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

点击翻译按钮获取中文摘要

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.