Publications by authors named "John T Lis"

139 Publications

RNA polymerase mapping in plants identifies intergenic regulatory elements enriched in causal variants.

G3 (Bethesda) 2021 Sep 6. Epub 2021 Sep 6.

Plant Breeding and Genetics, School of Integrative Plant Science, Cornell University, Ithaca, NY 14853, USA.

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.
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http://dx.doi.org/10.1093/g3journal/jkab273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8527479PMC
September 2021

Stress-induced transcriptional memory accelerates promoter-proximal pause release and decelerates termination over mitotic divisions.

Mol Cell 2021 04 29;81(8):1715-1731.e6. Epub 2021 Mar 29.

Faculty of Science and Engineering, Cell Biology, Åbo Akademi University, 20520 Turku, Finland; Turku Bioscience Centre, University of Turku and Åbo Akademi University, 20520 Turku, Finland. Electronic address:

Heat shock instantly reprograms transcription. Whether gene and enhancer transcription fully recover from stress and whether stress establishes a memory by provoking transcription regulation that persists through mitosis remained unknown. Here, we measured nascent transcription and chromatin accessibility in unconditioned cells and in the daughters of stress-exposed cells. Tracking transcription genome-wide at nucleotide-resolution revealed that cells precisely restored RNA polymerase II (Pol II) distribution at gene bodies and enhancers upon recovery from stress. However, a single heat exposure in embryonic fibroblasts primed a faster gene induction in their daughter cells by increasing promoter-proximal Pol II pausing and by accelerating the pause release. In K562 erythroleukemia cells, repeated stress refined basal and heat-induced transcription over mitotic division and decelerated termination-coupled pre-mRNA processing. The slower termination retained transcripts on the chromatin and reduced recycling of Pol II. These results demonstrate that heat-induced transcriptional memory acts through promoter-proximal pause release and pre-mRNA processing at transcription termination.
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http://dx.doi.org/10.1016/j.molcel.2021.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8054823PMC
April 2021

EmPC-seq: Accurate RNA-sequencing and Bioinformatics Platform to Map RNA Polymerases and Remove Background Error.

Bio Protoc 2021 Feb 20;11(4):e3921. Epub 2021 Feb 20.

The Hong Kong University of Science and Technology -Shenzhen Research Institute, Shenzhen, China.

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.
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http://dx.doi.org/10.21769/BioProtoc.3921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7952946PMC
February 2021

Distinct properties and functions of CTCF revealed by a rapidly inducible degron system.

Cell Rep 2021 02;34(8):108783

Division of Hematology, The Children's Hospital of Pennsylvania, Philadelphia, PA, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2021.108783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7999233PMC
February 2021

Pioneer-like factor GAF cooperates with PBAP (SWI/SNF) and NURF (ISWI) to regulate transcription.

Genes Dev 2021 01 10;35(1-2):147-156. Epub 2020 Dec 10.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.

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 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.
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http://dx.doi.org/10.1101/gad.341768.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7778264PMC
January 2021

Transcription imparts architecture, function and logic to enhancer units.

Nat Genet 2020 10 21;52(10):1067-1075. Epub 2020 Sep 21.

Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA.

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.
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http://dx.doi.org/10.1038/s41588-020-0686-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541647PMC
October 2020

RNA aptamer capture of macromolecular complexes for mass spectrometry analysis.

Nucleic Acids Res 2020 09;48(15):e90

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA.

Specific genomic functions are dictated by macromolecular complexes (MCs) containing multiple proteins. Affinity purification of these complexes, often using antibodies, followed by mass spectrometry (MS) has revolutionized our ability to identify the composition of MCs. However, conventional immunoprecipitations suffer from contaminating antibody/serum-derived peptides that limit the sensitivity of detection for low-abundant interacting partners using MS. Here, we present AptA-MS (aptamer affinity-mass spectrometry), a robust strategy primarily using a specific, high-affinity RNA aptamer against Green Fluorescent Protein (GFP) to identify interactors of a GFP-tagged protein of interest by high-resolution MS. Utilizing this approach, we have identified the known molecular chaperones that interact with human Heat Shock Factor 1 (HSF1), and observed an increased association with several proteins upon heat shock, including translation elongation factors and histones. HSF1 is known to be regulated by multiple post-translational modifications (PTMs), and we observe both known and new sites of modifications on HSF1. We show that AptA-MS provides a dramatic target enrichment and detection sensitivity in evolutionarily diverse organisms and allows identification of PTMs without the need for modification-specific enrichments. In combination with the expanding libraries of GFP-tagged cell lines, this strategy offers a general, inexpensive, and high-resolution alternative to conventional approaches for studying MCs.
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http://dx.doi.org/10.1093/nar/gkaa542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7470977PMC
September 2020

