Regulation of DNA methylation homeostasis in Arabidopsis

 描述（由申请人提供）：DNA甲基化对于包括基因组印记、X失活、转座因子沉默和基因组稳定性在内的基本过程至关重要。DNA甲基化模式是活跃的DNA甲基化和去甲基化过程的结果。拟南芥是一个很好的真核生物模型系统，因为它简单的遗传学和基因组学以及哺乳动物中这些过程的保守性，可以用来研究甲基化和去甲基化的功能和机制。拟南芥一直是表观遗传学领域新发现的持续来源。与促进DNA甲基化的过程相比，DNA去甲基化的功能和调节正在研究中，部分原因是活性DNA去甲基化的机制最近才被阐明。在植物中，DNA去甲基化由5-甲基胞嘧啶（5-mC）DNA糖基化酶介导，其通过碱基切除修复从DNA中去除5-mC，导致其被未修饰的胞嘧啶取代。5-mC DNA糖基化酶ROS 1的表达显著降低，以响应维持甲基化和RNA指导的DNA甲基化（RdDM）途径的组分中的突变。这表明在甲基化和去甲基化途径之间存在分子串扰，以实现这些拮抗酶活性的细胞平衡。这项研究的长期目标是确定在植物生长和发育过程中确保DNA甲基化稳态的分子机制，并确定扰乱这种稳态的功能后果。拟议的研究重点是ROS 1，这是一个多个表观遗传途径会聚的位点，以调节DNA去甲基化酶的表达，以响应DNA甲基化的全局和局部变化。我们将确定为什么以及如何ROS 1转录减少响应RdDM途径的中断。我们将通过一种新的遗传筛选来鉴定这一过程的调节因子，该筛选将鉴定在甲基化缺陷背景下促进ROS 1表达的基因。最后，我们将确定在野生型精子发育过程中发生的ROS 1表达降低是否是受精后建立正确的全基因组DNA甲基化模式的原因。这些努力将显著增加我们对表观遗传途径的活性如何响应表观基因组状态进行调节的理解。我们的发现将直接适用于哺乳动物和其他通过DNA甲基化机制控制其基因组的真核生物。

 DESCRIPTION (provided by applicant): DNA methylation is critical for essential processes including genomic imprinting, X-inactivation, transposable element silencing, and genome stability. DNA methylation patterns are the result of active DNA methylation and demethylation processes. Arabidopsis represents an excellent eukaryotic model system to dissect the functions and mechanisms of methylation and demethylation because of its facile genetics and genomics and the conservation of these processes with mammals. Arabidopsis has been a continued source of novel discoveries in the field of epigenetics. The function and regulation of DNA demethylation is under studied compared to processes that promote DNA methylation, partly because the mechanisms of active DNA demethylation have been elucidated only relatively recently. In plants, DNA demethylation is mediated by 5- methylcytosine (5-mC) DNA glycosylases that remove 5-mC from DNA by base excision repair, leading to its replacement with unmodified cytosine. Expression of the 5-mC DNA glycosylase ROS1 is significantly reduced in response to mutations in components of the maintenance methylation and RNA-directed DNA methylation (RdDM) pathways. This suggests that there is a molecular crosstalk between the methylation and demethylation pathways to achieve a cellular balance of these antagonistic enzymatic activities. The long-term goal of this research is to define the molecular mechanisms that ensure DNA methylation homeostasis during plant growth and development and to determine the functional consequences of perturbing that homeostasis. The proposed research focuses on ROS1, a locus where multiple epigenetic pathways converge to modulate DNA demethylase expression in response to global and local changes in DNA methylation. We will determine why and how ROS1 transcription is reduced in response to the disruption of the RdDM pathway. We will identify regulators of this process through a novel genetic screen that will identify genes that promote expression of ROS1 in a methylation deficient background. Finally, we will determine whether the decreased expression of ROS1 that occurs during wild type sperm development in response to reduced DNA methylation is responsible for establishing proper genome-wide DNA methylation patterns after fertilization. These efforts will significantly increase our understanding of how the activity of epigenetic pathways is modulated in response to the state of the epigenome. Our findings will be directly applicable to mammals and other eukaryotes that control their genome through DNA methylation based mechanisms.