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Genome-wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE48895 | GEO | 2014/03/21

SECONDARY ACCESSION(S): PRJNA212143

REPOSITORIES: GEO

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Genome-wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons

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.

2014-03-21 | E-GEOD-48895 | biostudies-arrayexpress

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.

2013-04-10 | E-GEOD-41323 | biostudies-arrayexpress

Signaling Pathways Deferentially Affect RNA Polymerase II Initiation, Pausing, and Elongation in Human Cells: Estrogen dependent signaling in MCF-7 cells

2013-04-10 | E-GEOD-41324 | biostudies-arrayexpress

Dynamic turnover of paused pol II complexes at human promoters

Project description:Paused RNA polymerase II that piles up near most human promoters is the target of mechanisms that control entry into productive elongation. Whether paused pol II is a stable or a dynamic target remains unresolved. We report that most 5’-paused pol II throughout the genome is turned over within 2 minutes. This process is revealed under hypertonic conditions that prevent pol II recruitment to promoters. This turnover requires cell viability but is not prevented by inhibiting transcription elongation suggesting that it is mediated at the level of termination. When initiation was prevented by triptolide during recovery from high salt, a novel pre-initiated state of pol II lacking the pausing factor Spt5 accumulated at transcription start sites. We propose that pol II occupancy near 5’ ends is governed by a cycle of ongoing assembly of pre-initiated complexes that transition to pause sites followed by eviction from the DNA template. This model suggests that mechanisms regulating the transition to productive elongation at pause sites operate on a dynamic population of pol II that is turning over at rates far higher than previously suspected. We suggest that a plausible alternative to elongation control via escape from a stable pause, is by escape from premature termination.

2018-09-14 | GSE117006 | GEO

Gene expression Microarray analysis for HEK293 WT and ELL KD with control and 1hr EGF stimulated conditions.

Project description:Transcription is a multi-stage process that coordinates several steps within the transcription cycle including chromatin reorganization, RNA polymerase II recruitment, initiation, promoter clearance and elongation. Recent advances have identified the super elongation complex (SEC), containing the eleven nineteen lysine rich leukemia protein (ELL), as a key regulator of transcriptional elongation. We show here that ELL plays a diverse and kinetically distinct role prior to its assembly into the SEC by stabilizing Pol II recruitment/initiation and entry into the pause site. Loss of ELL destabilizes the PIC complexes and results in disruption of early elongation and promoter proximal chromatin structure prior to recruitment of AFF4 and other SEC components. These changes result in significantly reduced transcriptional activation of rapidly induced genes. Thus, ELL plays an early and essential role during rapid high amplitude gene expression that is required for both Pol II pause site entry and release. Total RNA for unstimulated and for simulated with EGF (50 ng/mL) for 1hr from wild type and stably induced ELL KD in HEK293 cells were isolated and purified and submitted to LMT-Affymetrix microarray facility to obtain the raw data. Affymetrix Human Exon ST1.0 platform was used for the microarray analysis. Raw data in CEL files were preprocessed using Affymetrix Expression Console software.

2011-12-09 | E-GEOD-34104 | biostudies-arrayexpress

Gene expression Microarray analysis for HEK293 WT and ELL KD with control and 1hr EGF stimulated conditions.

2011-12-09 | GSE34104 | GEO

Signaling Pathways Deferentially Affect RNA Polymerase II Initiation, Pausing, and Elongation in Human Cells: Estrogen dependent signaling in MCF-7 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 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.

2013-04-10 | GSE41324 | GEO

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 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.

2013-04-10 | GSE41323 | GEO

Acetylation on Histone H3 Lysine 9 Mediates a Switch from Transcription Initiation to Elongation

Project description:The transition from transcription initiation to elongation is a key regulatory step in gene expression, which requires RNA polymerase II (Pol II) to escape promoter proximal pausing on chromatin. While elongation factors promote pause release leading to transcription elongation, the role of epigenetic modifications during this critical transition step is poorly understood. Two histone marks on histone H3, lysine 4 trimethylation (H3K4me3) and lysine 9 acetylation (H3K9ac), co-localize on active gene promoters and are associated with active transcription. H3K4me3 can promote transcription initiation, yet the functional role of H3K9ac is much less understood. We hypothesized that H3K9ac may function downstream of transcription initiation by recruiting specific proteins important for the next step of transcription. Here, we describe a functional role for H3K9ac in promoting Pol II pause release by directly recruiting the super elongation complex (SEC) to chromatin. H3K9ac serves as a substrate for direct binding of the SEC, as does acetylation of histone H4 lysine 5 (H4K5ac), to a lesser extent. Furthermore, lysine 9 on histone H3 is necessary for maximal Pol II pause release through SEC action, and loss of H3K9ac increases the Pol II pausing index on a subset of genes in HeLa cells. At select gene promoters, loss of H3K9ac or depletion of the SEC reduces gene expression and increases paused Pol II occupancy. We therefore propose that an ordered histone code drives progression through the transcription cycle, providing new mechanistic insight that SEC recruitment to certain acetylated histones promotes the subsequent release of paused Pol II needed for transcription elongation.

2017-07-11 | GSE99998 | GEO

CDK7 kinase activity promotes RNA polymerase II promoter escape by facilitating initiation factor release

Project description:Cyclin-dependent kinase 7 (CDK7), part of the general transcription factor TFIIH, promotes gene transcription by phosphorylating the C-terminal domain of RNA polymerase II (RNA Pol II). Here, we combine rapid CDK7 kinase inhibition with multi-omics analysis to unravel the direct functions of CDK7 in human cells. CDK7 inhibition causes RNA Pol II retention at promoters, leading to decreased RNA Pol II initiation and immediate global downregulation of transcript synthesis. Elongation, termination, and recruitment of co-transcriptional factors are not directly affected. Although RNA Pol II, initiation factors, and Mediator accumulate at promoters, RNA Pol II complexes can also proceed into gene bodies without promoter-proximal pausing while retaining initiation factors and Mediator. Further downstream, RNA Pol II phosphorylation increases and initiation factors and Mediator are released, allowing recruitment of elongation factors and an increase in RNA Pol II elongation velocity. Collectively, CDK7 kinase activity promotes the release of initiation factors and Mediator from RNA Pol II, facilitating RNA Pol II escape from the promoter.

2024-05-31 | PXD037842 | Pride

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