Precision Run-on Sequencing Research Articles

Mapping Active RNA Polymerases in Proliferating and Quiescent Fission Yeast Cells Using Precision Run-On Sequencing.

The development of next-generation sequencing (NGS) approaches to investigate the functioning of RNA polymerases has led to groundbreaking advances in the field of transcriptional regulation. One powerful method, Precision nuclear Run-On sequencing (PRO-seq), maps the locations of RNA polymerase active sites genome-wide at high resolution. PRO-seq provides a snapshot of strand-specific transcriptional activity and does not rely on immunoprecipitation of the polymerase of interest. Notably, this technique has been utilized to investigate the control of the RNA polymerase II transcription cycle in a variety of model systems. However, the initially published PRO-seq method required significant amounts of starting sample and was technically challenging, both of which were deterrents for its broader use. Recently, an improved and simplified version called qPRO-seq that reduced the length of the experiment and the quantity of necessary input sample was developed for human and Drosophila cell lines. Here we provide an updated, step-by-step protocol in which we have validated and optimized qPRO-seq for the fission yeast Schizosaccharomyces pombe. Importantly, we have implemented this method for assessing RNA polymerase activity in nutrient-limiting conditions, for both proliferating and nitrogen-depleted quiescent cells.

Precision run-on sequencing (PRO-seq) for microbiome transcriptomics.

Bacteria respond to environmental stimuli through precise regulation of transcription initiation and elongation. Bulk RNA sequencing primarily characterizes mature transcripts, so to identify actively transcribed loci we need to capture RNA polymerase (RNAP) complexed with nascent RNA. However, such capture methods have only previously been applied to culturable, genetically tractable organisms such as E. coli and B. subtilis. Here we apply precision run-on sequencing (PRO-seq) to profile nascent transcription in cultured E. coli and diverse uncultured bacteria. We demonstrate that PRO-seq can characterize the transcription of small, structured, or post-transcriptionally modified RNAs, which are often absent from bulk RNA-seq libraries. Applying PRO-seq to the human microbiome highlights taxon-specific RNAP pause motifs and pause-site distributions across non-coding RNA loci that reflect structure-coincident pausing. We also uncover concurrent transcription and cleavage of CRISPR guide RNAs and transfer RNAs. We demonstrate the utility of PRO-seq for exploring transcriptional dynamics in diverse microbial communities.

PRO-seq: Precise Mapping of Engaged RNA Pol II at Single-Nucleotide Resolution.

Gene regulation is dependent on the production of mRNAs and a repertoire of non-coding RNAs by RNA polymerase II (RNAPII). Precision run-on sequencing (PRO-seq) maps the position of engaged RNAPII complexes at single-nucleotide resolution and can reveal direct targets of regulation, locations of enhancers, and transcription mechanisms that are difficult or impossible to measure by analysis of total cellular RNA. Briefly, this method first involves permeabilizing cells with mild detergents to remove intracellular NTPs and halt transcription. Transcription is then resumed in the presence of biotin-NTPs and sarkosyl to allow transcriptional incorporation of a single biotinylated NTP by RNAPII. The biotin moiety is then bound to streptavidin beads to stringently enrich for nascent RNAs. Sequencing libraries are then generated such that the first base read corresponds to the 3' end of the nascent transcript. Here, we describe our current protocol for generating PRO-seq libraries from metazoan cells, including adaptations of previously published protocols to incorporate unique molecular identifiers, reduce ligation bias, and improve library yields. Additional commentary describes quality control and processing of PRO-seq data and references for more advanced downstream analysis such as gene and enhancer identification. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Cell permeabilization for PRO-seq Basic Protocol 2: Construction of PRO-seq libraries Support Protocol: Adenylation of 3' adapter.

Widespread priming of transcriptional regulatory elements by incipient accessibility or RNA polymerase II pause in early embryos of the sea urchin Strongylocentrotus purpuratus.

