N6-Methyldeoxyadenosine Marks Active Transcription Start Sites in Chlamydomonas

N6-methyldeoxyadenosine (6mA or m6A) is a DNA modification preserved in prokaryotes to eukaryotes. It is widespread in bacteria, and functions in DNA mismatch repair, chromosome segregation, and virulence regulation. In contrast, the distribution and function of 6mA in eukaryotes have been unclear. Here we present a comprehensive analysis of the 6mA landscape in the genome of Chlamydomonas using new sequencing approaches. We identified the 6mA modification in 84% of genes in Chlamydomonas. We found that 6mA mainly locates at ApT dinucleotides around transcription start sites (TSS) with a bimodal distribution, and appears to mark active genes. A periodic pattern of 6mA deposition was also observed at base resolution, which is associated with nucleosome distribution near the TSS, suggesting a possible role in nucleosome positioning. The new genome-wide mapping of 6mA and its unique distribution in the Chlamydomonas genome suggest potential regulatory roles of 6mA in gene expression in eukaryotic organisms. Multiple sequencing methods are developed to profile the distribution of 6mA in Chlamydomonas including MeDIP-Seq, enzyme-treated DNA-Seq, MNase-Seq and RNA-Seq.

N6-甲基脱氧腺苷（N6-methyldeoxyadenosine，简称6mA或m6A）是一种广泛存在于原核生物至真核生物中的保守DNA修饰。该修饰在细菌中分布广泛，参与DNA错配修复、染色体分离及毒力调控等重要生物学过程。与之形成鲜明对比的是，真核生物中6mA的分布模式与生物学功能长期以来尚未明确。本研究依托新型测序技术，对衣藻（Chlamydomonas）基因组中的6mA分布全景开展了全面解析。我们在衣藻84%的基因中检测到了6mA修饰。研究发现，6mA主要富集于转录起始位点（transcription start sites，TSS）附近的ApT二核苷酸区域，呈现双峰分布特征，且可作为活跃基因的表观遗传标记。此外，在碱基分辨率水平下还观察到6mA沉积的周期性模式，该模式与转录起始位点附近的核小体分布高度相关，提示6mA可能在核小体定位过程中发挥潜在调控作用。本研究获得的全新全基因组6mA图谱，及其在衣藻基因组中的独特分布特征，表明6mA在真核生物的基因表达调控中可能发挥关键的调节功能。本研究开发了多种测序方法以解析衣藻中6mA的全基因组分布，包括甲基化DNA免疫沉淀测序（MeDIP-Seq，Methylated DNA Immunoprecipitation Sequencing）、酶处理DNA测序（enzyme-treated DNA-Seq）、微球菌核酸酶测序（MNase-Seq，Micrococcal Nuclease Sequencing）以及RNA测序（RNA-Seq）。