Four SWI2/SNF2 chromatin remodeling proteins play roles as “double agents” in the tug of war between RNA-directed DNA methylation and active DNA demethylation in Arabidopsis [Bisulfite-seq]. Four SWI2/SNF2 chromatin remodeling proteins play roles as “double agents” in the tug of war between RNA-directed DNA methylation and active DNA demethylation in Arabidopsis [Bisulfite-seq]

Here we report that CLSY4 and its three paralogous SWI2/SNF2-type chromatin remodeling proteins function in both RdDM and active DNA demethylation for dynamic regulation of DNA methylation in Arabidopsis. We identified CLSY4 initially in a genetic screen for DNA demethylation factors and subsequently demonstrated that it also plays an important role in RdDM. Comprehensive genetic analyses using single and high order mutants of CLSY family proteins revealed their roles as double agents in the tug of war between methylation and demethylation reactions. The four CLSY proteins collectively are necessary for the canonical RdDM pathway; meanwhile, each CLSY also mediates active DNA demethylation at specific loci where DNA methylation is dependent on one or more of the other three CLSY proteins. Our results revealed dual functions of the four chromatin remodeling proteins in regulating genomic DNA methylation status, and thus provide new insights into the mechanisms underlying the dynamic regulation of DNA methylation in Arabidopsis. Overall design: whole genome wide DNA methylation data by bisulphite sequencing

本研究报道，CLSY4及其3个同源旁系的SWI2/SNF2型染色质重塑蛋白（SWI2/SNF2-type chromatin remodeling proteins），可同时参与RNA指导的DNA甲基化（RNA-directed DNA methylation, RdDM）与主动DNA去甲基化过程，以动态调控拟南芥的基因组DNA甲基化水平。本研究首先通过DNA去甲基化因子的遗传筛选鉴定得到CLSY4，后续实验证实其在RdDM通路中同样发挥关键调控作用。针对CLSY家族蛋白的单突变体及高阶突变体开展的综合遗传分析显示，这类蛋白充当了双重作用因子，参与甲基化与去甲基化反应之间的拉锯博弈。4个CLSY家族蛋白协同作用，是经典RdDM通路正常行使功能所必需的；与此同时，每个CLSY蛋白均可在特定基因组位点介导主动DNA去甲基化，而这些位点的DNA甲基化依赖于其余3个CLSY蛋白中的一个或多个。本研究结果揭示了这4种染色质重塑蛋白在调控基因组DNA甲基化状态中的双重功能，从而为解析拟南芥DNA甲基化动态调控的分子机制提供了全新视角。实验整体设计：通过亚硫酸氢盐测序获取全基因组范围的DNA甲基化数据