DNA N6-adenine methylation in Arabidopsis thaliana [SMRT-seq]

DNA methylation on N6-adenine (6mA) has recently been found as a potentially new epigenetic mark in several unicellular and multicellular eukaryotes. However, its distribution patterns and potential functions in land plants, which are primary producers for most ecosystems, remain completely unknown. Here we report global profiling of 6mA sites at single-nucleotide resolution in the genome of Arabidopsis thaliana using single-molecule real-time sequencing. 6mA sites are widely distributed across the Arabidopsis genome and enriched over the pericentromeric heterochromatin regions. Nearly 30% of 6mA sites are present in gene bodies. Further analysis of 6mA methylome and RNA-sequencing data demonstrates that 6mA frequency positively correlates with the gene expression level in Arabidopsis. Consistently, histone variants associated with actively expressed genes interact with 6mA DNA. Our results uncover 6mA as a DNA mark associated with actively expressed genes in Arabidopsis, suggesting that 6mA serves as a novel epigenetic mark in land plants. Overall design: SMRT sequencing in 3-week-old Arabidopsis thaliana (Col-0) wild-typeN6-腺嘌呤（N6-adenine，简称6mA）的DNA甲基化修饰，近期在多种单细胞与多细胞真核生物中被鉴定为一类潜在的新型表观遗传标记（epigenetic mark）。然而，作为多数生态系统初级生产者的陆生植物中，6mA的分布模式与潜在功能仍完全未知。本研究利用单分子实时测序（single-molecule real-time sequencing，SMRT测序）技术，对拟南芥（Arabidopsis thaliana）全基因组范围内的6mA位点进行了单核苷酸分辨率的全局图谱分析。6mA位点广泛分布于拟南芥基因组中，并富集于着丝粒周边异染色质（pericentromeric heterochromatin）区域；近30%的6mA位点位于基因编码区（gene bodies）内。进一步对6mA甲基化组（methylome）与RNA测序（RNA-sequencing）数据的分析表明，拟南芥中6mA的修饰频率与基因表达水平呈正相关。与之相符的是，与活跃表达基因相关的组蛋白变体（histone variants）可与6mA修饰的DNA发生相互作用。本研究结果证实6mA是拟南芥中与活跃表达基因相关的DNA修饰标记，表明6mA可作为陆生植物中的一类新型表观遗传标记。实验整体设计：对生长3周的野生型拟南芥（Arabidopsis thaliana，Col-0生态型）进行SMRT测序。