Genetics of DNA Methylation Patterning of Arabidopsis

描述(申请人提供)：胞嘧啶DMA甲基化是基因沉默的表观遗传标记，在许多基因调控系统中非常重要，包括基因组印迹、X染色体失活、转座子和其他包含正向或反向重复序列的DNA序列的沉默。甲基化在癌症生物学中很重要，因为肿瘤通常表现为全基因组范围的去甲基化和特定肿瘤抑制基因的高甲基化。我们正在研究模式植物拟南芥DNA甲基化控制的机制。拟南芥的DNA甲基化系统与哺乳动物的DNA甲基化系统有许多共同之处，显示出三种人类DNA甲基转移酶Dnmt1、2和3的同源基因。正向和反向遗传学都可以进行，拟南芥可以容忍几乎消除甲基化的突变，从而进行详细的分析。我们最近发现，RNA沉默成分包括DICER、依赖于RNA的RNA聚合酶和ArgAerte蛋白，是建立DNA甲基化和维持非CG环境中甲基化的机制的核心。我们提出了一系列的遗传和生化实验来进一步研究这一现象。正向重复序列和反向重复序列可以通过不同的机制触发DNA从头甲基化，我们建议研究这些差异。我们还提出了旨在分离在这些过程中重要的因子的突变筛选。ARGONAUTE4蛋白可能作用于小RNA和染色质修饰酶之间的界面，我们建议详细研究该蛋白的作用机制。由于DNA甲基化控制机制的许多方面在不同的真核生物中是相似的，在拟南芥中取得的进展很可能有助于揭示涉及基因组防御、发育生物学和人类疾病的一系列表观遗传调控系统。

DESCRIPTION (provided by applicant): Cytosine DMA methylation is an epigenetic mark for gene silencing that is important in many gene regulatory systems including genomic imprinting, X-chromosome inactivation, and the silencing of transposons and other DNA sequences containing either direct or inverted repeats. Methylation is important in cancer biology, as tumors often show both genome wide demethylation and hypermethylation of specific tumor suppressor genes. We are studying the mechanisms of DNA methylation control in the model plant Arabidopsis thaliana. Arabidopsis DNA methylation systems have much in common with mammalian systems, showing homologs of the three human DNA methyltransferases, Dnmtl, 2, and 3. Both forward and reverse genetics can be performed, and Arabidopsis can tolerate mutations that virtually eliminate methylation, allowing for detailed analysis. We have recently discovered that RNA silencing components including a DICER, an RNA-dependent RNA polymerase, and an ARGONAUTE protein, are central to the mechanisms by which DNA methylation is established, and by which methylation in non-CG contexts is maintained. We propose a series of genetic and biochemical experiments to further study this phenomena. Direct repeats and inverted repeats can trigger de novo DNA methylation by different mechanisms, and we propose to study these differences. We also propose mutant screens aimed at isolating factors important in these processes. The ARGONAUTE4 protein likely acts at the interface between small RNAs and chromatin modifying enzymes, and we propose to study the mechanism of action of this protein in detail. Since many aspects of DNA methylation control mechanisms are similar in diverse eukaryotic organisms, it is likely that progress made in Arabidopsis will shed light on a wide array of epigenetic regulatory systems involved in genome defense, developmental biology, and human disease.