Transcriptional regulatory elements (TREs) are the primary nodes that control developmental gene regulatory networks. In embryo stages, larvae, and adult differentiated red spherule cells of the sea urchin Strongylocentrotus purpuratus, transcriptionally engaged TREs are detected by Precision Run-On Sequencing (PRO-seq), which maps genome-wide at base pair resolution the location of paused or elongating RNA polymerase II (Pol II). In parallel, TRE accessibility is estimated by the Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-seq). Our analysis identifies surprisingly early and widespread TRE accessibility in 4-cell cleavage embryos that is not necessarily followed by concurrent or subsequent transcription. TRE transcriptional differences identified by PRO-seq provide more contrast among embryonic stages than ATAC-seq accessibility differences, in agreement with the apparent excess of accessible but inactive TREs during embryogenesis. Global TRE accessibility reaches a maximum around the 20-hour late blastula stage, which coincides with the consolidation of major embryo regionalizations and peak histone variant H2A.Z expression. A transcriptional potency model based on labile nucleosome TRE occupancy driven by DNA sequences and the prevalence of histone variants is proposed in order to explain the basal accessibility of transcriptionally inactive TREs during embryogenesis. However, our results would not reconcile well with labile nucleosome models based on simple A/T sequence enrichment. In addition, a large number of distal TREs become transcriptionally disengaged during developmental progression, in support of an early Pol II paused model for developmental gene regulation that eventually resolves in transcriptional activation or silencing. Thus, developmental potency in early embryos may be facilitated by incipient accessibility and transcriptional pause at TREs.

Inhibition of mitochondrial transcription by the neurotoxin MPP+

The neurotoxin MPP+ triggers cell death of dopamine neurons and induces Parkinson's disease symptoms in mice and men, but the immediate transcriptional response to this neurotoxin has not been studied. We therefore treated human SH-SY5Y cells with a low dose (0.1 mM) of MPP+ and measured the effect on nascent transcription by precision run-on sequencing (PRO-seq). We found that transcription of the mitochondrial genome was significantly reduced already after 30 min, whereas nuclear gene transcription was unaffected. Inhibition of respiratory complex I by MPP+ led to reduced ATP production, that may explain the diminished activity of mitochondrial RNA polymerase. Our results show that MPP+ has a direct effect on mitochondrial function and transcription, and that other gene expression or epigenetic changes induced by this neurotoxin are secondary effects that reflect a cellular adaptation program.

FT-6876, a Potent and Selective Inhibitor of CBP/p300, is Active in Preclinical Models of Androgen Receptor-Positive Breast Cancer.

Patients with triple-negative breast cancer (TNBC) expressing the androgen receptor (AR) respond poorly to neoadjuvant chemotherapy, although AR antagonists have shown promising clinical activity, suggesting these tumors are AR-dependent. cAMP responsive element binding protein (CREB)-binding protein (CBP) and p300 are transcriptional co-activators for the AR, a key driver of AR+ breast and prostate cancer, and may provide a novel therapeutic target in AR+ TNBC. The aim of this study was to determine the therapeutic potential of FT-6876, a new CBP/p300 bromodomain inhibitor, in breast cancer models with a range of AR levels in vitro and in vivo. Effects of FT-6876 on the CBP/p300 pathway were determined by combining chromatin immunoprecipitation (ChIP) with precision run-on sequencing (PRO-seq) complemented with H3K27 acetylation (Ac) and transcriptional profiling. The antiproliferative effect of FT-6876 was also measured in vitro and in vivo. We describe the discovery of FT-6876, a potent and selective CBP/p300 bromodomain inhibitor. The combination of ChIP and PRO-seq confirmed the reduction in H3K27Ac at specific promoter sites concurrent with a decrease in CBP/p300 on the chromatin and a reduction in nascent RNA and enhancer RNA. This was associated with a time- and concentration-dependent reduction in H3K37Ac associated with a decrease in AR and estrogen receptor (ER) target gene expression. This led to a time-dependent growth inhibition in AR+ models, correlated with AR expression. Tumor growth inhibition was also observed in AR+ tumor models of TNBC and ER+ breast cancer subtypes with consistent pharmacokinetics and pharmacodynamics. Our findings demonstrate FT-6876 as a promising new CBP/p300 bromodomain inhibitor, with efficacy in preclinical models of AR+ breast cancer.

Integrated genomics approaches identify transcriptional mediators and epigenetic responses to Afghan desert particulate matter in small airway epithelial cells.