Identifying Transcription Error-Enriched Genomic Loci Using Nuclear Run-on Circular-Sequencing Coupled with Background Error Modeling.

J Mol Biol 2020 06 20;432(13):3933-3949. Epub 2020 Apr 20.

The Hong Kong University of Science and Technology-Shenzhen Research Institute, Hi-Tech Park, Nanshan, Shenzhen 518057, China; Department of Chemistry, Centre of Systems Biology and Human Health, State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong; Bioengineering Graduate Program, Department of Biological and Chemical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jmb.2020.04.011DOI Listing
June 2020

Chemical roadblocking of DNA transcription for nascent RNA display.

J Biol Chem 2020 05 24;295(19):6401-6412. Epub 2020 Mar 24.

Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208.

Site-specific arrest of RNA polymerases (RNAPs) is fundamental to several technologies that assess RNA structure and function. Current 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 transcription reaction. In this work, we developed a chemical approach for halting transcription by 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-etheno-2'-deoxyadenosine base efficiently and stably halts 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 transcription reaction.
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http://dx.doi.org/10.1074/jbc.RA120.012641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212663PMC
May 2020

Chromatin conformation remains stable upon extensive transcriptional changes driven by heat shock.

Proc Natl Acad Sci U S A 2019 09 10;116(39):19431-19439. Epub 2019 Sep 10.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853;

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 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.
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http://dx.doi.org/10.1073/pnas.1901244116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6765289PMC
September 2019

A 50 year history of technologies that drove discovery in eukaryotic transcription regulation.

Authors:
John T Lis

Nat Struct Mol Biol 2019 09 22;26(9):777-782. Epub 2019 Aug 22.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA.

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.
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http://dx.doi.org/10.1038/s41594-019-0288-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7106917PMC
September 2019

Nascent RNA analyses: tracking transcription and its regulation.

Nat Rev Genet 2019 12 9;20(12):705-723. Epub 2019 Aug 9.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA.

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.
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http://dx.doi.org/10.1038/s41576-019-0159-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858503PMC
December 2019

Kinetics of -induced gene silencing can be predicted from combinations of epigenetic and genomic features.

Genome Res 2019 07 7;29(7):1087-1099. Epub 2019 Jun 7.

Otto Warburg Laboratories, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany.

To initiate X-Chromosome inactivation (XCI), the long noncoding RNA mediates chromosome-wide gene silencing of one X Chromosome in female mammals to equalize gene dosage between the sexes. The efficiency of gene silencing is highly variable across genes, with some genes even escaping XCI in somatic cells. A gene's susceptibility to -mediated silencing appears to be determined by a complex interplay of epigenetic and genomic features; however, the underlying rules remain poorly understood. We have quantified chromosome-wide gene silencing kinetics at the level of the nascent transcriptome using allele-specific Precision nuclear Run-On sequencing (PRO-seq). We have developed a Random Forest machine-learning model that can predict the measured silencing dynamics based on a large set of epigenetic and genomic features and tested its predictive power experimentally. The genomic distance to the locus, followed by gene density and distance to LINE elements, are the prime determinants of the speed of gene silencing. Moreover, we find two distinct gene classes associated with different silencing pathways: a class that requires -repeat A for silencing, which is known to activate the SPEN pathway, and a second class in which genes are premarked by Polycomb complexes and tend to rely on the B repeat in for silencing, known to recruit Polycomb complexes during XCI. Moreover, a series of features associated with active transcriptional elongation and chromatin 3D structure are enriched at rapidly silenced genes. Our machine-learning approach can thus uncover the complex combinatorial rules underlying gene silencing during X inactivation.
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http://dx.doi.org/10.1101/gr.245027.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6633258PMC
July 2019

An AR-ERG transcriptional signature defined by long-range chromatin interactomes in prostate cancer cells.

