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Mounting evidence supports the idea that transcriptional patterns serve as more specific identifiers of active enhancers than histone marks; however, the optimal strategy to identify active enhancers both experimentally and computationally has not been determined. Here, we compared 13 genome-wide RNA sequencing (RNA-seq) assays in K562 cells and show that nuclear run-on followed by cap-selection assay (GRO/PRO-cap) has advantages in enhancer RNA detection and active enhancer identification. We also introduce a tool, peak identifier for nascent transcript starts (PINTS), to identify active promoters and enhancers genome wide and pinpoint the precise location of 5' transcription start sites. Finally, we compiled a comprehensive enhancer candidate compendium based on the detected enhancer RNA (eRNA) transcription start sites (TSSs) available in 120 cell and tissue types, which can be accessed at https://pints.yulab.org . With knowledge of the best available assays and pipelines, this large-scale annotation of candidate enhancers will pave the way for selection and characterization of their functions in a time- and labor-efficient manner.

Promoter-proximal pausing of RNA polymerase II (Pol II) is a key regulatory step during transcription. To understand the evolution and function of pausing, we analyzed transcription in 20 organisms across the tree of life. Unicellular eukaryotes have a slow acceleration of Pol II near transcription start sites that matured into a longer and more focused pause in metazoans. Increased pause residence time coincides with the evolution of new subunits in the NELF and 7SK complexes. In mammals, depletion of NELF reverts a focal pause to a proto-paused-like state driven in part by DNA sequence. Loss of this focal pause compromises transcriptional activation for a set of heat shock genes. Overall, we discovered how pausing evolved and increased regulatory complexity in metazoans.

The first characterised FUSE Binding Protein family member, FUBP1, binds single-stranded DNA to activate MYC transcription. Psi, the sole FUBP protein in Drosophila, binds RNA to regulate P-element and mRNA splicing. Our previous work revealed pro-growth functions for Psi, which depend, in part, on transcriptional activation of Myc. Genome-wide functions for FUBP family proteins in transcriptional control remain obscure. Here, through the first genome-wide binding and expression profiles obtained for a FUBP family protein, we demonstrate that, in addition to being required to activate Myc to promote cell growth, Psi also directly binds and activates stg to couple growth and cell division. Thus, Psi knockdown results in reduced cell division in the wing imaginal disc. In addition to activating these pro-proliferative targets, Psi directly represses transcription of the growth inhibitor tolkin (tok, a metallopeptidase implicated in TGFβ signalling). We further demonstrate tok overexpression inhibits proliferation, while tok loss of function increases mitosis alone and suppresses impaired cell division caused by Psi knockdown. Thus, Psi orchestrates growth through concurrent transcriptional activation of the pro-proliferative genes Myc and stg, in combination with repression of the growth inhibitor tok.

Transcription at most promoters is divergent, initiating at closely spaced oppositely oriented core promoters to produce sense transcripts along with often unstable upstream antisense transcripts (uasTrx). How antisense transcription is regulated and to what extent it is coordinated with sense transcription is not well understood. Here, by combining acute degradation of the multi-functional transcription factor CTCF and nascent transcription measurements, we find that CTCF specifically suppresses antisense but not sense transcription at hundreds of divergent promoters. Primary transcript RNA-FISH shows that CTCF lowers burst fraction but not burst intensity of uasTrx and that co-bursting of sense and antisense transcripts is disfavored. Genome editing, chromatin conformation studies and high-resolution transcript mapping revealed that precisely positioned CTCF directly suppresses the initiation of uasTrx, in a manner independent of its architectural function. In sum, CTCF shapes the transcriptional landscape in part by suppressing upstream antisense transcription.

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.