Military Deployment to Southwest Asia and Afghanistan and exposure to toxic airborne particulates have been associated with an increased risk of developing respiratory disease, collectively termed deployment-related respiratory diseases (DRRDs). Our knowledge about how particulates mediate respiratory disease is limited, precluding the appropriate recognition or management. Central to this limitation is the lack of understanding of how exposures translate into dysregulated cell identity with dysregulated transcriptional programs. The small airway epithelium is involved in both the pathobiology of DRRD and fine particulate matter deposition. To characterize small airway epithelial cell epigenetic and transcriptional responses to Afghan desert particulate matter (APM) and investigate the functional interactions of transcription factors that mediate these responses, we applied two genomics assays, the assay for transposase accessible chromatin with sequencing (ATAC-seq) and Precision Run-on sequencing (PRO-seq). We identified activity changes in a series of transcriptional pathways as candidate regulators of susceptibility to subsequent insults, including signal-dependent pathways, such as loss of cytochrome P450 or P53/P63, and lineage-determining transcription factors, such as GRHL2 loss or TEAD3 activation. We further demonstrated that TEAD3 activation was unique to APM exposure despite similar inflammatory responses when compared with wood smoke particle exposure and that P53/P63 program loss was uniquely positioned at the intersection of signal-dependent and lineage-determining transcriptional programs. Our results establish the utility of an integrated genomics approach in characterizing responses to exposures and identifying genomic targets for the advanced investigation of the pathogenesis of DRRD.

The Drosophila BEAF insulator protein interacts with the polybromo subunit of the PBAP chromatin remodeling complex.

The Drosophila Boundary Element-Associated Factor of 32 kDa (BEAF) binds in promoter regions of a few thousand mostly housekeeping genes. BEAF is implicated in both chromatin domain boundary activity and promoter function, although molecular mechanisms remain elusive. Here, we show that BEAF physically interacts with the polybromo subunit (Pbro) of PBAP, a SWI/SNF-class chromatin remodeling complex. BEAF also shows genetic interactions with Pbro and other PBAP subunits. We examine the effect of this interaction on gene expression and chromatin structure using precision run-on sequencing and micrococcal nuclease sequencing after RNAi-mediated knockdown in cultured S2 cells. Our results are consistent with the interaction playing a subtle role in gene activation. Fewer than 5% of BEAF-associated genes were significantly affected after BEAF knockdown. Most were downregulated, accompanied by fill-in of the promoter nucleosome-depleted region and a slight upstream shift of the +1 nucleosome. Pbro knockdown caused downregulation of several hundred genes and showed a correlation with BEAF knockdown but a better correlation with promoter-proximal GAGA factor binding. Micrococcal nuclease sequencing supports that BEAF binds near housekeeping gene promoters while Pbro is more important at regulated genes. Yet there is a similar general but slight reduction of promoter-proximal pausing by RNA polymerase II and increase in nucleosome-depleted region nucleosome occupancy after knockdown of either protein. We discuss the possibility of redundant factors keeping BEAF-associated promoters active and masking the role of interactions between BEAF and the Pbro subunit of PBAP in S2 cells. We identify Facilitates Chromatin Transcription (FACT) and Nucleosome Remodeling Factor (NURF) as candidate redundant factors.

Abstract 5375: Precision run-on sequencing (PRO-seq) analysis of a treatment time course in ER+ breast cancer cell lines reveals the transcriptional changes underlying response to complete estrogen receptor antagonist (CERAN) OP-1250

Abstract OP-1250 is an orally-bioavailable complete estrogen receptor antagonist (CERAN) previously shown to shrink wild-type and Y537S mutant estrogen receptor-positive (ER+) tumors in multiple preclinical xenograft models. To better understand the effects of OP-1250 treatment on ER+ breast cancer cells, we used precision run-on sequencing (PRO-seq) to profile the direct transcriptional effects of OP-1250 in two commonly used breast cancer cell lines, MCF7 and CAMA-1. PRO-seq detects nascent RNA, and thus can detect changes at shorter timescales than traditional RNA-seq methods. We conducted PRO-seq 15 minutes, 1 hour, 6 hours and 24 hours after administration of OP-1250, estradiol, fulvestrant and tamoxifen to compare the effects of these compounds across the two cell lines. OP-1250 was administered in the absence of estradiol and after estradiol pretreatment to generate a detailed profile of the time course of its activity. We identified genes that display early transcriptional changes in response to the compounds tested, many of which have been previously identified as estrogen response genes. At later time points we observed transcriptional changes consistent with modulation of pathways involved in cell cycle progression. We show that OP-1250 treatment reverses the estradiol-induced transcriptional changes associated with estrogen receptor activation and chronicle the time course of these changes. Finally, we show greater similarity in the transcriptional changes induced by CERANs OP-1250 and fulvestrant compared to tamoxifen, as well as cell line-specific differences in estrogen response and response to anti-estrogen treatment. These data provide additional insights into the mechanisms of OP-1250 action on ER+ cell lines and patient tumors. OP-1250 is currently in a Phase I/II clinical trial for the treatment of ER+ breast cancer. Citation Format: Alison D. Parisian, Caitlin Miller, Fabian Ortega, Leslie Hodges-Gallagher, Susanna Barratt, Joey Azofeifa, Peter J. Kushner, David Kulp, Cyrus L. Harmon. Precision run-on sequencing (PRO-seq) analysis of a treatment time course in ER+ breast cancer cell lines reveals the transcriptional changes underlying response to complete estrogen receptor antagonist (CERAN) OP-1250 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5375.

