Reporter-ChIP-nexus reveals strong contribution of the Drosophila initiator sequence to RNA polymerase pausing

RNA polymerase II (Pol II) pausing is a general regulatory step in transcription, yet the stability of paused Pol II varies widely between genes. Although paused Pol II stability correlates with core promoter elements, the contribution of individual sequences remains unclear, in part because no rapid assay is available for measuring the changes in Pol II pausing as a result of altered promoter sequences. Here, we overcome this hurdle by showing that ChIP-nexus captures the endogenous Pol II pausing on transfected plasmids. Using this reporter-ChIP-nexus assay in Drosophila cells, we show that the pausing stability is influenced by downstream promoter sequences, but that the strongest contribution to Pol II pausing comes from the initiator sequence, in which a single nucleotide, a G at the +2 position, is critical for stable Pol II pausing. These results establish reporter-ChIP-nexus as a valuable tool to analyze Pol II pausing. ChIP-seq, ChIP-nexus or 5' RNA-seq was performed using transfected Drosophila Kc167 cells or Drosphila pseudoobscura ML83-63 cell line. Triptolide (TRI) treatment was used to assay the stability of paused Pol II.

RNA聚合酶II（RNA polymerase II, Pol II）暂停是转录过程中的通用调控步骤，但不同基因内暂停Pol II的稳定性差异悬殊。尽管暂停Pol II的稳定性与核心启动子元件相关，但单个序列的具体贡献仍不明确，部分原因在于尚无快速检测手段可用于测定启动子序列改变后Pol II暂停状态的变化。本研究通过证实ChIP-nexus技术可捕获转染质粒上的内源性Pol II暂停信号，突破了这一技术瓶颈。以果蝇细胞为模型开展reporter-ChIP-nexus检测后，本研究证实Pol II暂停稳定性受启动子下游序列调控，而对Pol II暂停影响最强的元件为起始子序列（initiator sequence），其中+2位的单核苷酸G对维持Pol II稳定暂停至关重要。上述研究结果证实reporter-ChIP-nexus可作为分析Pol II暂停状态的有效工具。本研究采用转染的果蝇Kc167细胞以及伪暗果蝇（Drosophila pseudoobscura）ML83-63细胞系，开展了ChIP-seq、ChIP-nexus及5' RNA-seq实验，并通过雷公藤内酯（triptolide, TRI）处理以检测暂停Pol II的稳定性。