Oligomerization of DNMT3A Controls the Mechanism of de Novo DNA Methylation
Oligomerization of DNMT3A Controls the Mechanism of de Novo DNA Methylation
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DOI：
10.1074/jbc.m111.284687
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发表时间：
2011-12-02
影响因子：
4.8
通讯作者：
Reich, Norbert O.
中科院分区：
文献类型：
--
作者：
Holz-Schietinger, Celeste;Matje, Douglas M.;Reich, Norbert O.
DNMT3A 是两种人类从头 DNA 甲基转移酶之一，对于通过细胞发育和分化调节基因表达至关重要。在这里，我们描述了 DNMT3A.DNMT3A 同四聚体和 DNMT3A.DNMT3L 异四聚体界面上单个氨基酸突变的后果，包括与急性髓系白血病 (AML) 和骨髓增生异常综合征的发展有关的后果。通过计算接口扫描开发了 DNMT3A 同四聚体的模型，并使用光散射和电泳迁移率变动测定进行了测试。使用荧光各向异性和稳态动力学对不同的寡聚状态进行功能表征。导致 DNMT3A 二聚体的残基替换（包括在 AML 患者中鉴定的残基）显示出甲基化活性的微小变化，但失去了对多位点 DNA 底物进行持续催化的能力，这与高度持续进行的野生型酶不同。我们的结果与体内 DNA 甲基化的双峰分布以及 AML 患者中聚集甲基化的丧失一致。与已知的相互作用伙伴 DNMT3L 进行四聚化，可挽救持续催化作用，证明 DNMT3A 四聚体界面上的蛋白质结合可以调节甲基化模式。我们的结果为 DNMT3A 活性和表观遗传印记的调节提供了结构机制。
DNMT3A is one of two human de novo DNA methyltransferases essential for regulating gene expression through cellular development and differentiation. Here we describe the consequences of single amino acid mutations, including those implicated in the development of acute myeloid leukemia (AML) and myelodysplastic syndromes, at the DNMT3A.DNMT3A homotetramer and DNMT3A.DNMT3L heterotetramer interfaces. A model for the DNMT3A homotetramer was developed via computational interface scanning and tested using light scattering and electrophoretic mobility shift assays. Distinct oligomeric states were functionally characterized using fluorescence anisotropy and steady-state kinetics. Replacement of residues that result in DNMT3A dimers, including those identified in AML patients, show minor changes in methylation activity but lose the capacity for processive catalysis on multisite DNA substrates, unlike the highly processive wild-type enzyme. Our results are consistent with the bimodal distribution of DNA methylation in vivo and the loss of clustered methylation in AML patients. Tetramerization with the known interacting partner DNMT3L, rescues processive catalysis, demonstrating that protein binding at the DNMT3A tetramer interface can modulate methylation patterning. Our results provide a structural mechanism for the regulation of DNMT3A activity and epigenetic imprinting.