Genome Res 2019 02 3;29(2):223-235. Epub 2019 Jan 3.

Genome Institute of Singapore, Singapore 138672.

The aberrant activities of transcription factors such as the androgen receptor (AR) underpin prostate cancer development. While the AR -regulation has been extensively studied in prostate cancer, information pertaining to the spatial architecture of the AR transcriptional circuitry remains limited. In this paper, we propose a novel framework to profile long-range chromatin interactions associated with AR and its collaborative transcription factor, erythroblast transformation-specific related gene (ERG), using chromatin interaction analysis by paired-end tag (ChIA-PET). We identified ERG-associated long-range chromatin interactions as a cooperative component in the AR-associated chromatin interactome, acting in concert to achieve coordinated regulation of a subset of AR target genes. Through multifaceted functional data analysis, we found that AR-ERG interaction hub regions are characterized by distinct functional signatures, including bidirectional transcription and cotranscription factor binding. In addition, cancer-associated long noncoding RNAs were found to be connected near protein-coding genes through AR-ERG looping. Finally, we found strong enrichment of prostate cancer genome-wide association study (GWAS) single nucleotide polymorphisms (SNPs) at AR-ERG co-binding sites participating in chromatin interactions and gene regulation, suggesting GWAS target genes identified from chromatin looping data provide more biologically relevant findings than using the nearest gene approach. Taken together, our results revealed an AR-ERG-centric higher-order chromatin structure that drives coordinated gene expression in prostate cancer progression and the identification of potential target genes for therapeutic intervention.
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http://dx.doi.org/10.1101/gr.230243.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6360806PMC
February 2019

Single-molecule nascent RNA sequencing identifies regulatory domain architecture at promoters and enhancers.

Nat Genet 2018 11 22;50(11):1533-1541. Epub 2018 Oct 22.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA.

Eukaryotic RNA polymerase II (Pol II) has been found at both promoters and distal enhancers, suggesting additional functions beyond mRNA production. To understand this role, we sequenced nascent RNAs at single-molecule resolution to unravel the interplay between Pol II initiation, capping and pausing genome-wide. Our analyses identify two pause classes that are associated with different RNA capping profiles. More proximal pausing is associated with less complete capping, less elongation and a more enhancer-like complement of transcription factors than later pausing. Unexpectedly, transcription start sites (TSSs) are predominantly found in constellations composed of multiple divergent pairs. TSS clusters are intimately associated with precise arrays of nucleosomes and correspond with boundaries of transcription factor binding and chromatin modification at promoters and enhancers. TSS architecture is largely unchanged during the dramatic transcriptional changes induced by heat shock. Together, our results suggest that promoter- and enhancer-associated Pol II is a regulatory nexus for integrating information across TSS ensembles.
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http://dx.doi.org/10.1038/s41588-018-0234-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6422046PMC
November 2018

Chromatin run-on and sequencing maps the transcriptional regulatory landscape of glioblastoma multiforme.

Nat Genet 2018 11 22;50(11):1553-1564. Epub 2018 Oct 22.

Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.

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.
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http://dx.doi.org/10.1038/s41588-018-0244-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204104PMC
November 2018

A Cdk9-PP1 switch regulates the elongation-termination transition of RNA polymerase II.

Nature 2018 06 13;558(7710):460-464. Epub 2018 Jun 13.

Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

The end of the RNA polymerase II (Pol II) transcription cycle is strictly regulated to prevent interference between neighbouring genes and to safeguard transcriptome integrity . The accumulation of Pol II downstream of the cleavage and polyadenylation signal can facilitate the recruitment of factors involved in mRNA 3'-end formation and termination , but how this sequence is initiated remains unclear. In a chemical-genetic screen, human protein phosphatase 1 (PP1) isoforms were identified as substrates of positive transcription elongation factor b (P-TEFb), also known as the cyclin-dependent kinase 9 (Cdk9)-cyclin T1 (CycT1) complex . Here we show that Cdk9 and PP1 govern phosphorylation of the conserved elongation factor Spt5 in the fission yeast Schizosaccharomyces pombe. Cdk9 phosphorylates both Spt5 and a negative regulatory site on the PP1 isoform Dis2 . Sites targeted by Cdk9 in the Spt5 carboxy-terminal domain can be dephosphorylated by Dis2 in vitro, and dis2 mutations retard Spt5 dephosphorylation after inhibition of Cdk9 in vivo. Chromatin immunoprecipitation and sequencing analysis indicates that Spt5 is dephosphorylated as transcription complexes traverse the cleavage and polyadenylation signal, concomitant with the accumulation of Pol II phosphorylated at residue Ser2 of the carboxy-terminal domain consensus heptad repeat . A conditionally lethal Dis2-inactivating mutation attenuates the drop in Spt5 phosphorylation on chromatin, promotes transcription beyond the normal termination zone (as detected by precision run-on transcription and sequencing ) and is genetically suppressed by the ablation of Cdk9 target sites in Spt5. These results suggest that the transition of Pol II from elongation to termination coincides with a Dis2-dependent reversal of Cdk9 signalling-a switch that is analogous to a Cdk1-PP1 circuit that controls mitotic progression .
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http://dx.doi.org/10.1038/s41586-018-0214-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6021199PMC
June 2018

Molecular mechanisms driving transcriptional stress responses.

Nat Rev Genet 2018 06;19(6):385-397

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA.

Proteotoxic stress, that is, stress caused by protein misfolding and aggregation, triggers the rapid and global reprogramming of transcription at genes and enhancers. Genome-wide assays that track transcriptionally engaged RNA polymerase II (Pol II) at nucleotide resolution have provided key insights into the underlying molecular mechanisms that regulate transcriptional responses to stress. In addition, recent kinetic analyses of transcriptional control under heat stress have shown how cells 'prewire' and rapidly execute genome-wide changes in transcription while concurrently becoming poised for recovery. The regulation of Pol II at genes and enhancers in response to heat stress is coupled to chromatin modification and compartmentalization, as well as to co-transcriptional RNA processing. These mechanistic features seem to apply broadly to other coordinated genome-regulatory responses.
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http://dx.doi.org/10.1038/s41576-018-0001-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6036639PMC
June 2018

Enhancer transcription: what, where, when, and why?

Genes Dev 2018 01;32(1):1-3

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.

Following the discovery of widespread enhancer transcription, enhancers and promoters have been found to be far more similar than previously thought. In this issue of , two studies (Henriques and colleagues [pp. 26-41] and Mikhaylichenko and colleagues [pp. 42-57]) shine new light on the transcriptional nature of promoters and enhancers in Together, these studies support recent work in mammalian cells that indicates that most active enhancers drive local transcription using factors and mechanisms similar to those of promoters. Intriguingly, enhancer transcription is shown to be coordinated by SPT5- and P-TEFb-mediated pause-release, but the pause half-life is shorter, and termination is more rapid at enhancers than at promoters. Moreover, bidirectional transcription from promoters is associated with enhancer activity, lending further credence to models in which regulatory elements exist along a spectrum of promoter-ness and enhancer-ness. We propose a general unified model to explain possible functions of transcription at enhancers.
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http://dx.doi.org/10.1101/gad.311605.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5828389PMC
January 2018

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

Nat Commun 2018 02 7;9(1):543. Epub 2018 Feb 7.

Department of Molecular Biology and Genetics, Cornell University, 107 Biotechnology Building, 526 Campus Road, Ithaca, NY, 14853-2703, USA.

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.
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http://dx.doi.org/10.1038/s41467-018-03006-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5803247PMC
February 2018

Dynamic evolution of regulatory element ensembles in primate CD4 T cells.

Nat Ecol Evol 2018 03 29;2(3):537-548. Epub 2018 Jan 29.

Simons Center for Quantitative Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA.

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.
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http://dx.doi.org/10.1038/s41559-017-0447-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5957490PMC
March 2018

CBP Regulates Recruitment and Release of Promoter-Proximal RNA Polymerase II.

