Abstract
Gene transcription can be activated by decreasing the duration of RNA polymerase II pausing in the promoter-proximal region, but how this is achieved remains unclear. Here we use a 'multi-omics' approach to demonstrate that the duration of polymerase pausing generally limits the productive frequency of transcription initiation in human cells ('pause-initiation limit'). We further engineer a human cell line to allow for specific and rapid inhibition of the P-TEFb kinase CDK9, which is implicated in polymerase pause release. CDK9 activity decreases the pause duration but also increases the productive initiation frequency. This shows that CDK9 stimulates release of paused polymerase and activates transcription by increasing the number of transcribing polymerases and thus the amount of mRNA synthesized per time. CDK9 activity is also associated with long-range chromatin interactions, suggesting that enhancers can influence the pause-initiation limit to regulate transcription.
Highlights
How can changes in the pause duration lead to synthesis of a different number of RNA transcripts per time? It has been suggested that a decreased pause duration goes along with a higher initiation frequency, because occupancy peaks for promoter-proximal Pol II can increase upon gene activation (Boehm et al, 2003) or can remain high even when pausing is impaired (Henriques et al, 2013)
We introduced a CDK9 analog sensitive mutation (CDK9as) into human Raji B cells by CRISPR-Cas9 (Materials and methods, Figure 1—figure supplement 2A–B)
Our results show that Pol II pausing can control transcription initiation and demonstrate the central role of CDK9 in controlling pause duration and thereby the productive initiation frequency
Summary
Transcription in metazoan cells is often regulated at the level of promoter-proximal pausing (Core et al, 2008; Day et al, 2016; Henriques et al, 2013; Nechaev et al, 2010; Rougvie and Lis, 1988; Strobl and Eick, 1992), which can be detected by measuring the occupancy with paused Pol II by ChIP-seq (Johnson et al, 2007), GRO-seq (Core et al, 2008), (m)NET-seq (Mayer et al, 2015; Nojima et al, 2015), or PRO-seq (Kwak et al, 2013). The mechanisms underlying how Pol II pausing can regulate RNA transcript synthesis remain unclear. The duration of pausing lies in the range of minutes (Jonkers et al, 2014) and does not considerably change the overall time it takes to complete a transcript. How can changes in the pause duration lead to synthesis of a different number of RNA transcripts per time? It has been suggested that a decreased pause duration goes along with a higher initiation frequency, because occupancy peaks for promoter-proximal Pol II can increase upon gene activation (Boehm et al, 2003) or can remain high even when pausing is impaired (Henriques et al, 2013)
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