DNA 5-methylcytosine regulates genome-wide formation of G-quadruplex structures [CUT&Tag]. DNA 5-methylcytosine regulates genome-wide formation of G-quadruplex structures [CUT&Tag]

G-quadruplex structures (G4s) have been identified in genomes of multiple organisms and proven to play important epigenetic regulatory roles in various cellular functions. However, the G4 formation mechanism remains largely unknown. Here, we found a negative correlation between DNA 5mC methylation and G4 abundance. The abundance of genomic G4s significantly increased when the whole-genome methylation level was reduced in DNMT1-knockout cells. This increase was then suppressed by DNMT1 over-expression. And more G4s were detected in the hypomethylated cancer cell line HepG2 and rectal cancer tissues. Besides, 5mC modification significantly inhibited G4 formation of the potential G-quadruplex sequences (PQSs). The transcription of genes with 5mC modification sites in their promoter PQSs was affected after treatment with G4 stabilizer pyridostatin or methylation inhibitor 5-aza-dC. The global reduction of genomic methylation elevates gene transcription levels through increased G4s. Taken together, DNA 5mC methylation prevents PQSs from folding into G4s in genomes. Overall design: ATAC for 293T cells and DNMT1-KO 293T cells

G-四链体结构（G-quadruplexes, G4s）已在多种生物的基因组中被鉴定，并被证实于诸多细胞功能中发挥重要的表观遗传调控作用。然而，G4的形成机制在很大程度上仍未明确。本研究发现DNA 5mC甲基化水平与G4丰度呈负相关。在DNMT1敲除细胞中，全基因组甲基化水平降低时，基因组G4的丰度显著升高；该升高效应可被DNMT1过表达所抑制。此外，在低甲基化癌细胞系HepG2及直肠癌组织中可检测到更多的G4。进一步研究显示，5mC修饰可显著抑制潜在G-四链体序列（potential G-quadruplex sequences, PQSs）形成G4结构。对于启动子区域PQSs携带5mC修饰位点的基因，经G4稳定剂吡哆司他汀（pyridostatin）或甲基化抑制剂5-氮杂-dC处理后，其转录会受到影响。全基因组甲基化水平降低可通过提升G4丰度来上调基因转录水平。综上，DNA 5mC甲基化可阻止基因组内的PQS折叠形成G4结构。整体实验设计：针对293T细胞及DNMT1敲除293T细胞开展ATAC实验。