Won So Youn, Li Shengben, Zheng Binglian, Zhao Yuanyuan, Li Dongming, Zhao Xin, Yi Huilan, Gao Lei, Dinh Thanh Theresa, Chen Xuemei
Department of Botany and Plant Sciences, Institute of Integrative Genome Biology, University of California, Riverside, CA, 92521, USA.
Silence. 2012 Jun 7;3(1):6. doi: 10.1186/1758-907X-3-6.
Cytosine methylation is an important chromatin modification that maintains genome integrity and regulates gene expression through transcriptional gene silencing. Major players in de novo methylation guided by siRNAs (known as RNA-directed DNA methylation, or RdDM), maintenance methylation, and active demethylation have been identified in Arabidopsis. However, active demethylation only occurs at a subset of RdDM loci, raising the question of how the homeostasis of DNA methylation is achieved at most RdDM loci. To identify factors that regulate the levels of cytosine methylation, we aimed to establish a transgenic reporter system that allows for forward genetic screens in Arabidopsis.
We introduced a dual 35 S promoter (d35S) driven luciferase reporter, LUCH, into Arabidopsis and isolated a line with a moderate level of luciferase activity. LUCH produced transgene-specific 24 nucleotide siRNAs and its d35S contained methylated cytosine in CG, CHG and CHH contexts. Treatment of the transgenic line with an inhibitor of cytosine methylation de-repressed luciferase activity. Mutations in several components of the RdDM pathway but not the maintenance methylation genes resulted in reduced d35S methylation, especially CHH methylation, and de-repression of luciferase activity. A mutation in MOM1, which is known to cooperate with RdDM to silence transposons, reduced d35S DNA methylation and de-repressed LUCH expression. A mutation in ROS1, a cytosine demethylation enzyme, increased d35S methylation and reduced LUCH expression.
We developed a luciferase-based reporter, LUCH, which reports both DNA methylation directed by small RNAs and active demethylation by ROS1 in Arabidopsis. The moderate basal level of LUCH expression allows for bi-directional genetic screens that dissect the mechanisms of DNA methylation as well as demethylation.
胞嘧啶甲基化是一种重要的染色质修饰，它通过转录基因沉默维持基因组完整性并调控基因表达。在拟南芥中，已鉴定出由小干扰RNA引导的从头甲基化（称为RNA指导的DNA甲基化，或RdDM）、维持甲基化和主动去甲基化过程中的主要作用因子。然而，主动去甲基化仅发生在一部分RdDM位点，这就引发了一个问题：在大多数RdDM位点，DNA甲基化的稳态是如何实现的。为了鉴定调控胞嘧啶甲基化水平的因子，我们旨在建立一个转基因报告系统，以便在拟南芥中进行正向遗传筛选。
我们将一个由双35S启动子（d35S）驱动的荧光素酶报告基因LUCH导入拟南芥，并分离出一个具有中等水平荧光素酶活性的株系。LUCH产生转基因特异性的24个核苷酸的小干扰RNA，其d35S在CG、CHG和CHH序列背景中含有甲基化的胞嘧啶。用胞嘧啶甲基化抑制剂处理转基因株系可解除对荧光素酶活性的抑制。RdDM途径的几个组分发生突变，但维持甲基化基因未突变，导致d35S甲基化减少，尤其是CHH甲基化减少，并解除对荧光素酶活性的抑制。已知与RdDM协同沉默转座子的MOM1发生突变，会降低d35S DNA甲基化并解除对LUCH表达的抑制。胞嘧啶去甲基化酶ROS1发生突变会增加d35S甲基化并降低LUCH表达。
我们开发了一种基于荧光素酶的报告基因LUCH，它可报告拟南芥中小RNA介导的DNA甲基化以及ROS1介导的主动去甲基化。LUCH表达的适度基础水平允许进行双向遗传筛选，以剖析DNA甲基化和去甲基化的机制。
