Oxidative stress rapidly stabilizes promoter-proximal paused Pol II across the human genome

Abstract

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.

Promoter-proximal pausing of RNA polymerase II: a nexus of gene regulation

“…A number of subsequent studies have made use of Trp to monitor pausing duration in a variety of cell types and conditions, using a broad repertoire of techniques to measure paused Pol II levels (Buckley et al 2014;Jonkers et al 2014;Chen et al 2015;Krebs et al 2017;Nilson et al 2017;Shao and Zeitlinger 2017;Erickson et al 2018). Despite these differences, all such studies have shown that Pol II displays highly variable, gene-specific profiles of stability.…”

“…Despite these differences, all such studies have shown that Pol II displays highly variable, gene-specific profiles of stability. Pol II signal was dramatically reduced at some genes by the earliest time point tested (often ∼2 min), whereas other genes displayed stably paused Pol II after 20 min or even 1 hr (Henriques et al 2013;Buckley et al 2014;Jonkers et al 2014;Chen et al 2015;Krebs et al 2017;Nilson et al 2017;Shao and Zeitlinger 2017;Erickson et al 2018;Steurer et al 2018). As a result of this striking variability among genes and the different strategies, numbers of genes, and thresholds used for data analysis in the individual studies, reported average half-lives of paused Pol II span from ∼2 to 30 min.…”

“…However, despite >30 yr of research, the mechanisms underlying the regulation of early elongation remain an area of vigorous study and even controversy to this day. In particular, active debates remain about how much of the paused elongation complex proceeds into productive RNA synthesis versus undergoing promoter-proximal termination (Henriques et al 2013;Jonkers et al 2014;Chen et al 2015;Krebs et al 2017;Nilson et al 2017;Shao and Zeitlinger 2017;Erickson et al 2018).…”

The Integrator complex terminates promoter-proximal transcription at protein-coding genes

“…Thus, these genes consistently require new transcription initiation in order to maintain appropriate levels of promoter Pol II. As such, it has been proposed that Pol II undergoes multiple iterative cycles of initiation, early elongation and premature termination at these genes, each time releasing a short, non-functional RNA (Erickson et al, 2018;Kamieniarz-Gdula and Proudfoot, 2019;Krebs et al, 2017;Nilson et al, 2017;Steurer et al, 2018). In contrast, a majority of genes were found to harbor a more stable Pol II, with paused polymerase levels persisting after Trp treatment.…”

“…We set out to determine the causes of differential stability of paused Pol II across mRNA genes. In particular, we were interested in defining factors that might render promoter Pol II susceptible to premature termination and the release of short, immature RNAs (Erickson et al, 2018;Henriques et al, 2013;Krebs et al, 2017;Nilson et al, 2017;Shao and Zeitlinger, 2017;Steurer et al, 2018). Strikingly, we discovered that the Integrator complex is enriched at mRNA promoters with unstable Pol II pausing.…”