Mechanism of transcriptional regulation by methyl-CpG binding protein MBD1
Mechanism of transcriptional regulation by methyl-CpG binding protein MBD1
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DOI：
10.1128/mcb.20.14.5107-5118.2000
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发表时间：
2000-07-01
影响因子：
5.3
通讯作者：
Nakao, M
中科院分区：
文献类型：
--
作者：
Fujita, N;Shimotake, N;Nakao, M
MBD 1是一种哺乳动物蛋白，结合对称甲基化的CpG序列，并调节与DNA甲基化相关的基因表达。该蛋白质在介导甲基化的生物学后果的甲基-CpG结合蛋白家族中具有保守序列，称为甲基-CpG结合结构域（MBD）。此外，由于选择性剪接事件，MBD 1至少有五种亚型，导致MBD 1亚型v1（MBD 1v 1）和MBD 1v 2中存在CXXC 1、CXXC 2和CXXC 3，MBD 1v 3和-v4中存在CXXC 1和CXXC 2。在本研究中，我们研究了MBD，CXXC和C-末端转录抑制结构域（TRD）在MBD 1中的意义。细菌表达的MBD有效地结合密集甲基化的DNA，而不是稀疏甲基化的DNA。在甲基化缺陷的果蝇SL 2细胞和哺乳动物CHO-K1细胞中，MBD 1v 1优先抑制未甲基化和稀疏甲基化启动子的转录，而MBD 1v 3抑制密集甲基化但不抑制未甲基化启动子的活性。MBD 1v 1中的CXXC 3序列负责结合未甲基化启动子的能力。另外，为了研究MBD 1中的MBD的结构与功能的相关性，构建了将功能上重要的残基Arg 22、Arg 30、Asp 32、Tyr 34、Arg 44、Ser 45、Tyr 52变更为丙氨酸的突变型MBD 1。除Ser 45和Tyr 52外，没有一个重组MBD突变体与密集甲基化或未甲基化的DNA结合，并且绿色荧光蛋白融合的MBD 1突变体不能正确地定位在细胞核中。所有MBD 1v 1和-v3突变体都失去了甲基化依赖的基因阻遏活性。基于这些发现，我们得出结论，MBD 1作为转录调节因子，依赖于甲基-CpG对的密度，通过MBD，CXXC和TRD序列的合作。
MBD1 is a mammalian protein that binds symmetrically methylated CpG sequences and regulates gene expression in association with DNA methylation. This protein possesses a conserved sequence, named methyl-CpG binding domain (MBD), among a family of methyl-CpG binding proteins that mediate the biological consequences of the methylation. In addition, MBD1 has at least five isoforms due to alternative splicing events, resulting in the presence of CXXC1, CXXC2, and CXXC3 in MBD1 isoforms v1 (MBD1v1) and MBD1v2, and CXXC1 and CXXC2 in MBD1v3 and -v4. In the present study, we have investigated the significance of MBD, CXXC, and the C-terminal transcriptional repression domain (TRD) in MBD1. A bacterially expressed MBD binds efficiently to densely methylated rather than to sparsely methylated DNAs. In both methylation-deficient Drosophila melanogaster SL2 cells and mammalian CHO-K1 cells, MBD1v1 represses transcription preferentially from both unmethylated and sparsely methylated promoters, while MBD1v3 inhibits densely methylated but not unmethylated promoter activities. The CXXC3 sequence in MBD1v1 is responsible for the ability to bind unmethylated promoter. Furthermore, we have constructed mutant-type MBD1s in which the functionally important residues Arg22, Arg30, Asp32, Tyr34, Arg44, Ser45, and Tyr52 are changed to alanine to investigate the correlation between the structure and function of the MBD in MBD1. Excepting those for Ser45 and Tyr52, none of the recombinant MBD mutants bound to the densely methylated or unmethylated DNAs, and green fluorescent protein-fused MBD1 mutants did not localize properly in the nucleus. All the MBD1v1 and -v3 mutants lost the activity of methylation-dependent gene repression. Based on these findings we have concluded;that MBD1 acts as a transcriptional regulator depending on the density of methyl-CpG pairs through the cooperation of MBD, CXXC, and TRD sequences.