Bisulfite-Seq and RNA-seq in rootstock of Arabidopsis thaliana grafts. Bisulfite-Seq and RNA-seq in rootstock of Arabidopsis thaliana grafts

RNA silencing is a mechanism for regulating gene expression at the transcriptional and post-transcriptional levels. Its functions include regulating endogenous gene expression and protecting the cell against viruses and invading transposable elements (TEs). A key component of the mechanism is small RNAs (sRNAs) of 21-24 nucleotides (nt) in length, which direct the silencing machinery in a sequence specific manner to target nucleic acids. sRNAs of 24 nt are involved in methylation of cytosine residues of target loci in three sequence contexts (CG, CHG and CHH), referred to as RNA-directed DNA methylation (RdDM). We previously demonstrated that 24 nt sRNAs are mobile from shoot to root in Arabidopsis thaliana. In this study we demonstrated that methylation of thousands of loci in root tissues is dependent upon mobile sRNAs from the shoot. Furthermore, we found that mobile sRNA-dependent DNA methylation occurs predominantly in non-CG contexts. These findings were made using base-resolution next generation sequencing approaches and genome wide analyses. Specific classes of short TEs are the predominant targets of mobile sRNA-dependent DNA methylation; classes typically found in gene-rich euchromatic regions. Mobile sRNA-regulated genes were also identified. Mechanistically, we demonstrate that mobile sRNA-dependent non-CG methylation is largely independent of the CMT2/3 RdDM pathway but dependent upon the DRM1/DRM2 RdDM pathway. This is in contrast to non-mobile sRNA-dependent DNA methylation, which predominantly depends upon the CMT2/3 RdDM pathway. These data are complementary to the small RNA sequencing data from Arabidopsis root grafts described in Molnar et al (Science, 2010 May 14;328(5980):872-5).

RNA沉默 (RNA silencing) 是在转录及转录后水平调控基因表达的核心机制。其功能涵盖内源基因表达调控，以及抵御病毒与入侵的转座因子 (transposable elements, TEs)。该机制的关键组分为长度21~24核苷酸 (nucleotides, nt) 的小RNA (small RNAs, sRNAs)，它们可通过序列特异性方式引导沉默复合体靶向结合核酸。

长度为24 nt的小RNA可参与三类序列环境（CG、CHG及CHH）下靶位点胞嘧啶残基的甲基化修饰，这一过程被称为RNA指导的DNA甲基化 (RNA-directed DNA methylation, RdDM)。我们此前的研究证实，拟南芥 (Arabidopsis thaliana) 中24 nt的小RNA可从地上部向根部移动。

本研究证实，根部组织中数千个基因座的甲基化依赖于来自地上部的移动小RNA。进一步研究发现，依赖移动小RNA的DNA甲基化主要发生在非CG序列环境中。上述结论均通过碱基分辨率级别的下一代测序 (next generation sequencing) 技术与全基因组分析获得。

特定类别的短转座因子是依赖移动小RNA的DNA甲基化的主要靶标，这类转座因子通常富集于基因密集的常染色质区域。我们同时鉴定出了受移动小RNA调控的基因。从机制层面而言，本研究证实依赖移动小RNA的非CG甲基化在很大程度上不依赖CMT2/3介导的RdDM通路，而依赖DRM1/DRM2介导的RdDM通路。这与依赖非移动小RNA的DNA甲基化形成鲜明对比，后者主要依赖CMT2/3介导的RdDM通路。

本研究数据与Molnar等人2010年5月14日发表于《科学》（Science, 2010 May 14;328(5980):872-5）的拟南芥根部嫁接实验的小RNA测序数据互为补充。