Identification of active transcriptional regulatory elements with GRO-seq
ABSTRACT: Transcriptional regulatory elements (TREs), including enhancers and promoters, determine the transcription levels of associated genes. We have recently shown that global run-on and sequencing (GRO-seq) with enrichment for 5'-capped RNAs reveals active TREs with high accuracy. Here, we demonstrate that active TREs can be identified by applying sensitive machine-learning methods to standard GRO-seq data. This approach allows TREs to be assayed together with gene expression levels and other transcriptional features in a single experiment. Our prediction method, called discriminative Regulatory Element detection from GRO-seq (dREG), summarizes GRO-seq read counts at multiple scales and uses support vector regression to identify active TREs. The predicted TREs are more strongly enriched for several marks of transcriptional activation, including eQTL, GWAS-associated SNPs, H3K27ac, and transcription factor binding than those identified by alternative functional assays. Using dREG, we survey TREs in eight human cell types and provide new insights into global patterns of TRE function. We analyzed GRO-seq or PRO-seq data from eight human cell lines. Please note that this study comprises new sample data plus reanalysis of old Sample data submitted by another user. Existing PRO-seq or GRO-seq data was combined as detailed in the GSE66031_readme.txt. See GSM1613181 and GSM1613182 Sample records for data processing information.
Project description:Production of mRNA depends critically on the rate of RNA polymerase II (Pol II) elongation. To dissect Pol II dynamics in mouse ES cells, we inhibited Pol II transcription at either initiation or promoter-proximal pause escape with Triptolide or Flavopiridol, and tracked Pol II kinetically using GRO-seq. Both inhibitors block transcription of more than 95% of genes, showing that pause escape, like initiation, is a ubiquitous and crucial step within the transcription cycle. Moreover, paused Pol II is relatively stable, as evidenced from half-life measurements at ~3200 genes. Finally, tracking the progression of Pol II after drug treatment establishes Pol II elongation rates at over 1,000 genes. Notably, Pol II accelerates dramatically while transcribing through genes, but slows at exons. Furthermore, intergenic variance in elongation rates is substantial, and is influenced by a positive effect of H3K79me2 and negative effects of exon density and CG content within genes. We isolated replicates of nuclei of untreated mESCs and cells treated for 2, 5, 12.5, 25 and 50 min with 300nM flavopiridol, as well as nuclei treated for 12.5, 25, and 50 min with 500nM triptolide and performed GRO-seq with these.
Project description:The transition in developmental control from maternal to zygotic gene products marks a critical step in early embryogenesis. Here, we use GRO-seq analysis to map the genome-wide RNA polymerase distribution during the Drosophila maternal to zygotic transition. This analysis unambiguously identifies the zygotic transcriptome, and provides insight into its mechanisms of regulation. Two replicates of GRO-seq at each time point.
Project description:Repair of DNA double-strand breaks (DSBs) by non-homologous end-joining is critical for neural development, and brain cells frequently contain somatic genomic variations that might involve DSB intermediates. We now use an unbiased, high-throughput approach to identify genomic regions harboring recurrent DSBs in primary neural stem/progenitor cells (NSPCs). We identify 27 recurrent DSB clusters (RDCs) and, remarkably, all occur within gene bodies. Most of these NSPC RDCs were detected only upon mild, aphidicolin-induced replication stress, providing a nucleotide-resolution view of replication-associated genomic fragile sites. The vast majority of RDCs occur in long, transcribed, and late-replicating genes. Moreover, almost 90% of identified RDC-containing genes are involved in synapse function and/or neural cell adhesion, with a substantial fraction also implicated in tumor suppression and/or mental disorders. Our characterization of NSPC RDCs reveals a basis of gene fragility and suggests potential impacts of DNA breaks on neurodevelopment and neural functions. We performed high-throughput, genome-wide, translocation sequencing (HTGTS) and GRO-seq in primary mouse neural stem/progenitor cells of the indicated genotypes.
Project description:Inflammation is associated with many cardiovascular pathologies, but the underlying mechanisms remain unclear. To explore this in more detail, we characterized the transcriptome of an immortalized adult human ventricular cardiomyocyte cell line (AC16) in response to tumor necrosis factor (TNFa). Using a combination of genomic approaches, including global nuclear run-on sequencing (GRO-seq) and chromatin immunoprecipitation coupled with sequencing (ChIP-seq), we identified ~30,000 transcribed regions in AC16 cells, which includes a set of RNA polymerases I and III (Pol I and Pol III) transcribed regions revealed in the presence of α-amanitin. The set of transcribed regions produces both protein-coding and non-coding RNAs, many of which have not been annotated previously, including enhancer RNAs originating from NF-κB binding sites. In addition, we observed that AC16 cells rapidly and dynamically reorganize their transcriptomes in response to TNFa stimulation in an NF-κB-dependent manner, switching from a basal state to a proinflammatory state affecting a spectrum of cardiac-associated protein-coding and non-coding genes. Moreover, we observed distinct Pol II dynamics for up- and downregulated genes, with a rapid release of Pol II into productive elongation for TNFa-stimulated genes. Our studies shed new light on the regulation of the cardiomyocyte transcriptome in response to a proinflammatory signal and help to clarify the link between inflammation and cardiomyocyte function at the transcriptional level. Using GRO-seq and ChIP-seq (p65 and RNA Pol II) over a time course of TNFα signaling in AC16 human cardiomyocytes.
