A Universal DNA Hemimethylation Probe

项目总结 胞嘧啶DNA甲基化是一种复制后的表观遗传修饰，它调节组织和 遗传信息的表达。DNA甲基化缺陷与许多疾病相关， 最显著的是基因组印记障碍、免疫缺陷和癌症。在脊椎动物中，胞嘧啶 甲基化发生在CpG上下文中。DNA复制产生一种短暂的半甲基化DNA 中级的。由于其转瞬即逝的性质和缺乏适当的工具，dna半甲基化仍然存在。 开发不足。耐人寻味的是，最近的研究揭示了持续DNA半甲基化的有趣例子。 这些研究表明，DNA甲基化的功能状态范围更广。开始解决这个问题 在我们对基因组表观遗传调控这一潜在关键因素的理解上存在差距，我们 建议修改uhrf1的集合和环指相关(SRA)结构域以产生DNA 用于体外和体内研究的半甲基化探针。紧凑的SRA域可以自给自足地进行绑定 半甲基化DNA具有很高的特异性。为了实现这项建议的总体目标，我们将首先 生产重组SRA结构域蛋白并开发分离和可视化的方法 体外复杂基因组中的半甲基化DNA。为了在体内研究DNA半甲基化，我们将表达一种 小鼠胚胎中SRA结构域与mScarlet荧光蛋白的融合蛋白 干细胞和转基因小鼠。我们将使用带有半甲基化DNA的细胞的实时成像来验证 SRA-mScarlet融合蛋白的染色质定位模式和动力学。这项研究将加强 我们对基因组在发育和疾病中的表观遗传调控的理解。

PROJECT SUMMARY Cytosine DNA methylation is a post-replicative epigenetic modification that regulates the organization and expression of the genetic information. Deficiencies in DNA methylation associate with numerous diseases, most notably genomic imprinting disorders, immunodeficiencies, and cancers. In the vertebrates, cytosine methylation happens in the CpG context. DNA replication produces a short-lived hemimethylated DNA intermediate. Due to its fleeting nature and the lack of adequate tools, DNA hemimethylation remains underexplored. Intriguingly, recent studies revealed fascinating examples of persistent DNA hemimethylation. These studies suggest a broader spectrum of functional states of DNA methylation. To begin to address this gap in our understanding of this potentially crucial element of the epigenetic regulation of the genome, we propose to adapt a SET- and RING Finger-Associated (SRA) domain of UHRF1 to generate a DNA hemimethylation probe for in vitro and in vivo studies. The compact SRA domain is self-sufficient to bind hemimethylated DNA with high specificity. To accomplish the overarching goal of this proposal, we will first produce recombinant SRA domain proteins and develop methods for the isolation and visualization of hemimethylated DNA in complex genomes in vitro. To study DNA hemimethylation in vivo, we will express a fusion protein comprised of the SRA domain and mScarlet fluorescent protein in mouse embryos, embryonic stem cells, and transgenic mice. We will use live imagining of cells with hemimethylated DNA to validate chromatin localization patterns and dynamics of the SRA-mScarlet fusion protein. This research will enhance our understanding of the epigenetic regulation of the genome in development and disease.