Mol Cell 2017 Nov 19;68(3):491-503.e5. Epub 2017 Oct 19.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 10691 Stockholm, Sweden. Electronic address:

Transcription activation involves RNA polymerase II (Pol II) recruitment and release from the promoter into productive elongation, but how specific chromatin regulators control these steps is unclear. Here, we identify a novel activity of the histone acetyltransferase p300/CREB-binding protein (CBP) in regulating promoter-proximal paused Pol II. We find that Drosophila CBP inhibition results in "dribbling" of Pol II from the pause site to positions further downstream but impedes transcription through the +1 nucleosome genome-wide. Promoters strongly occupied by CBP and GAGA factor have high levels of paused Pol II, a unique chromatin signature, and are highly expressed regardless of cell type. Interestingly, CBP activity is rate limiting for Pol II recruitment to these highly paused promoters through an interaction with TFIIB but for transit into elongation by histone acetylation at other genes. Thus, CBP directly stimulates both Pol II recruitment and the ability to traverse the first nucleosome, thereby promoting transcription of most genes.
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http://dx.doi.org/10.1016/j.molcel.2017.09.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5826544PMC
November 2017

Nascent RNA sequencing reveals a dynamic global transcriptional response at genes and enhancers to the natural medicinal compound celastrol.

Genome Res 2017 11 12;27(11):1816-1829. Epub 2017 Oct 12.

Simons Center for Quantitative Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.

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.
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http://dx.doi.org/10.1101/gr.222935.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5668940PMC
November 2017

The 4D nucleome project.

Nature 2017 09;549(7671):219-226

Department of Bioengineering, University of California San Diego, La Jolla, California 92093, USA.

The 4D Nucleome Network aims to develop and apply approaches to map the structure and dynamics of the human and mouse genomes in space and time with the goal of gaining deeper mechanistic insights into how the nucleus is organized and functions. The project will develop and benchmark experimental and computational approaches for measuring genome conformation and nuclear organization, and investigate how these contribute to gene regulation and other genome functions. Validated experimental technologies will be combined with biophysical approaches to generate quantitative models of spatial genome organization in different biological states, both in cell populations and in single cells.
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http://dx.doi.org/10.1038/nature23884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5617335PMC
September 2017

Transcriptional response to stress is pre-wired by promoter and enhancer architecture.

Nat Commun 2017 08 15;8(1):255. Epub 2017 Aug 15.

Faculty of Science and Engineering, Cell Biology, Åbo Akademi University, Turku, 20520, Finland.

Programs of gene expression are executed by a battery of transcription factors that coordinate divergent transcription from a pair of tightly linked core initiation regions of promoters and enhancers. Here, to investigate how divergent transcription is reprogrammed upon stress, we measured nascent RNA synthesis at nucleotide-resolution, and profiled histone H4 acetylation in human cells. Our results globally show that the release of promoter-proximal paused RNA polymerase into elongation functions as a critical switch at which a gene's response to stress is determined. Highly transcribed and highly inducible genes display strong transcriptional directionality and selective assembly of general transcription factors on the core sense promoter. Heat-induced transcription at enhancers, instead, correlates with prior binding of cell-type, sequence-specific transcription factors. Activated Heat Shock Factor 1 (HSF1) binds to transcription-primed promoters and enhancers, and CTCF-occupied, non-transcribed chromatin. These results reveal chromatin architectural features that orient transcription at divergent regulatory elements and prime transcriptional responses genome-wide.Heat Shock Factor 1 (HSF1) is a regulator of stress-induced transcription. Here, the authors investigate changes to transcription and chromatin organization upon stress and find that activated HSF1 binds to transcription-primed promoters and enhancers, and to CTCF occupied, untranscribed chromatin.
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http://dx.doi.org/10.1038/s41467-017-00151-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557961PMC
August 2017

Use of conditioned media is critical for studies of regulation in response to rapid heat shock.

Cell Stress Chaperones 2017 01 3;22(1):155-162. Epub 2016 Nov 3.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, 14850, USA.

