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The expansion of introns within mammalian genomes poses a challenge for the production of full-length messenger RNAs (mRNAs), with increasing evidence that these long AT-rich sequences present obstacles to transcription. Here, we investigate RNA polymerase II (RNAPII) elongation at high resolution in mammalian cells and demonstrate that RNAPII transcribes faster across introns. Moreover, we find that this acceleration requires the association of U1 snRNP (U1) with the elongation complex at 5′ splice sites. The role of U1 to stimulate elongation rate through introns reduces the frequency of both premature termination and transcriptional arrest, thereby dramatically increasing RNA production. We further show that changes in RNAPII elongation rate due to AT content and U1 binding explain previous reports of pausing or termination at splice junctions and the edge of CpG islands. We propose that U1-mediated acceleration of elongation has evolved to mitigate the risks that long AT-rich introns pose to transcript completion. [Display omitted] • AT content of the transcribed region strongly impacts RNAPII elongation rate • U1 snRNP enhances transcription initiation or elongation in a gene-dependent manner • RNAPII acceleration in AT-rich sequences requires U1 snRNP • Without U1 snRNP to stimulate elongation, RNAPII is prone to arrest and termination How does RNAPII transcribe long mammalian genes, avoiding premature termination or arrest within expansive introns? Mimoso and Adelman demonstrate that splicing factor U1 snRNP increases RNAPII elongation rate within AT-rich introns, thereby reducing the likelihood of RNAPII termination or arrest. U1 snRNP is thus critical for synthesis of long genes. [ABSTRACT FROM AUTHOR]

Widespread anti-inflammatory actions of glucocorticoid hormones are mediated by the glucocorticoid receptor (GR), a ligand-dependent transcription factor of the nuclear receptor superfamily. In conjunction with its corepressor GR-interacting protein-1 (GRIP1), GR tethers to the DNA-bound activator protein-1 and NF-κB and represses transcription of their target proinflammatory cytokine genes. However, these target genes fall into distinct classes depending on the step of the transcription cycle that is rate-limiting for their activation: Some are controlled through RNA polymerase II (PolII) recruitment and initiation, whereas others undergo signal-induced release of paused elongation complexes into productive RNA synthesis. Whether these genes are differentially regulated by GR is unknown. Here we report that, at the initiation-controlled inflammatory genes in primary macrophages, GR inhibited LPS-induced PolII occupancy. In contrast, at the elongation-controlled genes, GR did not affect PolII recruitment or transcription initiation but promoted, in a GRIP1-dependent manner, the accumulation of the pause-inducing negative elongation factor. Consistently, GR-dependent repression of elongation-controlled genes was abolished specifically in negative elongation factor-deficient macrophages. Thus, GR:GRIP1 use distinct mechanisms to repress inflammatory genes at different stages of the transcription cycle. [ABSTRACT FROM AUTHOR]

Summary: Recent genome-wide studies in metazoans have shown that RNA polymerase II (Pol II) accumulates to high densities on many promoters at a rate-limited step in transcription. However, the status of this Pol II remains an area of debate. Here, we compare quantitative outputs of a global run-on sequencing assay and chromatin immunoprecipitation sequencing assays and demonstrate that the majority of the Pol II on Drosophila promoters is transcriptionally engaged; very little exists in a preinitiation or arrested complex. These promoter-proximal polymerases are inhibited from further elongation by detergent-sensitive factors, and knockdown of negative elongation factor, NELF, reduces their levels. These results not only solidify the notion that pausing occurs at most promoters, but demonstrate that it is the major rate-limiting step in early transcription at these promoters. Finally, the divergent elongation complexes seen at mammalian promoters are far less prevalent in Drosophila, and this specificity in orientation correlates with directional core promoter elements, which are abundant in Drosophila. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Altering gene expression in response to stimuli is a pivotal mechanism through which organisms execute developmental programs and respond to changes in their environment. Packaging of promoter DNA into chromatin can greatly impact the ability of RNA polymerase II to access and transcribe a gene. Promoter chromatin environments thus play a central role in establishing transcriptional output appropriate for specific environmental conditions or developmental states. Recent genomic studies have illuminated general principles of chromatin organization and deepened our understanding of how promoter sequence and nucleosome architecture may impact gene expression. Concurrently, pausing of polymerase during early elongation has been recognized as an important event influencing transcription of genes within stimulus-responsive networks. Promoters regulated by pausing are now recognized to possess a distinct chromatin architecture that may facilitate the plasticity of gene expression in response to signaling events. Here we review advances in understanding chromatin and pausing, and explore how coupling Pol II pausing to distinct promoter architectures may help organisms achieve flexible yet precise transcriptional control. This article is part of a Special Issue entitled: Chromatin in time and space. [Copyright &y& Elsevier]

