多种真核生物都使用非编码RNA介导的组蛋白修饰或DNA甲基化来沉默转座子。在植物中这一过程被称为RNA介导的DNA甲基化（RdDM）,多年来一直是研究的热点。但是染色体结构怎样影响这一过程仍不清楚。我们实验室已有一个成熟的遗传筛选系统用于发现参与RdDM的基因。使用这个系统，我们发现ATP依赖的染色体重塑因子PKL在RdDM通路中起作用，并且促进四分之一RdDM位点的甲基化水平。此前研究表明PKL是一个重要的发育调控因子，并可在体外实验中通过其染色体重塑活性改变核小体位置。因此这一突变体为研究染色体结构对RdDM的影响提供了良好的契机。我们假设PKL通过其染色体重塑活性调节RdDM位点附近核小体位置来协调RNA Pol IV或Pol V的转录，从而影响全新甲基化。我们将采用高通量测序、生化和细胞生物学的手段来验证这个假设。该项目的研究成果可以帮助我们深入理解RNA介导DNA甲基化的分子机理

Noncoding RNA guided histone modification or DNA methylation is a common mechanism used by many eukaryotic organisms to silence transposons. In plants, this process is called RNA-directed DNA methylation. Due to its importance in help establishing de novo methylation patterns in the plant genome, this pathway has been extensively studied over the past years. However how chromatin structure could affect this process is unclear. Our lab has established a mature genetic screen system that identified genes involved in RNA-directed DNA methylation. Using this system we identified an ATP-dependent chromatin remodeling factor PICKLE (PKL) that functions in the RdDM pathway and is required to promote methylation at a quarter of the RdDM loci. Previous studies found that PKL is an important developmental regulator and it could change nucleosome positioning through its remodeling activity in in vitro assays. Thus the pkl mutant provides a good opportunity to study the effect of chromatin structure on RNA-directed DNA methylation. We hypothesize that PKL, through its chromatin remodeling activity, regulates nucleosome positioning around RdDM loci to mediate transcription by RNA Pol IV or Pol V, which in turn directs de novo DNA methylation. We will test this hypothesis using a combined means of high throughput sequencing, biochemistry and molecular biology. Findings of this proposal would further our understanding in the molecular mechanism of RNA-directed DNA methylation.

在植物中RNA介导的DNA甲基化（RdDM）是很多生物学过程所必需的一个表观遗传通路。在这一过程中两种非编码RNA（ncRNA）介导位点特异的DNA甲基化，但是非编码RNA本身的转录是怎样收到调控的仍然不很清楚。我们在两次独立的正向遗传筛选中发现CHD3类型的染色质重塑因子PKL是RD29A-LUC转基因的沉默因子。全基因组分析发现PKL是维持RdDM位点正常的DNA甲基化水平和小干扰RNA（siRNA）水平所必需的。此外，PKL也影响被RNA聚合酶V（Pol V）稳定的核小体定位，并影响依赖于Pol V的转录产物。这些结果提示PKL可能是Pol IV和Pol V的非编码RNA转录所必需的。转录组分析发现PKL和RdDM影响类似数量的转座子，但表现出去抑制的转座子并不具有显著的DNA甲基化水平改变。对特定位点的染色质免疫沉淀分析也未能发现与转录去抑制相关的组蛋白修饰变化。这些结果提示PKL也有与DNA甲基化无关的基因沉默的功能。在动物中CHD蛋白质在Pol II转录的起始、延伸和终止阶段都起重要的作用，可能植物特有的Pol IV和Pol V招募了保守的因子来进行非编码RNA的转录调控。对于PKL相互作用蛋白质和他们在异染色质上的活性分析将会为PKL影响DNA甲基化和转录沉默的分子机制提供重要线索。