Recent years have witnessed a sea change in our understanding of transcription regulation: whereas traditional models focused solely on the events that brought RNA polymerase II (Pol II) to a gene ...promoter to initiate RNA synthesis, emerging evidence points to the pausing of Pol II during early elongation as a widespread regulatory mechanism in higher eukaryotes. Current data indicate that pausing is particularly enriched at genes in signal-responsive pathways. Here the evidence for pausing of Pol II from recent high-throughput studies will be discussed, as well as the potential interconnected functions of promoter-proximally paused Pol II.
Elongation is becoming increasingly recognized as a critical step in eukaryotic transcriptional regulation. Although traditional genetic and biochemical studies have identified major players of ...transcriptional elongation, our understanding of the importance and roles of these factors is evolving rapidly through the recent advances in genome-wide and single-molecule technologies. Here, we focus on how elongation can modulate the transcriptional outcome through the rate-liming step of RNA polymerase II (Pol II) pausing near promoters and how the participating factors were identified. Among the factors we describe are the pausing factors--NELF (negative elongation factor) and DSIF (DRB sensitivity-inducing factor)--and P-TEFb (positive elongation factor b), which is the key player in pause release. We also describe the high-resolution view of Pol II pausing and propose nonexclusive models for how pausing is achieved. We then discuss Pol II elongation through the bodies of genes and the roles of FACT and SPT6, factors that allow Pol II to move through nucleosomes.
The heat shock response (HSR) is critical for survival of all organisms. However, its scope, extent, and the molecular mechanism of regulation are poorly understood. Here we show that the genome-wide ...transcriptional response to heat shock in mammals is rapid and dynamic and results in induction of several hundred and repression of several thousand genes. Heat shock factor 1 (HSF1), the “master regulator” of the HSR, controls only a fraction of heat shock-induced genes and does so by increasing RNA polymerase II release from promoter-proximal pause. Notably, HSF2 does not compensate for the lack of HSF1. However, serum response factor appears to transiently induce cytoskeletal genes independently of HSF1. The pervasive repression of transcription is predominantly HSF1-independent and is mediated through reduction of RNA polymerase II pause release. Overall, mammalian cells orchestrate rapid, dynamic, and extensive changes in transcription upon heat shock that are largely modulated at pause release, and HSF1 plays a limited and specialized role.
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•∼1,500 genes are up- and ∼8,000 are downregulated during the first hour of heat shock•HSF1 induces heat shock protein genes by increasing promoter-proximal pause release•Upon heat shock, SRF transiently induces immediate-early cytoskeletal genes•Broad repression during heat shock in mammals occurs by inhibiting pause release
Using PRO-seq, Mahat et al. identify hundreds of genes that are upregulated and thousands that are downregulated transcriptionally in mammals during heat shock. A majority of this regulation is independent of HSF1 and HSF2, and many rapidly and transiently upregulated cytoskeletal genes depend on SRF. Both upregulation and downregulation are modulated at promoter-proximal pause release.
RNA polymerase II (Pol II) transcribes hundreds of kilobases of DNA, limiting the production of mRNAs and lncRNAs. We used global run-on sequencing (GRO-seq) to measure the rates of transcription by ...Pol II following gene activation. Elongation rates vary as much as 4-fold at different genomic loci and in response to two distinct cellular signaling pathways (i.e., 17β-estradiol E2 and TNF-α). The rates are slowest near the promoter and increase during the first ∼15 kb transcribed. Gene body elongation rates correlate with Pol II density, resulting in systematically higher rates of transcript production at genes with higher Pol II density. Pol II dynamics following short inductions indicate that E2 stimulates gene expression by increasing Pol II initiation, whereas TNF-α reduces Pol II residence time at pause sites. Collectively, our results identify previously uncharacterized variation in the rate of transcription and highlight elongation as an important, variable, and regulated rate-limiting step during transcription.