Transcription of immediate early genes (IEGs) in neurons is highly sensitive to neuronal activity, but the mechanism underlying these early transcription events is largely unknown. We found that several IEGs, such as Arc (also known as Arg3.1), are poised for near-instantaneous transcription by the stalling of RNA polymerase II (Pol II) just downstream of the transcription start site in rat neurons. Depletion through RNA interference of negative elongation factor, a mediator of Pol II stalling, reduced the Pol II occupancy of the Arc promoter and compromised the rapid induction of Arc and other IEGs. In contrast, reduction of Pol II stalling did not prevent transcription of IEGs that were expressed later and largely lacked promoter-proximal Pol II stalling. Together, our data strongly indicate that the rapid induction of neuronal IEGs requires poised Pol II and suggest a role for this mechanism in a wide variety of transcription-dependent processes, including learning and memory. [ABSTRACT FROM AUTHOR]

Abstract: Proper regulation of gene expression is essential for the differentiation, development and survival of all cells and organisms. Recent work demonstrates that transcription of many genes, including key developmental and stimulus-responsive genes, is regulated after the initiation step, by pausing of RNA polymerase II during elongation through the promoter-proximal region. Thus, there is great interest in better understanding the events that follow transcription initiation and the ways in which the efficiency of early elongation can be modulated to impact expression of these highly regulated genes. Here we describe our current understanding of the steps involved in the transition from an unstable initially transcribing complex into a highly stable and processive elongation complex. We also discuss the interplay between factors that affect early transcript elongation and the potential physiological consequences for genes that are regulated through transcriptional pausing. [ABSTRACT FROM AUTHOR]

Several eukaryotic transcription factors have been shown to modulate the elongation rate of RNA polymerase II (Pol II) on naked or chromatin-reconstituted templates in vitro. However, none of the tested factors have been shown to directly affect the elongation rate of Pol II in vivo. We performed a directed RNAi knock-down (KD) screen targeting 141 candidate transcription factors and identified multiple factors, including Spt6, that alter the induced Hsp70 transcript levels in Drosophila S2 cells. Spt6 is known to interact with both nucleosome structure and Pol II, and it has properties consistent with having a role in elongation. Here, ChIP assays of the first wave of Pol II after heat shock in S2 cells show that KD of Spt6 reduces the rate of Pol II elongation. Also, fluorescence recovery after photobleaching assays of GFP-Pol II in salivary gland cells show that this Spt6-dependent effect on elongation rate persists during steady-state-induced transcription, reducing the elongation rate from ∼1100 to 500 bp/min. Furthermore, RNAi depletion of Spt6 reveals its broad requirement during different stages of development. [ABSTRACT FROM AUTHOR]

