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

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

eLife digest

Many different factors determine how quickly the DNA in genes is transcribed to produce molecules of messenger RNA. The start of the transcription process features two milestones: first, an enzyme called RNA Polymerase II starts the process; shortly afterwards, however, the process pauses and only starts again when other proteins are recruited. This provides two levels of control over the production of messenger RNA and, it also allows the transcription process to be interrupted in order to study the rate of transcription.

Here, Jonkers, Kwak and Lis used two drugs to block either the start of transcription or the release from the paused state in mouse cells. Both drugs prevented new transcription and disrupted about 95% of the total number of genes. However, RNA Polymerase II that was already copying DNA could continue to copy, and did so at an average rate of 2000 bases per minute. Transcription rates were, however, shown to vary between different genes—highly active genes are transcribed faster. Transcription rates also varied within individual genes, with the enzyme accelerating as it moves along the gene. This suggests that the transcription machinery, including other proteins that improve the enzyme’s efficiency, are recruited or modified after transcription has already started, and that these proteins help the enzyme to reach its maximum transcription speed.

Other factors also affected the transcription rate: the genetic code is written in four letters—A, C, G and T—and genes that contained more Cs and Gs were transcribed slower than those with lots of As and Ts. Genes also contain regions called exons that code for proteins, and regions called introns that do not: Jonkers, Kwak and Lis found that genes with lots of exons were transcribed slower. Furthermore, DNA is wrapped around proteins into a compacted structure, and genes that had certain chemical markings added to these proteins were transcribed faster.

The work of Jonkers, Kwak and Lis is the first in-depth look at how transcription is affected by gene structure, and leads the way to uncovering how transcription rates throughout genes are regulated to influence production of messenger RNA.

DOI: http://dx.doi.org/10.7554/eLife.02407.002