Abstract
Eukaryotic gene transcription is often controlled at the level of RNA polymerase II (Pol II) pausing in the promoter-proximal region. Pausing Pol II limits the frequency of transcription initiation (‘pause-initiation limit’), predicting that the pause duration must be decreased for transcriptional activation. To test this prediction, we conduct a genome-wide kinetic analysis of the heat shock response in human cells. We show that the pause-initiation limit restricts transcriptional activation at most genes. Gene activation generally requires the activity of the P-TEFb kinase CDK9, which decreases the duration of Pol II pausing and thereby enables an increase in the productive initiation frequency. The transcription of enhancer elements is generally not pause limited and can be activated without CDK9 activity. Our results define the kinetics of Pol II transcriptional regulation in human cells at all gene classes during a natural transcription response.
Highlights
Eukaryotic gene transcription is often controlled at the level of RNA polymerase II (Pol II) pausing in the promoter-proximal region
We show that inhibition of the P-TEFb kinase CDK9 impairs full activation upon heat shock, confirming that a decrease in Pol II pause duration is a critical step in gene activation
To obtain the productive initiation frequency I and the promoter-proximal pause duration d28, we carried out transcriptome sequencing (TT-seq) and mNET-seq of total Pol II in human K562 cells (Fig. 1a)
Summary
Eukaryotic gene transcription is often controlled at the level of RNA polymerase II (Pol II) pausing in the promoter-proximal region. Pausing Pol II limits the frequency of transcription initiation (‘pause-initiation limit’), predicting that the pause duration must be decreased for transcriptional activation. To test this prediction, we conduct a genome-wide kinetic analysis of the heat shock response in human cells. Pol II occupancy can be mapped using DNA by chromatin immunoprecipitation (IP) assays[23], and along nascent RNA transcripts by nuclear run-on assays[8,24] or by native elongating transcript sequencing (NET-seq)[25,26]. RNA is fragmented, and only newly synthesized RNA fragments are purified and sequenced
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