Regulation of gene expression is integral to the development and survival of all organisms. Transcription begins with the assembly of a pre-initiation complex at the gene promoter, followed by initiation of RNA synthesis and the transition to productive elongation. In many cases, recruitment of RNA polymerase II (Pol II) to a promoter is necessary and sufficient for activation of genes. However, there are a few notable exceptions to this paradigm, including heat shock genes and several proto-oncogenes, whose expression is attenuated by regulated stalling of polymerase elongation within the promoter-proximal region. To determine the importance of polymerase stalling for transcription regulation, we carried out a genome-wide search for Drosophila melanogaster genes with Pol II stalled within the promoter-proximal region. Our data show that stalling is widespread, occurring at hundreds of genes that respond to stimuli and developmental signals. This finding indicates a role for regulation of polymerase elongation in the transcriptional responses to dynamic environmental and developmental cues. [ABSTRACT FROM AUTHOR]

It is widely assumed that the key rate-limiting step in gene activation is the recruitment of RNA polymerase II (Pol II) to the core promoter. Although there are well-documented examples in which Pol II is recruited to a gene but stalls, a general role for Pol II stalling in development has not been established. We have carried out comprehensive Pol II chromatin immunoprecipitation microarray (ChIP-chip) assays in Drosophila embryos and identified three distinct Pol II binding behaviors: active (uniform binding across the entire transcription unit), no binding, and stalled (binding at the transcription start site). The notable feature of the ∼10% genes that are stalled is that they are highly enriched for developmental control genes, which are either repressed or poised for activation during later stages of embryogenesis. We propose that Pol II stalling facilitates rapid temporal and spatial changes in gene activity during development. [ABSTRACT FROM AUTHOR]

Uninduced heat shock genes are poised for rapid activation, with RNA polymerase II (Pol II) transcriptionally engaged, but paused or stalled, within the promoter-proximal region. Upon heat shock, this Pol II is promptly released from the promoter region and additional Pol II and transcription factors are robustly recruited to the gene. Regulation of the heat shock response relies upon factors that modify the efficiency of elongation through the initially transcribed sequence. Here, we report that Pol II is susceptible to transcription arrest within the promoter-proximal region of Drosophila hsp70 and that transcript cleavage factor TFIIS is essential for rapid induction of hsp70 RNA. Moreover, using a tandem RNAi-ChIP assay, we discovered that TFIIS is not required to establish the stalled Pol II, but that TFIIS is critical for efficient release of Pol II from the hsp70 promoter region and the subsequent recruitment of additional Pol II upon heat induction. [Copyright &y& Elsevier]

Focuses on uniform kinetic behavior of Escherichia coli RNA polymerase molecules by using single-molecule measurements. Display of transient pauses by RNA polymerase molecules.

Presents information on a study which demonstrated that while the presence of MotA and AsiA increases the initial recruitment of RNA polymerase to a T4 middle promoter, it does not alter the intrinsic stability of the discrete complexes formed. Materials and methods; Techniques used in the study; Discussion on the research findings.

By precisely tracking the waves of elongating RNA polymerase II (Pol II) during gene activation, Danko et al. (2013), in this issue of Molecular Cell, discovered a surprising diversity of elongation rates among and along human genes. [ABSTRACT FROM AUTHOR]

DNA packaging into chromatin presents a strong barrier to RNA polymerase II transcription. In this issue of Molecular Cell, Kireeva et al. describe a minimal system to examine polymerase II transcription through a positioned nucleosome and show, surprisingly, that transcription leads to the displacement of an H2A·H2B dimer from the nucleosome without altering nucleosome position. [Copyright &y& Elsevier]

Abstract: Transcription is a sophisticated multi-step process in which RNA polymerase II (Pol II) transcribes a DNA template into RNA in concert with a broad array of transcription initiation, elongation, capping, termination, and histone modifying factors. Recent global analyses of Pol II distribution have indicated that many genes are regulated during the elongation phase, shedding light on a previously underappreciated mechanism for controlling gene expression. Understanding how various factors regulate transcription elongation in living cells has been greatly aided by chromatin immunoprecipitation (ChIP) studies, which can provide spatial and temporal resolution of protein–DNA binding events. The coupling of ChIP with DNA microarray and high-throughput sequencing technologies (ChIP-chip and ChIP-seq) has significantly increased the scope of ChIP studies and genome-wide maps of Pol II or elongation factor binding sites can now be readily produced. However, while ChIP-chip/ChIP-seq data allow for high-resolution localization of protein–DNA binding sites, they are not sufficient to dissect protein function. Here we describe techniques for coupling ChIP-chip/ChIP-seq with genetic, chemical, and experimental manipulation to obtain mechanistic insight from genome-wide protein–DNA binding studies. We have employed these techniques to discern immature promoter-proximal Pol II from productively elongating Pol II, and infer a critical role for the transition between initiation and full elongation competence in regulating development and gene induction in response to environmental signals. [Copyright &y& Elsevier]

