Maintenance and modification of epigenetic information: Coordination of DNA replication and methylation during the cell cycle and in development

DNA甲基转移酶在表观遗传信息的建立、维护和修改中起着核心作用。我们以前已经证明维持酶DNMT1与复制机制有关，而从头酶DNMT3a和Dnmt3b则不相关。我们现在想利用活细胞显微镜和荧光漂白技术，在存在或不存在5-aza-DC等基于机制的抑制剂的情况下，分析Dnmt1与体内复制机制及其DNA底物的相互作用的动力学。为了阐明相互作用因素的作用，我们将比较活细胞中特定Dnmt1突变体的动态特性，并测试它们重新甲基化/拯救Dnmt缺陷ES细胞的能力。虽然有明确的证据表明受精后父亲的基因组主动去甲基化，但对植入前发育过程中的被动去甲基化知之甚少。我们已经证明，Dnmt1保留在细胞质中，从而与复制机制及其DNA底物分离。我们将Dnmt1/3a/3b注射到受精卵的细胞核中，并确定其对甲基化模式和发育的影响。我们将分析核Dnmt1是否阻止甲基化的丢失，以及从头甲基化是否需要额外的因素。这些实验应该有助于阐明DNA甲基化和表观遗传重编程在发育和疾病中的调节。

DNA methyltransferases play a central role in the establishment, maintenance and modification of epigenetic information. We have previously shown that the maintenance enzyme, Dnmt 1, is associated with the replication machinery while the de novo enzymes, Dnmt3a and Dnmt3b, are not. We now want to analyse the dynamics of Dnmt1 interaction with the replication machinery and its DNA substrate in vivo using live cell microscopy and fluorescence bleaching techniques in the presence or absence of mechanism based inhibitors like 5-Aza-dC. To elucidate the role of interacting factors, we will compare the dynamic properties of specific Dnmt1 mutants in living cells and will test their ability to remethylate/rescue Dnmt-deficient ES cells. While there is clear evidence for active demethylation of the paternal genome after fertilization, only little is known about passive demethylation during preimplantation development. We have shown that Dnmt1 is retained in the cytoplasm and thus separated from the replication machinery and its DNA substrate. We will inject Dnmt1/3a/3b into nuclei of zygotes and determine the effect on methylation patterns and development. We will analyse whether nuclear Dnmt1 prevents the loss of methylation and whether additional factors are required for de novo methylation. These experiments should help to elucidate the regulation of DNA methylation and epigenetic reprogramming in development and disease.