G-quadruplexes are promoter elements controlling nucleosome exclusion and RNA polymerase II pausing

Despite their central role in transcription, it has been difficult to define universal sequences associated with eukaryotic promoters. Within the chromatin context, recruitment of transcriptional machinery requires promoter opening, but how DNA elements contribute to this process is unclear. Here we show that G-quadruplex (G4) secondary DNA structures are highly enriched at mammalian promoters. G4s are located at the deepest point of nucleosome exclusion at promoters and correlate with maximum promoter activity. We found that experimental G4s exclude nucleosomes in vivo and in vitro while favouring strong positioning. At model promoters, impairing G4s affected both transcriptional activity and chromatin opening. G4 destabilization also resulted in an inactive promoter state and affected the transition to effective RNA production. Finally, G4 stabilization resulted in global reduction of proximal promoter pausing. Altogether, our data introduce G4s as bona fide promoter elements allowing nucleosome exclusion and facilitating pause-release by RNA polymerase II. MNase-seq and ChIP-seq of RNA-Polymerase II (Pol II) were used to address how G-quadruplexes (G4s) influence chromatin organisation and transcription. Functional analyses were performed using pyridostatin, a drug that stabilize G4s and alpha-amanitin that inhibits Pol II.

尽管真核启动子在转录过程中发挥核心作用，但要界定与之相关的通用序列始终是一项难题。在染色质环境中，转录机器的招募需要启动子区域染色质开放，但DNA元件如何参与这一过程目前仍不明确。本研究证实，G-四链体（G-quadruplex, G4）这种DNA二级结构在哺乳动物启动子区域高度富集。G4位于启动子区域核小体排除的最深位点，并与启动子的最大活性呈正相关。我们发现，人工构建的G4结构在体内和体外均可排除核小体，同时能促进核小体的精准定位。在模式启动子实验中，破坏G4结构会同时影响转录活性与染色质开放状态。G4结构的不稳定还会导致启动子处于失活状态，并影响向有效RNA合成的过渡过程。最后，稳定G4结构可在全局范围内减少近端启动子处的RNA聚合酶暂停现象。综上，本研究数据表明G4是一类确凿的启动子元件，能够介导核小体排除，并助力RNA聚合酶II（RNA-Polymerase II, Pol II）完成暂停释放。本研究采用微球菌核酸酶测序（MNase-seq）以及RNA聚合酶II（Pol II）的染色质免疫共沉淀测序（ChIP-seq）技术，探究G-四链体（G4s）对染色质组织与转录过程的调控机制。功能分析实验使用了两种工具：可稳定G4结构的吡啶司他汀（pyridostatin），以及能够抑制Pol II活性的α-鹅膏蕈碱（alpha-amanitin）。