Physical interactions between viral and host proteins are responsible for almost all aspects of the viral life cycle and the host’s immune response. Studying viral-host protein-protein interactions is thus crucial for identifying strategies for treatment and prevention of viral infection. Here, we use high-throughput yeast two-hybrid and affinity purification followed by mass spectrometry to generate a comprehensive SARS-CoV-2-human protein-protein interactome network consisting of both binary and co-complex interactions. We report a total of 739 high-confidence interactions, showing the highest overlap of interaction partners among published datasets as well as the highest overlap with genes differentially expressed in samples (such as upper airway and bronchial epithelial cells) from patients with SARS-CoV-2 infection. Showcasing the utility of our network, we describe a novel interaction between the viral accessory protein ORF3a and the host zinc finger transcription factor ZNF579 to illustrate a SARS-CoV-2 factor mediating a direct impact on host transcription. Leveraging our interactome, we performed network-based drug screens for over 2,900 FDA-approved/investigational drugs and obtained a curated list of 23 drugs that had significant network proximities to SARS-CoV-2 host factors, one of which, carvedilol, showed promising antiviral properties. We performed electronic health record-based validation using two independent large-scale, longitudinal COVID-19 patient databases and found that carvedilol usage was associated with a significantly lowered probability (17%-20%, P < 0.001) of obtaining a SARS-CoV-2 positive test after adjusting various confounding factors. Carvedilol additionally showed anti-viral activity against SARS-CoV-2 in a human lung epithelial cell line [half maximal effective concentration (EC50) value of 4.1 µM], suggesting a mechanism for its beneficial effect in COVID-19. Our study demonstrates the value of large-scale network systems biology approaches for extracting biological insight from complex biological processes.

Transcriptionally silent genes must be activated throughout development. This requires nucleosomes be removed from promoters and enhancers to allow transcription factor (TF) binding and recruitment of coactivators and RNA polymerase II (Pol II). Specialized pioneer TFs bind nucleosome-wrapped DNA to perform this chromatin opening by mechanisms that remain incompletely understood. Here, we show that GAGA factor (GAF), a Drosophila pioneer-like factor, functions with both SWI/SNF and ISWI family chromatin remodelers to allow recruitment of Pol II and entry to a promoter-proximal paused state, and also to promote Pol II's transition to productive elongation. We found that GAF interacts with PBAP (SWI/SNF) to open chromatin and allow Pol II to be recruited. Importantly, this activity is not dependent on NURF as previously proposed; however, GAF also synergizes with NURF downstream from this process to ensure efficient Pol II pause release and transition to productive elongation, apparently through its role in precisely positioning the +1 nucleosome. These results demonstrate how a single sequence-specific pioneer TF can synergize with remodelers to activate sets of genes. Furthermore, this behavior of remodelers is consistent with findings in yeast and mice, and likely represents general, conserved mechanisms found throughout eukarya.

The role of histone modifications in transcription remains incompletely understood. Here, we examine the relationship between histone modifications and transcription using experimental perturbations combined with sensitive machine-learning tools. Transcription predicted the variation in active histone marks and complex chromatin states, like bivalent promoters, down to single-nucleosome resolution and at an accuracy that rivaled the correspondence between independent ChIP-seq experiments. Blocking transcription rapidly removed two punctate marks, H3K4me3 and H3K27ac, from chromatin indicating that transcription is required for active histone modifications. Transcription was also required for maintenance of H3K27me3, consistent with a role for RNA in recruiting PRC2. A subset of DNase-I-hypersensitive sites were refractory to prediction, precluding models where transcription initiates pervasively at any open chromatin. Our results, in combination with past literature, support a model in which active histone modifications serve a supportive, rather than an essential regulatory, role in transcription.

Distal enhancers play pivotal roles in development and disease yet remain one of the least understood regulatory elements. We used massively parallel reporter assays to perform functional comparisons of two leading enhancer models and find that gene-distal transcription start sites are robust predictors of active enhancers with higher resolution than histone modifications. We show that active enhancer units are precisely delineated by active transcription start sites, validate that these boundaries are sufficient for capturing enhancer function, and confirm that core promoter sequences are necessary for this activity. We assay adjacent enhancers and find that their joint activity is often driven by the stronger unit within the cluster. Finally, we validate these results through functional dissection of a distal enhancer cluster using CRISPR–Cas9 deletions. In summary, definition of high-resolution enhancer boundaries enables deconvolution of complex regulatory loci into modular units. A massively parallel reporter assay (eSTARR-seq) shows that gene-distal transcription start sites can delineate active enhancers with higher resolution than histone modifications.

