Regulation of RNA Polymerase II Pausing during Transcription Elongation

Fei Chen

doi:https://doi.org/10.21985/N2P484

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Regulation of RNA Polymerase II Pausing during Transcription Elongation

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Transcription by RNA polymerase II (Pol II) in metazoans is regulated in multiple steps, including preinitiation complex (PIC) formation, initiation, Pol II escape, productive elongation, cotranscriptional RNA processing, and termination. Genome-wide studies have demonstrated that the phenomenon of promoter-bound Pol II pausing is widespread, especially for genes that respond to environmental and developmental cues. However, a mechanistic understanding of the paused Pol II state at promoters is limited. For example, at a global level, it is unclear to what extent the engaged paused Pol II is stably tethered to the promoter or undergoes rapid cycles of initiation and termination. Here we used the small molecule triptolide (TPL), an XPB/TFIIH inhibitor, to block transcriptional initiation and then measured Pol II occupancy by chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (ChIP-seq). This inhibition of initiation enabled us to investigate different states of paused Pol II. Specifically, our global analysis revealed that most genes with paused Pol II, as defined by a pausing index, show significant clearance of Pol II during the period of TPL treatment. Our study further identified a group of genes with unexpectedly stably paused Pol II, indicating the divergence in the dynamics of paused Pol II and possibly distinct mechanisms of pausing regulation. Moreover, we identified that Pol-II-associated factor 1 (PAF1) possesses an evolutionarily conserved function in metazoans in the regulation of promoter-proximal pausing. Reduction in PAF1 levels leads to an increased release of paused Pol II into gene bodies at thousands of genes. PAF1 depletion results in increased nascent and mature transcripts and increased levels of phosphorylation of Pol II's C-terminal domain on serine 2 (Ser2P). These changes can be explained by the recruitment of the Ser2P kinase super elongation complex (SEC) effecting increased release of paused Pol II into productive elongation, thus establishing PAF1 as a regulator of promoter-proximal pausing by Pol II. In addition, we found that PAF1 occupies transcriptional enhancers and restrains hyperactivation of a subset of them. Enhancer activation upon PAF1 loss leads to a major release of Pol II from paused promoters of nearby PAF1 target genes, which provides mechanistic insights in the regulation of pausing by PAF1. In particular, knockout of PAF1-regulated enhancers attenuates the release of paused Pol II on PAF1 target genes without major interference in the establishment of pausing at their cognate promoters. Thus, we propose that enhancers can primarily modulate gene expression by controlling the release of paused Pol II in a PAF1-dependent manner.

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