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► Pol II rates vary by 4-fold between genes, cell types, and induction conditions ► Rates correlate strongly with gene expression but weakly with nucleosome occupancy ► Rate variation has important consequences for mRNA and lncRNA production ► E2 signaling stimulates Pol II initiation; TNF-α signaling reduces Pol II pause time
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.
RNA polymerases are highly regulated molecular machines. We present a method (global run-on sequencing, GRO-seq) that maps the position, amount, and orientation of transcriptionally engaged RNA ...polymerases genome-wide. In this method, nuclear run-on RNA molecules are subjected to large-scale parallel sequencing and mapped to the genome. We show that peaks of promoter-proximal polymerase reside on ~30% of human genes, transcription extends beyond pre-messenger RNA 3' cleavage, and antisense transcription is prevalent. Additionally, most promoters have an engaged polymerase upstream and in an orientation opposite to the annotated gene. This divergent polymerase is associated with active genes but does not elongate effectively beyond the promoter. These results imply that the interplay between polymerases and regulators over broad promoter regions dictates the orientation and efficiency of productive transcription.
Transcription regulation occurs frequently through promoter-associated pausing of RNA polymerase II (Pol II). We developed a precision nuclear run-on and sequencing (PRO-seq) assay to map the ...genome-wide distribution of transcriptionally engaged Pol II at base pair resolution. Pol II accumulates immediately downstream of promoters, at intron-exon junctions that are efficiently used for splicing, and over 3′ polyadenylation sites. Focused analyses of promoters reveal that pausing is not fixed relative to initiation sites, nor is it specified directly by the position of a particular core promoter element or the first nucleosome. Core promoter elements function beyond initiation, and when optimally positioned they act collectively to dictate the position and strength of pausing. This "complex interaction" model was tested with insertional mutagenesis of the Drosophila Hsp70 core promoter.
Despite the conventional distinction between them, promoters and enhancers share many features in mammals, including divergent transcription and similar modes of transcription factor binding. Here we ...examine the architecture of transcription initiation through comprehensive mapping of transcription start sites (TSSs) in human lymphoblastoid B cell (GM12878) and chronic myelogenous leukemic (K562) ENCODE Tier 1 cell lines. Using a nuclear run-on protocol called GRO-cap, which captures TSSs for both stable and unstable transcripts, we conduct detailed comparisons of thousands of promoters and enhancers in human cells. These analyses identify a common architecture of initiation, including tightly spaced (110 bp apart) divergent initiation, similar frequencies of core promoter sequence elements, highly positioned flanking nucleosomes and two modes of transcription factor binding. Post-initiation transcript stability provides a more fundamental distinction between promoters and enhancers than patterns of histone modification and association of transcription factors or co-activators. These results support a unified model of transcription initiation at promoters and enhancers.
Transcription regulation is critical to organism development and homeostasis. Control of expression of the 20,000 genes in human cells requires many hundreds of proteins acting through sophisticated ...multistep mechanisms. In this Historical Perspective, I highlight the progress that has been made in elucidating eukaryotic transcriptional mechanisms through an array of disciplines and approaches, and how this concerted effort has been driven by the development of new technologies.
Following the discovery of widespread enhancer transcription, enhancers and promoters have been found to be far more similar than previously thought. In this issue of
, two studies (Henriques and ...colleagues pp. 26-41 and Mikhaylichenko and colleagues pp. 42-57) shine new light on the transcriptional nature of promoters and enhancers in
Together, these studies support recent work in mammalian cells that indicates that most active enhancers drive local transcription using factors and mechanisms similar to those of promoters. Intriguingly, enhancer transcription is shown to be coordinated by SPT5- and P-TEFb-mediated pause-release, but the pause half-life is shorter, and termination is more rapid at enhancers than at promoters. Moreover, bidirectional transcription from promoters is associated with enhancer activity, lending further credence to models in which regulatory elements exist along a spectrum of promoter-ness and enhancer-ness. We propose a general unified model to explain possible functions of transcription at enhancers.
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