Measuring the precise position of transitions in transcription with PRO‐IP‐seq

Transcription is a fundamental process for all known organisms. Transcription by RNA Polymerase II (Pol II), the enzyme responsible for mRNA synthesis, is highly regulated by protein factors at every step. In multicellular eukaryotes, Pol II pauses 20‐60 bases downstream of the transcription start site in nearly all genes. This promoter‐proximal pausing is established as an important step in transcription regulation and is dependent on several pausing and elongation factors. The protein complexes DSIF (DRB Sensitivity Inducing Factor) and NELF (Negative Elongation Factor) bind to Pol II early in transcription to induce pausing. Pause release occurs upon phosphorylation of Pol II, DSIF and NELF by the Cdk9, Cyclin T1 heterodimer P‐TEFb (Positive Transcription Elongation Factor b). Although Pol II pausing is a key step in transcription regulation and has been extensively studied, the molecular mechanisms of pause and pause release are incompletely understood. Furthermore, recent studies have implicated controlled termination early in the gene as a mechanism to control transcription. A challenge to studying these key regulatory steps is that they occur in a narrow window: from initiation to Pol II pausing and release occurs in 20‐60 base range. Current approaches to study transcription factor interactions are unable to provide high enough resolution or sensitivity to address precisely where key transitions in transcription occur.To address this problem, I am developing an assay to measure the exact position of Pol II, when it is bound by a factor of interest. The technique couples our lab’s Precision Run‐on sequencing technique (PRO‐seq), which measures the position of active Pol II with single base‐pair resolution, and an immunoprecipitation enrichment step. Initial PRO‐IP‐seq (Precision Run‐on Immunoprecipitation sequencing) data shows that we can enrich for populations of Pol II bound by specific transcription factors. Utilizing this tool, we will be able to measure genomically where both stable and more transient transcription factors interact with Pol II at near single‐base resolution.

Characterizing RNA stability genome-wide through combined analysis of PRO-seq and RNA-seq data

BackgroundThe concentrations of distinct types of RNA in cells result from a dynamic equilibrium between RNA synthesis and decay. Despite the critical importance of RNA decay rates, current approaches for measuring them are generally labor-intensive, limited in sensitivity, and/or disruptive to normal cellular processes. Here, we introduce a simple method for estimating relative RNA half-lives that is based on two standard and widely available high-throughput assays: Precision Run-On sequencing (PRO-seq) and RNA sequencing (RNA-seq).ResultsOur method treats PRO-seq as a measure of transcription rate and RNA-seq as a measure of RNA concentration, and estimates the rate of RNA decay required for a steady-state equilibrium. We show that this approach can be used to assay relative RNA half-lives genome-wide, with good accuracy and sensitivity for both coding and noncoding transcription units. Using a structural equation model (SEM), we test several features of transcription units, nearby DNA sequences, and nearby epigenomic marks for associations with RNA stability after controlling for their effects on transcription. We find that RNA splicing-related features are positively correlated with RNA stability, whereas features related to miRNA binding and DNA methylation are negatively correlated with RNA stability. Furthermore, we find that a measure based on U1 binding and polyadenylation sites distinguishes between unstable noncoding and stable coding transcripts but is not predictive of relative stability within the mRNA or lincRNA classes. We also identify several histone modifications that are associated with RNA stability.ConclusionWe introduce an approach for estimating the relative half-lives of individual RNAs. Together, our estimation method and systematic analysis shed light on the pervasive impacts of RNA stability on cellular RNA concentrations.

Co-transcriptional splicing regulates 3′ end cleavage during mammalian erythropoiesis

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.