Nucleic acid crosslinkers that covalently join complementary strands of DNA/RNA have applications in both pharmaceuticals and as biochemical probes. Psoralen is a popular crosslinking moiety that reacts with double stranded DNA and RNA upon exposure to longwave UV light. The commercially available compound EZ-link psoralen-PEG3-biotin has been used in numerous studies to crosslink DNA and double-stranded RNA for genome-wide investigations. Here we present a new probe, AP3B, which uses the psoralen derivative, 4′-aminomethyltrioxsalen, to crosslink and biotinylate nucleic acids. We show that AP3B is 4 to 5 times more effective at labeling DNA in cells and produces a comparable number of crosslinks with over 100 times less compound and less exposure to UV light in vitro than EZ-link psoralen-PEG3-biotin.

Physical interactions between viral and host proteins are responsible for almost all aspects of the viral life cycle and the host’s immune response. Studying viral-host protein-protein interactions is thus crucial for identifying strategies for treatment and prevention of viral infection. Here, we use high-throughput yeast two-hybrid and affinity purification followed by mass spectrometry to generate a comprehensive SARS-CoV-2-human protein-protein interactome network consisting of both binary and co-complex interactions. We report a total of 739 high-confidence interactions, showing the highest overlap of interaction partners among published datasets as well as the highest enrichment for genes differentially expressed in samples (such as upper airway and bronchial epithelial cells) from patients with SARS-CoV-2 infection. Showcasing the utility of our network, we describe a novel interaction between the viral accessory protein ORF3a and the host zinc finger transcription factor ZNF579 to illustrate one of the first examples of a viral factor mediating a direct impact on host transcription. Leveraging our interactome, we performed network-based drug screens for over 2,900 FDA-approved/investigational drugs and obtained a curated list of 21 drugs that had significant network proximities to SARS-CoV-2 host factors, one of which, carvedilol, showed promising antiviral properties. We performed electronic health record-based validation using two independent large-scale, longitudinal COVID-19 patient databases and found that carvedilol usage was associated with a significantly lowered probability (17%-20%, P < 0.001) of obtaining a SARS-CoV-2 positive test after adjusting various confounding factors. Carvedilol additionally showed anti-viral activity against SARS-CoV-2 in a human lung epithelial cell line (EC50 value of 4.1 µM), suggesting a mechanism for its beneficial effect in COVID-19. Our study demonstrates the value of large-scale network systems biology approaches for extracting biological insight from complex biological processes.

Physical interactions between viral and host proteins are responsible for almost all aspects of the viral life cycle and the host's immune response. Studying viral-host protein-protein interactions is thus crucial for identifying strategies for treatment and prevention of viral infection. Here, we use high-throughput yeast two-hybrid and affinity purification followed by mass spectrometry to generate a comprehensive SARS-CoV-2-human protein-protein interactome network consisting of both binary and co-complex interactions. We report a total of 739 high-confidence interactions, showing the highest overlap of interaction partners among published datasets as well as the highest overlap with genes differentially expressed in samples (such as upper airway and bronchial epithelial cells) from patients with SARS-CoV-2 infection. Showcasing the utility of our network, we describe a novel interaction between the viral accessory protein ORF3a and the host zinc finger transcription factor ZNF579 to illustrate a SARS-CoV-2 factor mediating a direct impact on host transcription. Leveraging our interactome, we performed network-based drug screens for over 2,900 FDA-approved/investigational drugs and obtained a curated list of 23 drugs that had significant network proximities to SARS-CoV-2 host factors, one of which, carvedilol, showed promising antiviral properties. We performed electronic health record-based validation using two independent large-scale, longitudinal COVID-19 patient databases and found that carvedilol usage was associated with a significantly lowered probability (17%-20%

Transcription at most promoters is divergent, initiating at closely spaced oppositely oriented core promoters to produce sense transcripts along with often unstable upstream antisense (uasTrx). How antisense transcription is regulated and to what extent it is coordinated with sense transcription is largely unknown. Here by combining acute degradation of the multi-functional transcription factor CTCF and nascent transcription measurements, we find that CTCF specifically suppresses antisense but not sense transcription at hundreds of divergent promoters, the great majority of which bear proximal CTCF binding sites. Genome editing, chromatin conformation studies, and high-resolution transcript mapping revealed that precisely positioned CTCF directly suppresses the initiation of uasTrx, in a manner independent of its chromatin architectural function. Primary transcript RNA FISH revealed co-bursting of sense and anti-sense transcripts is disfavored, suggesting CTCF-regulated competition for transcription initiation. In sum, CTCF shapes the transcriptional landscape in part by suppressing upstream antisense transcription.

