RNA polymerase is poised for activation across the genome

Regulation of gene expression is integral to the development and survival of all organisms. Transcription begins with the assembly of a pre-initiation complex at the gene promoter, followed by initiation of RNA synthesis and the transition to productive elongation. In many cases, recruitment of RNA polymerase II (Pol II) to a promoter is necessary and sufficient for activation of gene. However, there are a few notable exceptions to this paradigm, including heat shock genes and several proto-oncogenes, whose expression is attenuated by regulated stalling of polymerase elongation within the promoter-proximal region. To determine the importance of polymerase stalling for transcription regulation, we performed a genome-wide search for Drosophila genes with promoter-proximally stalled Pol II. Our data reveal that stalling is widespread, occurring at hundreds of genes that respond to stimuli and developmental signals, indicating a role for regulation of polymerase elongation in the transcriptional responses to dynamic environmental and developmental cues. Keywords: gene expression, ChIP-chip, transcriptional regulation Drosophila Schneider cells (S2) were untreated or treated with dsRNA against LacZ or NELF for 96 hours. RNA isolation and immunoprecipitations (IPs) were performed for each of the test samples. Each treatment group (untreated, dsRNA-LacZ, dsRNA-NELF) was cultured in duplicate. Each RNA sample was amplified according to Affymetrix Eukaryotic One-cycle Protocol and hybridized to an Affymetrix Drosophila Genome 2.0 array, resulting in two biological replicates for each treatment group. Immunoprecipitations were performed either in the presence of Pol II Rpb3 or Ser2P Pol II CTD antibody or mock-immunoprecipitated with Protein-A agarose in the absence of antibody. Each IP was paired with a genomic DNA sample obtained at the time of the experiment. A separate labeling reaction was completed for each array type on which the IP/genomic DNA comparison was made (Agilent Dm3, Dm7 and Whole Drosophila Genome 2 chip set) and each of the two biological replicates were assayed.

基因表达调控是所有生物体发育与存活的核心组成环节。转录起始于基因启动子区域前起始复合物的组装，随后启动RNA合成并过渡至有效延伸阶段。多数情况下，将RNA聚合酶II（RNA polymerase II, Pol II）招募至启动子区，足以激活基因的表达。然而该调控范式存在若干显著例外，包括热休克基因与多种原癌基因，它们的表达通过启动子近端区域内聚合酶延伸的调控性暂停而受到衰减。为探究聚合酶暂停对转录调控的重要性，我们开展了全基因组范围的筛选，以鉴定携带启动子近端暂停Pol II的果蝇基因。本研究数据显示，聚合酶暂停现象广泛存在，发生于数百个响应刺激与发育信号的基因中，这表明聚合酶延伸的调控在生物体响应动态环境与发育信号的转录应答中发挥着关键作用。
关键词：基因表达、染色质免疫沉淀芯片（ChIP-chip）、转录调控
果蝇Schneider细胞（S2）分为未处理组，以及分别使用靶向LacZ或NELF的双链RNA（double-stranded RNA, dsRNA）处理96小时的组别。对各测试样本开展RNA分离与免疫沉淀（immunoprecipitations, IPs）实验。每个处理组（未处理组、dsRNA-LacZ组、dsRNA-NELF组）均设置双重复培养。每份RNA样本均按照Affymetrix真核生物单循环实验方案进行扩增，并与Affymetrix果蝇基因组2.0芯片进行杂交，每个处理组获得两份生物学重复样本。免疫沉淀实验分别使用Pol II Rpb3抗体、Ser2P Pol II C端结构域抗体开展，同时设置以蛋白A琼脂糖珠在无抗体条件下进行的模拟免疫沉淀对照组。每份免疫沉淀样本均与实验同期获取的基因组DNA样本配对分析。针对用于免疫沉淀/基因组DNA对比分析的各类芯片（Agilent Dm3、Dm7及全果蝇基因组2.0芯片组）分别完成独立的标记反应，并对两份生物学重复样本均进行检测。