Abstract
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.
Highlights
RNA polymerase II (Pol II) pausing is a key regulatory step in metazoan gene regulation
To explore whether Pol II pausing can be detected on this plasmid, we first cloned in the super core promoter (SCP) (Figure 1B and Table S1)
Genome-wide correlations have suggested a role for promoter sequences in Pol II pausing (Chen et al, 2013; Gaertner et al, 2012; Gilchrist et al, 2010; Hendrix et al, 2008; Nechaev et al, 2010; Shao and Zeitlinger, 2017), but functional data supporting a causal role for these sequences had been largely lacking
Summary
RNA polymerase II (Pol II) pausing is a key regulatory step in metazoan gene regulation. Genome-wide profiling of Pol II and nascent transcripts suggest that pausing is widespread across the genome (Core et al, 2008; Muse et al, 2007; Nechaev et al, 2010; Zeitlinger et al, 2007), and inhibition of p-TEFb blocks the productive elongation of the majority of active genes (Chao and Price, 2001; Jonkers et al, 2014; Ni et al, 2008) Together, these results suggest that pause release is a general and obligatory step in transcription. Despite the evidence that Pol II pausing depends on the promoter sequence, how core promoter elements regulate Pol II pausing remains elusive
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