Promoter G-quadruplex folding precedes transcription and is controlled by chromatin

Four stranded DNA G-quadruplex (G4) structures are common features of the human genome that are primarily found in active promoters associated with elevated transcription. Here, we explore the relationship between the folding of G4s in promoters, transcription and chromatin state. Transcriptional inhibition by DRB or by triptolide reveals that promoter G4 formation, as assessed G4 ChIP-seq, is not reliant on transcriptional activity. Establishing a link between G4 formation and chromatin accessibility, we demonstrate that chromatin compaction leads to loss of promoter G4s accompanied by a corresponding loss of RNA polymerase II (Pol II). Furthermore, pre-treatment of cells with a G4-stabilising ligand can mitigate Pol II loss at promoters induced by chromatin compaction. Overall, our findings show that G4 formation is fostered in accessible chromatin and does not require active transcription. Furthermore, our findings suggest that G4s have a role to recruit Pol II to promote transcription. Overall design: K562 cells profiling by G4-ChIP-seq upon DRB, TPL; K562 cells profiling by G4-ChIP-seq, Pol2-ChIP-seq and Atac-seq upon pyPDS, Hypoxia, Hypoxia plus pyPDS or DMSO, Normoxia plus pyPDS or DMSO. U2OS cells profiling by G4-ChIP-seq in Hypoxia and Normoxia in triplicates.

四链DNA G-四链体（G-quadruplex, G4）结构是人类基因组的常见特征，主要富集于与转录激活增强相关的活性启动子区域。本研究探讨了启动子区域G4折叠、转录与染色质状态三者之间的内在关联。通过DRB或雷公藤内酯（triptolide）进行转录抑制实验结果显示，经G4染色质免疫共沉淀测序（G4 ChIP-seq）检测的启动子G4形成，并不依赖于转录活性。在明确G4形成与染色质可及性之间的关联后，本研究证实染色质浓缩会导致启动子区域G4水平下降，并伴随RNA聚合酶II（RNA polymerase II, Pol II）的同步流失。此外，使用G4稳定配体预处理细胞，可缓解染色质浓缩诱导的启动子区域Pol II流失现象。综上，本研究结果表明，G4形成依赖于染色质可及性，且无需活跃的转录过程。此外，本研究结果提示G4可通过招募Pol II以促进转录过程。
实验设计：对经DRB、TPL处理的K562细胞开展G4-ChIP-seq测序分析；对经pyPDS、缺氧、缺氧联合pyPDS、常氧联合pyPDS或DMSO处理的K562细胞，分别进行G4-ChIP-seq、Pol2-ChIP-seq及Atac-seq测序分析；对在缺氧及常氧条件下培养的U2OS细胞进行三次生物学重复的G4-ChIP-seq测序分析。