Genome-wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons.

Iris Jonkers,Hojoong Kwak,John T Lis

doi:10.7554/elife.02407

Iris Jonkers, Hojoong Kwak + Show 1 more

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https://doi.org/10.7554/elife.02407

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Journal: eLife	Publication Date: Apr 29, 2014
Citations: 514	License type: CC BY 3.0

Affiliation: Howard Hughes Medical Institute, Cornell University

Abstract

Production of mRNA depends critically on the rate of RNA polymerase II (Pol II) elongation. To dissect Pol II dynamics in mouse ES cells, we inhibited Pol II transcription at either initiation or promoter-proximal pause escape with Triptolide or Flavopiridol, and tracked Pol II kinetically using GRO-seq. Both inhibitors block transcription of more than 95% of genes, showing that pause escape, like initiation, is a ubiquitous and crucial step within the transcription cycle. Moreover, paused Pol II is relatively stable, as evidenced from half-life measurements at ∼3200 genes. Finally, tracking the progression of Pol II after drug treatment establishes Pol II elongation rates at over 1000 genes. Notably, Pol II accelerates dramatically while transcribing through genes, but slows at exons. Furthermore, intergenic variance in elongation rates is substantial, and is influenced by a positive effect of H3K79me2 and negative effects of exon density and CG content within genes.DOI: http://dx.doi.org/10.7554/eLife.02407.001.

Highlights

Many steps throughout the transcription cycle of RNA polymerase II (Pol II) can be regulated, and modulation of any step has the potential to alter the timing and output of mRNA production
When a gene is activated, P-TEFb kinase is recruited to promoters and phosphorylates the paused Pol II·negative elongation factor (NELF)·DISF complex, allowing paused Pol II to more rapidly escape into productive elongation
While transcription elongation rates often seem to be taken for granted, we show that elongation rates are surprisingly diverse, both at the promoter proximal region, within the gene body, and between genes

Summary

Introduction

Many steps throughout the transcription cycle of RNA polymerase II (Pol II) can be regulated, and modulation of any step has the potential to alter the timing and output of mRNA production. The escape of Pol II from the paused state into productive elongation can be rate-limiting, and is dependent on the positive elongation factor P-TEFb, which consists of the Cdk kinase and CyclinT1 (Marshall et al, 1996; Lis et al, 2000; Ni et al, 2008). P-TEFb is recruited directly or indirectly to the paused Pol II complex by transcription activators, where it phosphorylates the C-Terminal domain (CTD), as well as DSIF and NELF, transforming DSIF into a positive elongation factor and evicting NELF (Peterlin and Price, 2006). P-TEFb appears to be both necessary and sufficient for paused Pol II escape into productive elongation; blocking P-TEFb kinase activity with the drug flavopiridol (FP) (Chao and Price, 2001) in Drosophila causes an increase of promoter proximal Pol II at the majority of genes (Henriques et al, 2013).

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