Nilson Kyle A, Lawson Christine K, Mullen Nicholas J, Ball Christopher B, Spector Benjamin M, Meier Jeffery L, Price David H
Department of Biochemistry, University of Iowa, Iowa City, IA 52242, USA.
Molecular and Cellular Biology Program, University of Iowa, Iowa City, IA 52242, USA.
Nucleic Acids Res. 2017 Nov 2;45(19):11088-11105. doi: 10.1093/nar/gkx724.
Oxidative stress has pervasive effects on cells but how they respond transcriptionally upon the initial insult is incompletely understood. We developed a nuclear walk-on assay that semi-globally quantifies nascent transcripts in promoter-proximal paused RNA polymerase II (Pol II). Using this assay in conjunction with ChIP-Seq, in vitro transcription, and a chromatin retention assay, we show that within a minute, hydrogen peroxide causes accumulation of Pol II near promoters and enhancers that can best be explained by a rapid decrease in termination. Some of the accumulated polymerases slowly move or 'creep' downstream. This second effect is correlated with and probably results from loss of NELF association and function. Notably, both effects were independent of DNA damage and ADP-ribosylation. Our results demonstrate the unexpected speed at which a global transcriptional response can occur. The findings provide strong support for the residence time of paused Pol II elongation complexes being much shorter than estimated from previous studies.
氧化应激对细胞具有广泛影响，但细胞在遭受初始损伤时如何进行转录应答尚不完全清楚。我们开发了一种核内游动分析方法，可半全局定量启动子近端暂停的RNA聚合酶II（Pol II）中的新生转录本。结合使用该分析方法与染色质免疫沉淀测序（ChIP-Seq）、体外转录和染色质保留分析，我们发现，在一分钟内，过氧化氢会导致Pol II在启动子和增强子附近积累，这最能通过终止的快速减少来解释。一些积累的聚合酶会缓慢向下游移动或“蠕动”。这第二种效应与NELF结合和功能丧失相关，可能是由其导致的。值得注意的是，这两种效应均独立于DNA损伤和ADP-核糖基化。我们的结果证明了全局转录应答能够发生的意外速度。这些发现为暂停的Pol II延伸复合物的停留时间比先前研究所估计的要短得多提供了有力支持。