Control of gene expression is fundamental at every level of cell function. Promoter-proximal pausing and divergent transcription at promoters and enhancers, which are prominent features in animals, have only been studied in a handful of research experiments in plants. PRO-Seq analysis in cassava (Manihot esculenta) identified peaks of transcriptionally engaged RNA polymerase at both the 5′ and 3′ end of genes, consistent with paused or slowly moving Polymerase. In addition, we identified divergent transcription at intergenic sites. A full genome search for bi-directional transcription using an algorithm for enhancer detection developed in mammals (dREG) identified many intergenic regulatory element (IRE) candidates. These sites showed distinct patterns of methylation and nucleotide conservation based on genomic evolutionary rate profiling (GERP). SNPs within these IRE candidates explained significantly more variation in fitness and root composition than SNPs in chromosomal segments randomly ascertained from the same intergenic distribution, strongly suggesting a functional importance of these sites. Maize GRO-Seq data showed RNA polymerase occupancy at IREs consistent with patterns in cassava. Furthermore, these IREs in maize significantly overlapped with sites previously identified on the basis of open chromatin, histone marks, and methylation, and were enriched for reported eQTL. Our results suggest that bidirectional transcription can identify intergenic genomic regions in plants that play an important role in transcription regulation and whose identification has the potential to aid crop improvement.

Mounting evidence supports the idea that transcriptional patterns serve as more specific identifiers of active enhancers than histone marks1,2; however, the optimal strategy to identify active enhancers both experimentally and computationally has not been determined. In this study, we compared 13 genome-wide RNA sequencing assays in K562 cells and showed that the nuclear run-on followed by cap-selection assay (namely, GRO/PRO-cap) has significant advantages in eRNA detection and active enhancer identification. We also introduced a new analytical tool, Peak Identifier for Nascent-Transcript Sequencing (PINTS), to identify active promoters and enhancers genome-wide and pinpoint the precise location of the 5’ transcription start sites (TSSs) within these regulatory elements. Finally, we compiled a comprehensive enhancer candidate compendium based on the detected eRNA TSSs available in 120 cell and tissue types. To facilitate the exploration and prioritization of these enhancer candidates, we also built a user-friendly web server (https://pints.yulab.org) for the compendium with various additional genomic and epigenomic annotations. With the knowledge of the best available assays and pipelines, this large-scale annotation of candidate enhancers will pave the road for selection and characterization of their functions in a time-, labor-, and cost-effective manner in the future.

The glucocorticoid receptor (GR) regulates transcription through binding to specific DNA motifs, particularly at enhancers. While the motif to which it binds is constant across cell types, GR has cell type-specific binding at genomic loci, resulting in regulation of different genes. The presence of other bound transcription factors (TFs) is hypothesized to strongly influence where GR binds. Here, we addressed the roles of other TFs in the glucocorticoid response by comparing changes in GR binding and nascent transcription at promoters and distal candidate cis-regulatory elements (CCREs) in two distinct human cancer cell types. We found that after glucocorticoid treatment, GR binds to thousands of genomic loci that are primarily outside of promoter regions and are potentially enhancers. The majority of these GR binding sites are cell-type specific, and they are associated with pioneer factor binding. A small fraction of GR occupied regions (GORs) displayed increased bidirectional nascent transcription, which is a characteristic of many active enhancers, after glucocorticoid treatment. Non-promoter GORs with increased transcription were specifically enriched for AP-1 binding prior to glucocorticoid treatment. These results support a model of transcriptional regulation in which multiple classes of TFs are required. The pioneer factors increase chromatin accessibility, facilitating the binding of GR and additional factors. AP-1 binding poises a fraction of accessible sites to be rapidly transcribed upon glucocorticoid-induced GR binding. The coordinated activity of multiple TFs then results in cell type-specific changes in gene expression. We anticipate that many models of inducible gene expression also require multiple distinct TFs that act at multiple steps of transcriptional regulation.

