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4sUDRB-seq: measuring transcription elongation and initiation genomewide

ABSTRACT: A new method to measure elongation and intitiation rates

ORGANISM(S): Homo sapiens

PROVIDER: GSE57116 | GEO | 2014/05/09

SECONDARY ACCESSION(S): PRJNA245468

REPOSITORIES: GEO

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4sUDRB-seq: measuring transcription elongation and initiation genomewide

Project description:A new method to measure elongation and intitiation rates Reversal inhibition of transcription with DRB and tagging newly transcribed RNA with 4-thiouridine (4sU)

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A Chemical Kinetic Basis for Measuring Translation Initiation and Elongation Rates from Ribosome Profiling data

Project description:Analysis methods based on simulations and optimization have been previously developed to estimate relative translation rates from next-generation sequencing data. Translation involves molecules and chemical reactions; hence, bioinformatics methods consistent with the laws of chemistry and physics are more likely to produce accurate results. Here, we derive simple equations based on chemical kinetic principles to measure the translation-initiation rate, transcriptome-wide elongation rate, and individual codon translation rates from ribosome profiling experiments. Our methods reproduce the known rates from ribosome profiles generated from detailed simulations of translation. Applying our methods to data from S. cerevisiae and mouse embryonic stem cells we find that the extracted rates reproduce previously reported correlations with various molecular quantities. A codon can exhibit up to 26-fold variability in its translation rate depending upon its context with in a transcript. This broad distribution means that the average translation rate of a codon is not representative of the rate at which most instances of that codon are translated. We also find that mouse embryonic stem cells have a global translation speed that is almost two-fold faster than previously reported. This large variability in translation rates suggests that translational regulation might be used by cells to a greater degree than previously thought.

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4sUDRB-seq: measuring genomewide transcriptional elongation rates and initiation frequencies within cells.

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Signaling Pathways Deferentially Affect RNA Polymerase II Initiation, Pausing, and Elongation in Human Cells: TNF-alpha dependent signaling in AC16 cells

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 TNFM-NM-1). 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 TNFM-NM-1 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.

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Gene body H2B1ub regulates RNA Polymerase II pause release and is not needed for transcription elongation

Project description:Histone H2B monoubiquitylation (H2Bub1) is localized to transcribed regions of genes and spread in parallel with the progress of RNA polymerase II (Pol II). H2Bub1 levels are highly correlated both with transcription and elongation rates of mammalian genes. Although H2Bub1 correlate with elongation rates, it is not clear whether the correlation is due to causative role of H2Bub1 in regulating elongation rate or the vice versa. By utilizing our recently developed method to measure genomewide elongation rate – 4sUDRB-seq, we tested genomewide elongation rates in H2Bub1 depleted cells. Our results show that H2Bub1, although widely appreciate as an elongation factor, is not needed for proper transcription elongation. Although H2Bub1 depletion does not affect transcription elongation, its depletion results in the upregulation of more than 1000 genes. Our findings show that H2Bub1 regulates the pause release of Pol II at these genes. This specificity might be mediated through the relatively high proximity of H2Bub1 to paused Pol II at these genes. Overall, our data shed light on the regulation of transcription by H2Bub1 and suggest that the role of H2Bub1 in transcription elongation should be reconsidered. ------------------------------------------- see also: Fuchs G, Voichek Y, Benjamin S, Gilad S et al. 4sUDRB-seq: measuring genomewide transcriptional elongation rates and initiation frequencies within cells. Genome Biol 2014 May 9;15(5):R69. PMID: 24887486 see also Dataset GSE57116, Samples GSM1375518 – GSM1375525

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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.

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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.

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Project description:We performed PRO-Seq to determine Pol II elongation rates by measruing the distance of Pol II travels during 25 minutes after Pol II being relased from DRB induced pausing. We allowed Pol II to release in the presence of 1-NM-PP1 or DMSO after DRB being wahsed out in order to assess the effects of CDK12 and/or CDK13 inhibtion on transcriptional elongation rates.

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