Heat shock response (HSR) maintains and restores protein homeostasis when cells are exposed to proteotoxic heat stress. Heat shock (HS) triggers a rapid and robust change in genome-wide transcription, protein synthesis, and chaperone activity; and therefore, the HSR has been widely used as a model system in these studies. The conventional method of performing instantaneous HS in the laboratory uses heated fresh media to induce HSR when added to cells. However, addition of fresh media to cells may evoke additional cellular responses and signaling pathways. Here, we compared the change in global transcription profile when HS is performed with either heated fresh media or heated conditioned media. We found that the use of heated fresh media induces transcription of hundreds of genes that HS alone does not induce, and masks or partially masks HS-mediated downregulation of thousands of genes. The fresh-media-dependent upregulated genes encode ribosomal subunit proteins involved in translation and RNA processing factors. More importantly, fresh media also induce transcription of several heat shock protein genes (Hsps) in a heat shock factor 1 (HSF1)-independent manner. Thus, we conclude that a conventional method of HS with heated fresh media causes changes in transcription regulation that confound the actual change caused solely by elevated temperature of cells.
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http://dx.doi.org/10.1007/s12192-016-0737-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5225056PMC
January 2017

Cotranscriptional folding of a riboswitch at nucleotide resolution.

Nat Struct Mol Biol 2016 Dec 31;23(12):1124-1131. Epub 2016 Oct 31.

Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, USA.

RNAs can begin to fold immediately as they emerge from RNA polymerase. During cotranscriptional folding, interactions between nascent RNAs and ligands are able to direct the formation of alternative RNA structures, a feature exploited by noncoding RNAs called riboswitches to make gene-regulatory decisions. Despite their importance, cotranscriptional folding pathways have yet to be uncovered with sufficient resolution to reveal how cotranscriptional folding governs RNA structure and function. To access cotranscriptional folding at nucleotide resolution, we extended selective 2'-hydroxyl acylation analyzed by primer-extension sequencing (SHAPE-seq) to measure structural information of nascent RNAs during transcription. Using cotranscriptional SHAPE-seq, we determined how the cotranscriptional folding pathway of the Bacillus cereus crcB fluoride riboswitch undergoes a ligand-dependent bifurcation that delays or promotes terminator formation via a series of coordinated structural transitions. Our results directly link cotranscriptional RNA folding to a genetic decision and establish a framework for cotranscriptional analysis of RNA structure at nucleotide resolution.
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http://dx.doi.org/10.1038/nsmb.3316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5497173PMC
December 2016

High-Resolution Mapping of RNA Polymerases Identifies Mechanisms of Sensitivity and Resistance to BET Inhibitors in t(8;21) AML.

Cell Rep 2016 08 4;16(7):2003-16. Epub 2016 Aug 4.

Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA; Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. Electronic address:

Bromodomain and extra-terminal domain (BET) family inhibitors offer an approach to treating hematological malignancies. We used precision nuclear run-on transcription sequencing (PRO-seq) to create high-resolution maps of active RNA polymerases across the genome in t(8;21) acute myeloid leukemia (AML), as these polymerases are exceptionally sensitive to BET inhibitors. PRO-seq identified over 1,400 genes showing impaired release of promoter-proximal paused RNA polymerases, including the stem cell factor receptor tyrosine kinase KIT that is mutated in t(8;21) AML. PRO-seq also identified an enhancer 3' to KIT. Chromosome conformation capture confirmed contacts between this enhancer and the KIT promoter, while CRISPRi-mediated repression of this enhancer impaired cell growth. PRO-seq also identified microRNAs, including MIR29C and MIR29B2, that target the anti-apoptotic factor MCL1 and were repressed by BET inhibitors. MCL1 protein was upregulated, and inhibition of BET proteins sensitized t(8:21)-containing cells to MCL1 inhibition, suggesting a potential mechanism of resistance to BET-inhibitor-induced cell death.
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http://dx.doi.org/10.1016/j.celrep.2016.07.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4996374PMC
August 2016

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

Genes Dev 2016 08 4;30(15):1731-46. Epub 2016 Aug 4.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14835, USA;

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.
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http://dx.doi.org/10.1101/gad.284430.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5002978PMC
August 2016
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