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. This historical Perspective by John Lis summarizes the array of complementary biochemical, genetic, optical and genome-wide approaches that have enabled dissection of eukaryotic transcriptional mechanisms in their native, cellular environment, and considers the future insights offered by emerging technologies of ever-increasing sensitivity and resolution.

The programmes that direct an organism's development and maintenance are encoded in its genome. Decoding of this information begins with regulated transcription of genomic DNA into RNA. Although transcription and its control can be tracked indirectly by measuring stable RNAs, it is only by directly measuring nascent RNAs that the immediate regulatory changes in response to developmental, environmental, disease and metabolic signals are revealed. Multiple complementary methods have been developed to quantitatively track nascent transcription genome-wide at nucleotide resolution, all of which have contributed novel insights into the mechanisms of gene regulation and transcription-coupled RNA processing. Here we critically evaluate the array of strategies used for investigating nascent transcription and discuss the recent conceptual advances they have provided.

Transcription is correlated with a variety of post-translational modifications to core histones, yet we still know relatively little about the precise and causal nature of their relationship. In particular, whether histone modifications serve a regulatory role, for example by marking functional elements for later activation, remains debated. Here we trained a support vector regression to infer the genomic distribution of 11 widely studied histone modifications using nascent transcription as input. Transcription captured the variation in active histone modifications, including punctate promoter and enhancer marks (e.g., H3K4me3, H3K4me1, and H3K27ac) and elongation marks (e.g., H3K36me3), down to single-nucleosome resolution at an accuracy that rivaled the correspondence between independent ChIP-seq experiments. The relationship between active histone modifications and transcription was conserved in all cell types examined. Surprisingly, transcription also accurately predicted the repressive chromatin mark H3K27me3. However, unlike active marks, H3K27me3 was found in two distinct patterns in different cell types: one pattern in fully differentiated cells covered broad regions with low levels of transcription, and a second pattern in stem cells was associated with transcription start sites of weakly transcribed genes. Transcription accurately identified bivalent promoters in embryonic stem cells, as well as other complex chromatin states identified by chromHMM. Although transcription accurately predicted nearly all histone modifications, we found a subset of DNase-I hypersensitive sites that were refractory to prediction, precluding models in which transcription initiates pervasively as a consequence of open chromatin. Our results demonstrate that histone modifications are tightly correlated with transcription, and provides a new tool for genome annotation using a single experiment.

The role of histone modifications in transcription remains incompletely understood. Here, we examine the relationship between histone modifications and transcription using experimental perturbations combined with sensitive machine-learning tools. Transcription predicted the variation in active histone marks and complex chromatin states, like bivalent promoters, down to single-nucleosome resolution and at an accuracy that rivaled the correspondence between independent ChIP-seq experiments. Blocking transcription rapidly removed two punctate marks, H3K4me3 and H3K27ac, from chromatin indicating that transcription is required for active histone modifications. Transcription was also required for maintenance of H3K27me3, consistent with a role for RNA in recruiting PRC2. A subset of DNase-I-hypersensitive sites were refractory to prediction, precluding models where transcription initiates pervasively at any open chromatin. Our results, in combination with past literature, support a model in which active histone modifications serve a supportive, rather than an essential regulatory, role in transcription.

SUMMARY CCCTC-binding factor (CTCF) is a conserved zinc finger transcription factor implicated in a wide range of functions, including genome organization, transcription activation, and elongation. To explore the basis for CTCF functional diversity, we coupled an auxin-induced degron system with precision nuclear run-on. Unexpectedly, oriented CTCF motifs in gene bodies are associated with transcriptional stalling in a manner independent of bound CTCF. Moreover, CTCF at different binding sites (CBSs) displays highly variable resistance to degradation. Motif sequence does not significantly predict degradation behavior, but location at chromatin boundaries and chromatin loop anchors, as well as co-occupancy with cohesin, are associated with delayed degradation. Single-molecule tracking experiments link chromatin residence time to CTCF degradation kinetics, which has ramifications regarding architectural CTCF functions. Our study highlights the heterogeneity of CBSs, uncovers properties specific to architecturally important CBSs, and provides insights into the basic processes of genome organization and transcription regulation., Graphical Abstract, In brief Using an inducible CTCF degradation approach, Luan et al. show that sensitivity to degradation of CTCF on chromatin is highly variable, reflecting its diverse roles in chromatin architecture and transcription.

