Role of Uhrf1 and histone H3 ubiquitylation in replicative DNA methylation

基因组和表观基因组需要被忠实地复制，以在世代之间保持遗传信息和细胞身份。DNA和染色质复制中不可修复的错误会导致基因组不稳定，从而导致癌症等几种疾病。表观遗传标记包括DNA中胞嘧啶的甲基化和核小体中组蛋白尾部的多种翻译后修饰(PTM)，这些修饰控制染色质的可及性和调节基因的活性。Gambus Lab研究了DNA复制反应中染色质上泛素化的蛋白质，发现组蛋白H3在赖氨酸18和23(K18和K23)上泛素化是非洲爪哇卵提取液中DNA复制过程中最丰富的泛素化蛋白质。据报道，这种修饰是uhrf1泛素连接酶作用的结果，DNA甲基酶DNMT1在复制后需要完全甲基化半甲基化的DNA。虽然uhrf1和Dnmt1在DNA甲基化模式遗传中的作用是明确的，但确切的机制仍有待剖析。我这个项目的目的是了解在DNA复制过程中，uhrf1和组蛋白H3泛素化导致DNA甲基化维持的机制。为了研究这一机制，我将使用非洲爪哇卵子提取模型系统，该系统允许在非常同步的设置下对DNA复制反应进行生化操作。

Genome and epigenome need to be faithfully duplicated to maintain genetic information and cell identity through generations. Unrepaired errors in DNA and chromatin replication lead to genomic instability that causes several diseases as cancer. Epigenetic marks comprise methylation of cytosine within DNA and many types of post-translational modifications (PTMs) of histones tails within nucleosomes, that control chromatin accessibility and regulate gene activity. Examining proteins ubiquitylated on chromatin during DNA replication reaction, Gambus lab identified histone H3 ubiquitylation on lysine 18 and 23 (K18 and K23) as the most abundant ubiquitylated protein during DNA replication in Xenopus egg extract. This modification has been reported by others to be a result of action of Uhrf1 ubiquitin ligase and be required for DNA methylase DNMT1 to fully methylate hemi-methylated DNA after replication. Although the role of Uhrf1 and Dnmt1 in inheritance of DNA methylation patterns is clear, the exact mechanism remains to be dissected. The aim of my project is understanding the mechanism by which Uhrf1 and histone H3 ubiquitylation leads to maintenance of DNA methylation during DNA replication. To investigate this mechanism, I am going to use Xenopus laevis egg extract model system, which allows for biochemical manipulation of DNA replication reaction in very synchronous setting.