Pre-mRNA processing steps are tightly coordinated with transcription in many organisms. To determine how co-transcriptional splicing is integrated with transcription elongation and 3′ end formation in mammalian cells, we performed long-read sequencing of individual nascent RNAs and precision run-on sequencing (PRO-seq) during mouse erythropoiesis. Splicing was not accompanied by transcriptional pausing and was detected when RNA polymerase II (Pol II) was within 75–300 nucleotides of 3′ splice sites (3′SSs), often during transcription of the downstream exon. Interestingly, several hundred introns displayed abundant splicing intermediates, suggesting that splicing delays can take place between the two catalytic steps. Overall, splicing efficiencies were correlated among introns within the same transcript, and intron retention was associated with inefficient 3′ end cleavage. Remarkably, a thalassemia patient-derived mutation introducing a cryptic 3′SS improved both splicing and 3′ end cleavage of individual β-globin transcripts, demonstrating functional coupling between the two co-transcriptional processes as a determinant of productive gene output. • Most introns in differentiating murine erythroblasts are spliced co-transcriptionally • Long-read sequencing of nascent RNA reveals coordinated removal of multiple introns • Pol II pausing is not significantly detected at splice sites • Co-transcriptional splicing efficiency influences 3′ end cleavage efficiency Does rapid splicing after intron transcription promote mammalian gene expression? Reimer et al. report that most introns are removed when Pol II is located within the downstream exon, suggesting that proximity to the spliceosome supports regulation. Introns within single transcripts were coordinately spliced, and efficient splicing promoted 3′ end cleavage. [ABSTRACT FROM AUTHOR]

RNA polymerase II (RNAPII) pausing in early elongation is critical for gene regulation. Paused RNAPII can be released into productive elongation by the kinase P-TEFb or targeted for premature termination by the Integrator complex. Integrator comprises endonuclease and phosphatase activities, driving termination by cleavage of nascent RNA and removal of stimulatory phosphorylation. We generated a degron system for rapid Integrator endonuclease (INTS11) depletion to probe the direct consequences of Integrator-mediated RNA cleavage. Degradation of INTS11 elicits nearly universal increases in active early elongation complexes. However, these RNAPII complexes fail to achieve optimal elongation rates and exhibit persistent Integrator phosphatase activity. Thus, only short transcripts are significantly upregulated following INTS11 loss, including transcription factors, signaling regulators, and non-coding RNAs. We propose a uniform molecular function for INTS11 across all RNAPII-transcribed loci, with differential effects on particular genes, pathways, or RNA biotypes reflective of transcript lengths rather than specificity of Integrator activity. [Display omitted] • Rapid degradation of INTS11 globally increases early elongation complexes • In the absence of INTS11, RNAPII released into genes is deficient in elongation • Loss of INTS11 endonuclease does not disrupt Integrator-PP2A phosphatase function • Predominantly short genes and non-coding RNAs are upregulated upon INTS11 degradation Stein et al. show that Integrator broadly balances levels of RNAPII pause release with premature termination. Acute depletion of Integrator endonuclease allows for increased RNAPII release into coding and non-coding RNAs. However, RNAPII elongation is inefficient, and only short RNAs are upregulated, including several transcription factors that initiate signaling pathways. [ABSTRACT FROM AUTHOR]

