DNA Methylation and Gene Silencing in Arabidopsis

描述（由申请人提供）：在包括哺乳动物和植物在内的真核生物中，胞嘧啶甲基化和相关的异染色质形成在调节基因表达和基因组完整性方面起关键作用。胞嘧啶甲基化失调导致人类癌症和许多遗传疾病。胞嘧啶甲基化也是抑制转座子表达和运动的关键。我们利用实验室植物拟南芥中一组重复甲基化的内源基因作为模型系统，研究胞嘧啶甲基化和基因沉默的建立和维持。在该系统中，甲基化印记是由基于双链RNA （dsRNA）的信号建立的。这种甲基化靶向模式最初在植物中被阐明，但现在很清楚，rna定向异染色质形成的相关机制发生在真菌和包括哺乳动物在内的动物中。作为本提案的目标之一，我们将利用RNA加工/结合突变体的遗传研究和RNA的分子分析相结合，进一步表征DMA甲基化的RNA信号，重点了解短干扰RNA （sirna）在指导甲基化模式中的作用。作为第二个目标，我们将使用一种新的遗传筛选来特异性地维持dsRNA源位点甲基化的因素。我们还确定了维持rna定向DNA甲基化印记的关键因素是胞嘧啶甲基转移酶CMT3和组蛋白H3赖氨酸9 （K9）甲基转移酶SUVH4/KYP。胞嘧啶甲基化和组蛋白H3 K9甲基化之间的这种关系也发生在真菌和哺乳动物系统中。在本提案的第三个目标中，我们将利用生化、遗传和转基因方法表征SUVH4/KYP和部分冗余相关SUVH蛋白的结构和功能，并以理解RNA、组蛋白甲基化和DNA甲基化之间的联系为长期目标。

DESCRIPTION (provided by applicant): In eukaryotes including mammals and plants, cytosine methylation and associated heterochromatin formation play a key role in regulating gene expression and genome integrity. Dysregulation of cytosine methylation contributes to human cancers and a number of genetic disorders. Cytosine methylation is also critical for the suppression of transposon expression and movement. We use a group of duplicated methylated endogenous genes in the laboratory plant Arabidopsis as a model system to study the establishment and maintenance of cytosine methylation and gene silencing. In this system, the methylation imprint is established by a double-stranded RNA (dsRNA)-based signal. This mode of methylation targeting was first elucidated in plants, but it is now clear that related mechanisms for RNA-directed heterochromatin formation occur in fungi and animals including mammals. As one aim of this proposal, we will further characterize the RNA signal for DMA methylation using a combination of genetic studies with RNA processing/binding mutants and molecular analysis of RNA, with a focus on understanding the role of short interfering RNAs (siRNAs) in guiding methylation patterning. As a second aim, we will use a novel genetic screen for factors that specifically maintain methylation at a dsRNA source locus. We have also determined that key factors for maintaining the RNA-directed DNA methylation imprint are a cytosine methyltransferase CMT3 and a histone H3 lysine 9 (K9) methyltransferase SUVH4/KYP. This relationship between cytosine methylation and histone H3 K9 methylation also occurs in fungal and mammlian systems. In the third aim of this proposal, we will characterize the stucture and function of SUVH4/KYP and partially redundant related SUVH proteins using biochemical, genetic, and transgenic approaches, with the long-term goal of understanding the connections between RNA, histone methylation, and DNA methylation.