Abstract P5-05-16: Role of estrogen receptor alpha acetylation in estrogen-dependent gene regulation in breast cancers

Abstract Estrogens, such as 17β-estradiol (E2), act through estrogen receptor alpha (ERα), a ligand-regulated transcription factor that binds across the genome to promote enhancer formation and regulate gene expression. ERα is expressed in approximately 70% of breast cancers, where it regulates the transcription of genes involved in mitogenic and inflammatory pathways. We are exploring the the acetylation of ERα on lysines 266 and 268, a modification that enhances the DNA binding and transcriptional activities of ERα. ERα acetylation is catalyzed by the lysine acetyltransferases p300 and CBP in an E2- and steroid receptor coregulator (SRC)-dependent manner. Acetylation-dependent activation of ERα has potential implications in breast cancers associated with enhanced coregulator interactions, such as SRC-3/Amplified in Breast (AIB1) gene amplifications and gain-of-function ERα mutations in endocrine resistant metastatic tumors, such as Y537S and D538G. Increased association of ERα with SRCs and p300/CBP leading to enhanced ERα acetylation may promote gain-of-function ERα activity. Our hypothesis is that acetylation of ERα alters its function by increasing E2-responsive gene transcription and signaling in breast cancers. Our current efforts are focused on investigating the role of ERα acetylation on chromatin binding and accessibility, enhancer activity, and target gene transcription. We are using the ER-positive MCF-7 breast cancer cell line with a knockdown/re-expression strategy with biochemical mimics of acetylated (K266/268Q) or unacetylated (K266/268R) ERα. Genomic ERα binding profiles using chromatin immunoprecipitation-sequencing (ChIP-seq) has defined overlapping, but unique, sets of transcriptional targets and recruitment kinetics for the ERα mutants. Current efforts are focused on defining global transcriptional responses associated with the unique binding activities using precision run-on sequencing (PRO-seq). Ultimately, our goal is to define how acetylation affects the binding of ERα to chromatin and its effect on the transcriptional activity of E2-target genes in breast cancers in the context of gain-of-function ERα mutations and coregulator amplifications. Supported in part by a postdoctoral fellowship from the Lalor Foundation to YMV, and grants from the NIDDK, NICHD, and CPRIT to WLK, as well as support from the Cecil H. and Ida Green Center for Reproductive Biology Sciences Endowment. Citation Format: Vasquez YM, Setlem R, Murakami S, Kraus WL. Role of estrogen receptor alpha acetylation in estrogen-dependent gene regulation in breast cancers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-16.

Nascent RNA sequencing analysis provides insights into enhancer-mediated gene regulation

BackgroundEnhancers are distal cis-regulatory elements that control gene expression. Despite an increasing appreciation of the importance of enhancers in cellular function and disease, our knowledge of enhancer-regulated transcription is very limited. Nascent RNA sequencing technologies, such as global nuclear run-on sequencing (GRO-seq) and precision run-on sequencing (PRO-seq), not only provide a direct and reliable measurement of enhancer activity, but also allow for quantifying transcription of enhancers and target genes simultaneously, making these technologies extremely useful for exploring enhancer-mediated regulation.ResultsNascent RNA sequencing analysis (NRSA) provides a comprehensive view of enhancer-mediated gene regulation. NRSA not only outperforms existing methods for enhancer identification, but also enables annotation and quantification of active enhancers, and prediction of their target genes. Furthermore, NRSA identifies functionally important enhancers by integrating 1) nascent transcriptional changes in enhancers and their target genes and 2) binding profiles from regulator(s) of interest. Applied to wildtype and histone deacetylase 3 (Hdac3) knockout mouse livers, NRSA showed that HDAC3 regulates RNA polymerase recruitment through both proximal (promoter) and distal (enhancer) regulatory elements. Integrating ChIP-seq with PRO-seq data, NRSA prioritized enhancers based on their potential contribution to mediating HDAC3 regulation.ConclusionsNRSA will greatly facilitate the usage of nascent RNA sequencing techniques and accelerate the study of enhancer-mediated regulation.