Project description:Dosage Compensation is required to correct for uneven gene dose between the sexes. We utilized global run-on sequencing (GRO-seq) to examine how Caenorhabditis elegans dosage compensation reduces transcription of X-linked genes. To facilitate these experiments, we required accurate 5’-ends of genes that have been missing due to a co-transcriptional trans-splicing event common in nematodes. We developed a modified GRO-seq protocol to identify TSSs that are supported by transcription, and determined that TSSs lie more than 1 kb upstream of the previously annotated TSS for nearly one-quarter of all genes. We then investigated the changes that occur in transcriptionally engaged RNA Polymerase when dosage compensation is disrupted, and find that dosage compensation controls recruitment of RNA Polymerase to X-linked genes. GRO-seq experiments (two biological replicates) were performed in nuclei from many wild-type states and a dosage compensation mutant
Project description:We used GRO-seq to examine the effect of Myc activation on RNA transcription in U2OS cells. We measure in duplicates gene transcription rates in U2OS cells containing an inducible Myc expression vector that were induced or mock-treated in duplicates for 5 hours.
Project description:The majority of transcription studies examine steady-state RNA . However steady-state RNA is not a true reflection of the transcriptome, because the RNA levels are affected by both transcription rate and degradation rate. In this experiment we measured the amount of transcription occurring in HCT116 colon cancer cells, regardless of degradation, using GRO-seq (global nuclear run-on sequencing). This information demonstrates that many genes have a pile-up of transcriptionally-engaged polymerase near their 5'-end. Nuclei were prepared from HCT116 cells (treated for 1hr with DMSO as control for additional GRO-seq experiments to be reported separately). Transcription run-on was performed (as per Core, L.J., Waterfall, J.J., and Lis, J.T. (2008). Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322, 1845-1848) and nascent RNAs were purified and sequenced.
Project description:We used Global Run-on and Sequencing (GRO-seq) to measure the rate of transcription elongation by RNA polymerase II (Pol II) following gene activation. We observed that Pol II elongation rates can vary as much as 4-fold at different genomic loci and in response to two distinct cellular signaling pathways (i.e., estrogen and TNFα). Elongation rates are slowest near the promoter and increase during the first ~15 kb transcribed into the gene body. Gene body elongation rates correlate with the density of Pol II, consequently resulting in systematically higher rates of transcript production at genes with higher Pol II density. By monitoring Pol II dynamics following short inductions, we found that E2 stimulates gene expression by increasing Pol II initiation, whereas TNFα stimulates the release of Pol II from promoter proximal pause sites. Collectively, our results identify previously uncharacterized variation in the rate of Pol II elongation and highlight elongation as an important, variable, and regulated rate limiting step in the transcription cycle. Using GRO-seq over a time course (0, 10, 25, and 40 min) of estrogen signaling in ER-alpha positive MCF-7 human breast cancer cells.
Project description:Global Run-On sequencing (GRO-seq) was carried out on nuclei isolated from HeLa cells after 30 minutes of treatment with CDK9 inhibitors KM05382 and DRB and from two untreated controls. Libraries of nascent RNAs were generated and sequenced using Illumina Hi-seq technology. GRO-seq reads were processed, deduplicated, mapped to human genome and normalised to reads in the 5’ ETS of the 45S rRNA gene.
Project description:Rosiglitazone (rosi) is a powerful insulin sensitizer, but serious toxicities have curtailed its widespread clinical use. Rosi functions as a high-affinity ligand for PPARg, the adipocyte-predominant nuclear receptor (NR). The classic model, involving binding of ligand to the NR on DNA, explains positive regulation of gene expression, but ligand-dependent repression is not well understood. We have now addressed this issue by studying the direct effects of rosiglitazone on gene transcription, using global run-on sequencing (GRO-seq). Rosi-induced changes in gene body transcription were pronounced after 10 minutes and correlated with steady-state mRNA levels as well as with transcription at nearby enhancers (eRNAs). Upregulated eRNAs occurred almost exclusively at PPARg binding sites, to which rosi treatment recruited the coactivator MED1. By contrast, transcriptional repression by rosi involved a loss of MED1 from eRNA sites devoid of PPARg and enriched for other TFs including AP-1 factors and C/EBPs. Thus, rosi activates and represses transcription by fundamentally different mechanisms that could inform the future development of antidiabetic drugs. 3T3-L1 matuer adipocyte were treated with rosi, and nascent transcripts were measured at various time points using GRO-seq. ChIP-seq experiments for various coactivators, corepressor, and transcription factors also have been done to monitor initial occupancy or change before and after treatment.