞中的一种次要 DNA 甲基转移酶亚型。
Tissue- and gene-specific patterns of cytosine-DNA methylation are characteristic features of vertebrate genomes, The generation and proper maintenance of DNA methylation patterns are essential for embryonic development, as demonstrated by the lethal phenotypes of mice with either a targeted disruption of Dnmt1, the gene responsible for the maintenance of DNA methylation, or targeted disruption of Dnmt3a or Dnmt3b, the genes involved in generation of newly formed methylation patterns. Recently, a novel mRNA, Dnmt1b, resulting from alternative splicing of Dnmt1 was identified (Hsu, D. W., Lin, M. J., Lee, T. L., Wen, S. C., Chen, X., and Shen, C. K., (1999) Proc. Natl. Acad Sci. U. S. A. 96, 9751-9756). The abundance of Dnmt1b mRNA was estimated by semiquantitative reverse transcription polymerase chain reaction and was suggested to encode a major C-5 DNA methyltransferase isoform, Here we report characterization of this novel DNA methyltransferase transcript, Dnmt1b, and its protein product in human cell lines and in freshly isolated human peripheral blood mononuclear cells. The abundance of Dnmt1b transcript, as determined by quantitative RNase protection analysis, was determined to range from 6% to 25% of Dnmt1 in human cells. Second generation antisense inhibitors targeted to the 5'- and 3'-ends of Dnmt1 inhibited the accumulation of both Dnmt1 and Dnmt1b in cells. Dnmt1b protein purified from a baculovirus expression system was demonstrated to be a functional DNA methyltransferase, and to have Michaelis constants for both DNA and S-adenosyl-L-methionine similar to baculovirus-expressed Dnmt1, However, antibodies raised against Dnmt1b epitopes demonstrated that Dnmt1b protein was present at approximately 2-5% of the level of Dnmt1 and therefore represents only a minor DNA methyltransferase isoform in human cells.