R1 retrotransposons in the nucleolar organizers of Drosophila melanogaster are transcribed by RNA polymerase I upon heat shock

ABSTRACTThe ribosomal RNA genes (rDNA) of Drosophila melanogaster reside within centromere-proximal nucleolar organizers on both the X and Y chromosomes. Each locus contains between 200–300 tandem repeat rDNA units that encode 18S, 5.8S, 2S, and 28S ribosomal RNAs (rRNAs) necessary for ribosome biogenesis. In arthropods like Drosophila, about 60% of the rDNA genes have R1 and/or R2 retrotransposons inserted at specific sites within their 28S regions; these units likely fail to produce functional 28S rRNA. We showed earlier that R2 expression increases upon nucleolar stress caused by the loss of the ribosome assembly factor, Nucleolar Phosphoprotein of 140 kDa (Nopp140). Here we show that R1 expression is selectively induced by heat shock. Actinomycin D, but not α-amanitin, blocked R1 expression in S2 cells upon heat shock, indicating that R1 elements are transcribed by Pol I. A series of RT-PCRs established read-through transcription by Pol I from the 28S gene region into R1. Sequencing the RT-PCR products confirmed the 28S-R1 RNA junction and the expression of R1 elements within nucleolar rDNA rather than R1 elements known to reside in centromeric heterochromatin. Using a genome-wide precision run-on sequencing (PRO-seq) data set available at NCBI-GEO, we show that Pol I activity on R1 elements is negligible under normal non-heat shock conditions but increases upon heat shock. We propose that prior to heat shock Pol I pauses within the 5ʹ end of R1 where we find a consensus “pause button”, and that heat shock releases Pol I for read-through transcription farther into R1.

Cdk9 regulates a promoter-proximal checkpoint to modulate RNA polymerase II elongation rate in fission yeast

Post-translational modifications of the transcription elongation complex provide mechanisms to fine-tune gene expression, yet their specific impacts on RNA polymerase II regulation remain difficult to ascertain. Here, in Schizosaccharomyces pombe, we examine the role of Cdk9, and related Mcs6/Cdk7 and Lsk1/Cdk12 kinases, on transcription at base-pair resolution with Precision Run-On sequencing (PRO-seq). Within a minute of Cdk9 inhibition, phosphorylation of Pol II-associated factor, Spt5 is undetectable. The effects of Cdk9 inhibition are more severe than inhibition of Cdk7 and Cdk12, resulting in a shift of Pol II toward the transcription start site (TSS). A time course of Cdk9 inhibition reveals that early transcribing Pol II can escape promoter-proximal regions, but with a severely reduced elongation rate of only ~400 bp/min. Our results in fission yeast suggest the existence of a conserved global regulatory checkpoint that requires Cdk9 kinase activity.

Transcription factors GAF and HSF act at distinct regulatory steps to modulate stress-induced gene activation.

The coordinated regulation of gene expression at the transcriptional level is fundamental to development and homeostasis. Inducible systems are invaluable when studying transcription because the regulatory process can be triggered instantaneously, allowing the tracking of ordered mechanistic events. Here, we use precision run-on sequencing (PRO-seq) to examine the genome-wide heat shock (HS) response in Drosophila and the function of two key transcription factors on the immediate transcription activation or repression of all genes regulated by HS. We identify the primary HS response genes and the rate-limiting steps in the transcription cycle that GAGA-associated factor (GAF) and HS factor (HSF) regulate. We demonstrate that GAF acts upstream of promoter-proximally paused RNA polymerase II (Pol II) formation (likely at the step of chromatin opening) and that GAF-facilitated Pol II pausing is critical for HS activation. In contrast, HSF is dispensable for establishing or maintaining Pol II pausing but is critical for the release of paused Pol II into the gene body at a subset of highly activated genes. Additionally, HSF has no detectable role in the rapid HS repression of thousands of genes.

Divergence of a conserved elongation factor and transcription regulation in budding and fission yeast.

Complex regulation of gene expression in mammals has evolved from simpler eukaryotic systems, yet the mechanistic features of this evolution remain elusive. Here, we compared the transcriptional landscapes of the distantly related budding and fission yeast. We adapted the Precision Run-On sequencing (PRO-seq) approach to map the positions of RNA polymerase active sites genome-wide in Schizosaccharomyces pombe and Saccharomyces cerevisiae. Additionally, we mapped preferred sites of transcription initiation in each organism using PRO-cap. Unexpectedly, we identify a pause in early elongation, specific to S. pombe, that requires the conserved elongation factor subunit Spt4 and resembles promoter-proximal pausing in metazoans. PRO-seq profiles in strains lacking Spt4 reveal globally elevated levels of transcribing RNA Polymerase II (Pol II) within genes in both species. Messenger RNA abundance, however, does not reflect the increases in Pol II density, indicating a global reduction in elongation rate. Together, our results provide the first base-pair resolution map of transcription elongation in S. pombe and identify divergent roles for Spt4 in controlling elongation in budding and fission yeast.