Transcription errors can substantially affect metabolic processes in organisms by altering the epigenome and causing misincorporations in mRNA, which is translated into aberrant mutant proteins. Moreover, within eukaryotic genomes there are specific Transcription Error-Enriched genomic Loci (TEELs) which are transcribed by RNA polymerases with significantly higher error rates and hypothesized to have implications in cancer, aging, and diseases such as Down syndrome and Alzheimer’s. Therefore, research into transcription errors is of growing importance within the field of genetics. Nevertheless, methodological barriers limit the progress in accurately identifying transcription errors. Pro-Seq and NET-Seq can purify nascent RNA and map RNA polymerases along the genome but cannot be used to identify transcriptional mutations. Here we present background Error Model-coupled Precision nuclear run-on Circular-sequencing (EmPC-seq), a method combining a nuclear run-on assay and circular sequencing with a background error model to precisely detect nascent transcription errors and effectively discern TEELs within the genome.

Psi, the sole FUSE Binding Protein (FUBP) family single stranded DNA/RNA binding protein inDrosophila, is essential for proper cell and tissue growth, however its mechanism of function remains unclear. Here we use Targeted DamID combined with RNA-sequencing to generate the first genome-wide binding and expression profiles for Psi. Surprisingly, we demonstrate Psi drives growth in theDrosophilawing through transcriptional repression of key developmental pathways (e.g. Wnt, Notch and TGFβ). Thus, Psi patterns tissue growth by directly repressing transcription of developmental growth suppressors. Analysis of direct Psi targets identified novel growth inhibitors, includingTolkin(Zinc metallopeptidase implicated in TGFβ signalling),Ephexin(Rho-GEF) andemp(CD36 scavenger receptor-related protein). Their depletion not only suppressed impaired growth associated with Psi knockdown, but alone was sufficient to drive wing overgrowth. Thus, Psi drives wing growth twofold, through direct activation ofMycand through transcriptional repression of growth inhibitors comprising core developmental pathways.

SummaryTranscriptionally silent genes must be activated throughout development. This requires nucleosomes be removed from promoters and enhancers to allow transcription factor binding (TFs) and recruitment of coactivators and RNA Polymerase II (Pol II). Specialized pioneer TFs bind nucleosome-wrapped DNA to perform this chromatin opening by mechanisms that remain incompletely understood1–3. Here, we show that GAGA-factor (GAF), a Drosophila pioneer factor4, interacts with both SWI/SNF and ISWI family chromatin remodelers to allow recruitment of Pol II and entry to a promoter-proximal paused state, and also to promote Pol II’s transition to productive elongation. We found that GAF functions with PBAP (SWI/SNF) to open chromatin and allow Pol II to be recruited. Importantly this activity is not dependent on NURF as previously proposed5–7; however, GAF also functions with NURF downstream of this process to ensure efficient Pol II pause release and transition to productive elongation apparently through its role in precisely positioning the +1 nucleosome. These results demonstrate how a single sequence-specific pioneer TF can synergize with remodelers to activate sets of genes. Furthermore, this behavior of remodelers is consistent with findings in yeast8–10 and mice11–13, and likely represents general, conserved mechanisms found throughout Eukarya.

Site-specific arrest of RNA polymerases (RNAPs) is fundamental to several technologies that assess RNA structure and function. Current in vitro transcription “roadblocking” approaches inhibit transcription elongation by blocking RNAP with a protein bound to the DNA template. One limitation of protein-mediated transcription roadblocking is that it requires inclusion of a protein factor extrinsic to the minimal in vitro transcription reaction. In this work, we developed a chemical approach for halting transcription by Escherichia coli RNAP. We first established a sequence-independent method for site-specific incorporation of chemical lesions into dsDNA templates by sequential PCR and translesion synthesis. We then show that interrupting the transcribed DNA strand with an internal desthiobiotin-triethylene glycol modification or 1,N(6)-etheno-2′-deoxyadenosine base efficiently and stably halts Escherichia coli RNAP transcription. By encoding an intrinsic stall site within the template DNA, our chemical transcription roadblocking approach enables display of nascent RNA molecules from RNAP in a minimal in vitro transcription reaction.