Summary: Metazoan gene expression is often regulated after the recruitment of RNA polymerase II (Pol II) to promoters, through the controlled release of promoter-proximally paused Pol II into productive RNA synthesis. Despite the prevalence of paused Pol II, very little is known about the dynamics of these early elongation complexes or the fate of the short transcription start site-associated (tss) RNAs they produce. Here, we demonstrate that paused elongation complexes can be remarkably stable, with half-lives exceeding 15 min at genes with inefficient pause release. Promoter-proximal termination by Pol II is infrequent, and released tssRNAs are targeted for rapid degradation. Further, we provide evidence that the predominant tssRNA species observed are nascent RNAs held within early elongation complexes. We propose that stable pausing of polymerase provides a temporal window of opportunity for recruitment of factors to modulate gene expression and that the nascent tssRNA represents an appealing target for these interactions. [Copyright &y& Elsevier]

Summary: Lysine acetylation regulates transcription by targeting histones and nonhistone proteins. Here we report that the central regulator of transcription, RNA polymerase II, is subject to acetylation in mammalian cells. Acetylation occurs at eight lysines within the C-terminal domain (CTD) of the largest polymerase subunit and is mediated by p300/KAT3B. CTD acetylation is specifically enriched downstream of the transcription start sites of polymerase-occupied genes genome-wide, indicating a role in early stages of transcription initiation or elongation. Mutation of lysines or p300 inhibitor treatment causes the loss of epidermal growth-factor-induced expression of c-Fos and Egr2, immediate-early genes with promoter-proximally paused polymerases, but does not affect expression or polymerase occupancy at housekeeping genes. Our studies identify acetylation as a new modification of the mammalian RNA polymerase II required for the induction of growth factor response genes. [Copyright &y& Elsevier]

Emerging evidence indicates that gene expression in higher organisms is regulated by RNA polymerase II stalling during early transcription elongation. To probe the mechanisms responsible for this regulation, we developed methods to isolate and characterize short RNAs derived from stalled RNA polymerase II in Drosophila cells. Significant levels of these short RNAs were generated from more than one-third of all genes, indicating that promoter-proximal stalling is a general feature of early polymerase elongation. Nucleotide composition of the initially transcribed sequence played an important role in promoting transcriptional stalling by rendering polymerase elongation complexes highly susceptible to backtracking and arrest. These results indicate that the intrinsic efficiency of early elongation can greatly affect gene expression. [ABSTRACT FROM AUTHOR]

The anti-σ factor Asia effects a change in promoter specificity of the Escherichia coli RNA polymerase via interactions with two conserved regions of the σ[sup 70] subunit, denoted 4.1 and 4.2. Free Asia is a symmetrical homodimer. Here, we show that Asia is monomeric when bound to σ[sup 70] and that a subset of the residues that contribute to the homodimer interface also contributes to the interface with σ[sup 70]. Asia interacts primarily with C-terminal sections of regions 4.1 and 4.2, which show remarkable sequence similarity. An Asia monomer can simultaneously, and apparently cooperatively, bind both isolated regions 4.1 and 4.2 at preferred, distinct subsites, whereas region 4.1 alone or region 4.2 alone can interact with either subsite. These results suggest structural and functional plasticity in the interaction of Asia with σ[sup 70] and support the notion of discrete roles for regions 4.1 and 4.2 in transcription regulation by AsiA. Furthermore, we show that Asia inhibits recognition of the -35 consensus promoter element by region 4 of σ[sup 70] indirectly, as the residues on region 4 responsible for Asia binding are distinct from those involved in DNA binding. Finally, we show that Asia must directly disrupt the interaction of region 4 with the RNA polymerase β subunit flap domain, resulting in a distance change between region 2 and region 4 of σ[sup 70]. Thus, a new paradigm for transcription regulation by Asia is emerging, whereby the distance between the DNA binding domains in σ[sup 70] is regulated, and promoter recognition specificity is modulated, by mediating the interactions of the o region 4 with the β subunit flap domain. [ABSTRACT FROM AUTHOR]

