Science for Life Laboratory, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Tomtebodavägen 23, 17165 Stockholm, Sweden.
Ming Wai Lau Centre for Reparative Medicine, Stockholm node, Karolinska Institutet, Solnavägen 9, 17165 Stockholm, Sweden.
Nucleic Acids Res. 2022 Feb 22;50(3):e13. doi: 10.1093/nar/gkab1073.
Single-stranded genomic DNA can fold into G-quadruplex (G4) structures or form DNA:RNA hybrids (R loops). Recent evidence suggests that such non-canonical DNA structures affect gene expression, DNA methylation, replication fork progression and genome stability. When and how G4 structures form and are resolved remains unclear. Here we report the use of Cleavage Under Targets and Tagmentation (CUT&Tag) for mapping native G4 in mammalian cell lines at high resolution and low background. Mild native conditions used for the procedure retain more G4 structures and provide a higher signal-to-noise ratio than ChIP-based methods. We determine the G4 landscape of mouse embryonic stem cells (ESC), observing widespread G4 formation at active promoters, active and poised enhancers. We discover that the presence of G4 motifs and G4 structures distinguishes active and primed enhancers in mouse ESCs. Upon differentiation to neural progenitor cells (NPC), enhancer G4s are lost. Further, performing R-loop CUT&Tag, we demonstrate the genome-wide co-occurrence of single-stranded DNA, G4s and R loops at promoters and enhancers. We confirm that G4 structures exist independent of ongoing transcription, suggesting an intricate relationship between transcription and non-canonical DNA structures.
单链基因组 DNA 可以折叠成 G-四链体 (G4) 结构或形成 DNA:RNA 杂交体 (R 环)。最近的证据表明，这种非典型的 DNA 结构会影响基因表达、DNA 甲基化、复制叉进展和基因组稳定性。G4 结构形成和解决的时间和方式仍不清楚。在这里，我们报告了使用靶向切割和标签化 (CUT&Tag) 技术以高分辨率和低背景在哺乳动物细胞系中绘制天然 G4 的方法。该方法使用温和的天然条件保留了更多的 G4 结构，并提供了比基于 ChIP 的方法更高的信号与噪声比。我们确定了小鼠胚胎干细胞 (ESC) 的 G4 图谱，观察到活跃启动子、活跃和静止增强子处广泛形成 G4。我们发现 G4 基序和 G4 结构的存在可以区分小鼠 ESC 中的活跃和初始增强子。在分化为神经祖细胞 (NPC) 后，增强子 G4 消失。此外，通过进行 R 环 CUT&Tag，我们证明了启动子和增强子上单链 DNA、G4 和 R 环的全基因组共发生。我们证实 G4 结构的存在独立于正在进行的转录，这表明转录和非典型 DNA 结构之间存在复杂的关系。