Nucleic acid crosslinkers that covalently join commentary strands have applications as both pharmaceuticals and biochemical probes. Psoralen is a popular crosslinker moiety that reacts with double stranded DNA and RNA upon irradiation with long wave UV light. A commercially available compound EZ-Link Psoralen-PEG3-Biotin has been used in many studies to crosslink DNA and double strand RNA for genome-wide investigations. Here we present a novel probe, AP3B, which uses a psoralen derivative, 4’-aminomethyltrioxsalen, to biotinylate nucleic acids. We show that this compound is 8-fold more effective at labeling DNA in cells and several hundred-fold more effective at crosslinking two strands of DNA in vitro than the commercially available compound EZ-Link Psoralen-PEG3-Biotin.

The human genome encodes a variety of poorly understood RNA species that remain challenging to identify using existing genomic tools. We developed chromatin run-on and sequencing (ChRO-seq) to map the location of RNA polymerase for almost any input sample, including samples with degraded RNA that are intractable to RNA sequencing. We used ChRO-seq to map nascent transcription in primary human glioblastoma (GBM) brain tumors. Enhancers identified in primary GBMs resemble open chromatin in the normal human brain. Rare enhancers that are activated in malignant tissue drive regulatory programs similar to the developing nervous system. We identified enhancers that regulate groups of genes that are characteristic of each known GBM subtype and transcription factors that drive them. Finally we discovered a core group of transcription factors that control the expression of genes associated with clinical outcomes. This study characterizes the transcriptional landscape of GBM and introduces ChRO-seq as a method to map regulatory programs that contribute to complex diseases.

Site-specific arrest of RNA polymerase is fundamental to several technologies that measure RNA structure and function. Currentin vitrotranscription ‘roadblocking’ approaches inhibit transcription elongation using a protein blockade bound to the DNA template. One limitation of protein-mediated transcription roadblocking is that it requires the inclusion of a protein factor that is extrinsic to the minimalin vitrotranscription reaction. In this work, we show that interrupting the transcribed DNA strand with an internal desthiobiotin-triethylene glycol modification efficiently and stably haltsEscherichia coliRNA polymerase transcription. To facilitate diverse applications of chemical transcription roadblocking, we establish a simple and sequence-independent method for the preparation of internally modified double-stranded DNA templates by sequential PCR and translesion synthesis. By encoding an intrinsic stall site within the template DNA, our chemical transcription roadblocking approach enables nascent RNA molecules to be displayed from RNA polymerase in a minimalin vitrotranscription reaction.

RNA polymerase transcribes certain genomic loci with higher errors rates. These transcription error-enriched genomic loci (TEELs) have implications in disease. Current deep-sequencing methods cannot distinguish TEELs from post-transcriptional modifications, stochastic transcription errors, and technical noise, impeding efforts to elucidate the mechanisms linking TEELs to disease. Here, we describe background error model-coupled precision nuclear run-on circular-sequencing (EmPC-seq) to discern genomic regions enriched for transcription misincorporations. EmPC-seq innovatively combines a nuclear run-on assay for capturing nascent RNA before post-transcriptional modifications, a circular-sequencing step that sequences the same nascent RNA molecules multiple times to improve accuracy, and a statistical model for distinguishing error-enriched regions among stochastic polymerase errors. Applying EmPC-seq to the ribosomal RNA transcriptome, we show that TEELs of RNA polymerase I are not randomly distributed but clustered together, with higher error frequencies at nascent transcript 3' ends. Our study establishes a reliable method of identifying TEELs with nucleotide precision, which can help elucidate their molecular origins.

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., While post-translational modifications of the RNA polymerase II complex regulate gene expression, the exact mechanism remains unknown. Here, the authors use altered-specificity kinase mutations to examine the role of Cdk9, Mcs6/Cdk7, and Lsk1/Cdk12 on transcription at base-pair resolution in yeast.