Transcriptional regulation in eukaryotes occurs at promoter-proximal regions wherein transcriptionally engaged RNA polymerase II (Pol II) pauses before proceeding toward productive elongation. The role of chromatin in pausing remains poorly understood. Here, we demonstrate that the histone deacetylase SIRT6 binds to Pol II and prevents the release of the negative elongation factor (NELF), thus stabilizing Pol II promoter-proximal pausing. Genetic depletion of SIRT6 or its chromatin deficiency upon glucose deprivation causes intragenic enrichment of acetylated histone H3 at lysines 9 (H3K9ac) and 56 (H3K56ac), activation of cyclin-dependent kinase 9 (CDK9)—that phosphorylates NELF and the carboxyl terminal domain of Pol II—and enrichment of the positive transcription elongation factors MYC, BRD4, PAF1, and the super elongation factors AFF4 and ELL2. These events lead to increased expression of genes involved in metabolism, protein synthesis, and embryonic development. Our results identified SIRT6 as a Pol II promoter-proximal pausing-dedicated histone deacetylase. • The histone deacetylase SIRT6 binds to Pol II and promotes transcriptional pausing • SIRT6 retains NELF by deacetylating intragenically H3K9 and H3K56 • Removal of SIRT6 from chromatin increases K9/K56 acetylation • Recruitment of elongation factors promotes transcriptional elongation of SIRT6 targets Historically, histone deacetylases have been described as inhibitors of transcription initiation by compacting chromatin at the transcriptional starting site. Etchegaray et al. identified the histone deacetylase SIRT6 as a critical modulator of transcriptional pausing and elongation, acting downstream of recruitment of Pol II. [ABSTRACT FROM AUTHOR]

The expression of many metazoan genes is regulated through controlled release of RNA polymerase II (Pol II) that has paused during early transcription elongation. Pausing is highly enriched at genes in stimulus-responsive pathways, where it has been proposed to poise downstream targets for rapid gene activation. However, whether this represents the major function of pausing in these pathways remains to be determined. To address this question, we analyzed pausing within several stimulus-responsive networks in Drosophila and discovered that paused Pol II is much more prevalent at genes encoding components and regulators of signal transduction cascades than at inducible downstream targets. Within immune-responsive pathways, we found that pausing maintains basal expression of critical network hubs, including the key NF-κB transcription factor that triggers gene activation. Accordingly, loss of pausing through knockdown of the pause-inducing factor NELF leads to broadly attenuated immune gene activation. Investigation of murine embryonic stem cells revealed that pausing is similarly widespread at genes encoding signaling components that regulate self-renewal, particularly within the MAPK/ERK pathway. We conclude that the role of pausing goes well beyond poising-inducible genes for activation and propose that the primary function of paused Pol II is to establish basal activity of signal-responsive networks. [ABSTRACT FROM AUTHOR]

The Negative Elongation Factor (NELF) is a transcription regulatory complex that induces stalling of RNA polymerase II (Pol II) during early transcription elongation and represses expression of several genes studied to date, including Drosophila Hsp70, mammalian proto-oncogene junB, and HIV RNA. To determine the full spectrum of NELF target genes in Drosophila, we performed a microarray analysis of S2 cells depleted of NELF and discovered that NELF RNAi affects many rapidly inducible genes involved in cellular responses to stimuli. Surprisingly, only one-third of NELF target genes were, like Hsp70, up-regulated by NELF-depletion, whereas the majority of target genes showed decreased expression levels upon NELF RNAi. Our data reveal that the presence of stalled Pol II at this latter group of genes enhances gene expression by maintaining a permissive chromatin architecture around the promoter-proximal region, and that loss of Pol II stalling at these promoters is accompanied by a significant increase in nucleosome occupancy and a decrease in histone H3 Lys 4 trimethylation. These findings identify a novel, positive role for stalled Pol II in regulating gene expression and suggest that there is a dynamic interplay between stalled Pol II and chromatin structure. [ABSTRACT FROM AUTHOR]