How evolutionary changes at enhancers affect the transcription of target genes remains an important open question. Previous comparative studies of gene expression have largely measured the abundance of messenger RNA, which is affected by post-transcriptional regulatory processes, hence limiting inferences about the mechanisms underlying expression differences. Here, we directly measured nascent transcription in primate species, allowing us to separate transcription from post-transcriptional regulation. We used precision run-on and sequencing to map RNA polymerases in resting and activated CD4+ T cells in multiple human, chimpanzee and rhesus macaque individuals, with rodents as outgroups. We observed general conservation in coding and non-coding transcription, punctuated by numerous differences between species, particularly at distal enhancers and non-coding RNAs. Genes regulated by larger numbers of enhancers are more frequently transcribed at evolutionarily stable levels, despite reduced conservation at individual enhancers. Adaptive nucleotide substitutions are associated with lineage-specific transcription and at one locus, SGPP2, we predict and experimentally validate that multiple substitutions contribute to human-specific transcription. Collectively, our findings suggest a pervasive role for evolutionary compensation across ensembles of enhancers that jointly regulate target genes.

Most studies of responses to transcriptional stimuli measure changes in cellular mRNA concentrations. By sequencing nascent RNA instead, it is possible to detect changes in transcription in minutes rather than hours and thereby distinguish primary from secondary responses to regulatory signals. Here, we describe the use of PRO-seq to characterize the immediate transcriptional response in human cells to celastrol, a compound derived from traditional Chinese medicine that has potent anti-inflammatory, tumor-inhibitory, and obesity-controlling effects. Celastrol is known to elicit a cellular stress response resembling the response to heat shock, but the transcriptional basis of this response remains unclear. Our analysis of PRO-seq data for K562 cells reveals dramatic transcriptional effects soon after celastrol treatment at a broad collection of both coding and noncoding transcription units. This transcriptional response occurred in two major waves, one within 10 min, and a second 40–60 min after treatment. Transcriptional activity was generally repressed by celastrol, but one distinct group of genes, enriched for roles in the heat shock response, displayed strong activation. Using a regression approach, we identified key transcription factors that appear to drive these transcriptional responses, including members of the E2F and RFX families. We also found sequence-based evidence that particular transcription factors drive the activation of enhancers. We observed increased polymerase pausing at both genes and enhancers, suggesting that pause release may be widely inhibited during the celastrol response. Our study demonstrates that a careful analysis of PRO-seq time-course data can disentangle key aspects of a complex transcriptional response, and it provides new insights into the activity of a powerful pharmacological agent.

Distal enhancers remain one of the least understood regulatory elements with pivotal roles in development and disease. We used massively parallel reporter assays to perform functional comparisons of two leading enhancer models and find that gene-distal transcription start sites (TSSs) are robust predictors of enhancer activity with higher resolution and specificity than histone modifications. We show that active enhancer units are precisely delineated by active TSSs, validate that these boundaries are sufficient to capture enhancer function, and confirm that core promoter sequences are required for this activity. Finally, we assay pairs of adjacent units and find that their cumulative activity is best predicted by the strongest unit within the pair. Synthetic fusions of enhancer units demonstrate that adjacency imposes winner-takes-all logic, revealing a simple design for a maximum-activity filter of enhancer unit outputs. Together, our results define fundamental enhancer units and a principle of non-cooperativity between adjacent units.

Heat shock (HS) initiates rapid, extensive, and evolutionarily conserved changes in transcription that are accompanied by chromatin decondensation and nucleosome loss at HS loci. Here we have employed in situ Hi-C to determine how heat stress affects long-range chromatin conformation in human and Drosophila cells. We found that compartments and topologically associating domains (TADs) remain unchanged by an acute HS. Knockdown of Heat Shock Factor 1 (HSF1), the master transcriptional regulator of the HS response, identified HSF1-dependent genes and revealed that up-regulation is often mediated by distal HSF1 bound enhancers. HSF1-dependent genes were usually found in the same TAD as the nearest HSF1 binding site. Although most interactions between HSF1 binding sites and target promoters were established in the nonheat shock (NHS) condition, a subset increased contact frequency following HS. Integrating information about HSF1 binding strength, RNA polymerase abundance at the HSF1 bound sites (putative enhancers), and contact frequency with a target promoter accurately predicted which up-regulated genes were direct targets of HSF1 during HS. Our results suggest that the chromatin conformation necessary for a robust HS response is preestablished in NHS cells of diverse metazoan species.