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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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE41323 | GEO | 2013/04/10

SECONDARY ACCESSION(S): PRJNA176559

REPOSITORIES: GEO

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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.

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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

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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 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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2022-12-12 | PXD035092 | Pride

Allosteric transcription stimulation by super elongation complex

Project description:The super elongation complex (SEC) contains the positive transcription elongation factor b (P-TEFb) and a subcomplex, ELL2-EAF1, which stimulates transcription elongation by RNA polymerase II (Pol II). Here we report the cryo-EM structure of ELL2-EAF1 bound to a Pol II elongation complex at 2.8 Å resolution. The ELL2-EAF1 dimerization module directly binds the Pol II lobe, explaining how SEC delivers P-TEFb to Pol II. The same site on the lobe also binds the initiation factor TFIIF, consistent with SEC binding only after the transition from transcription initiation to elongation. Structure-guided functional analysis shows that elongation stimulation requires the dimerization module and an ELL2 protein linker that tethers this module to the Pol II protrusion. Our results show that SEC stimulates elongation allosterically and indicate that this stimulation involves stabilization of a further closed conformation of the Pol II active center cleft.

2021-07-15 | PXD024457 | Pride

THOC5 complexes with DDX5, DDX17 and CDK12 to regulate R loop structures and transcription elongation rate

Project description:THOC5, a member of the THO complex, is essential for the 3´processing of some inducible genes, the export of a subset of genes and stem cell self-renewal. Utilising nanopore mRNA-sequencing we show that when THOC5 is depleted 50-60% of mRNAs undergo altered 3´end cleavage. Further, THOC5 depletion leads to increased RNA Polymerase II (Pol II) presence at the start site, on the gene body and close to the 3’end. Moreover, THOC5 is recruited close to high density Pol II sites suggesting that THOC5 is involved in transcriptional elongation. Indeed, DRB/TTchem-seq that measures elongation rates in vivo revealed an accumulation of released Pol II near to TSS and a decrease of elongation rates in THOC5 depleted cells. Furthermore, THOC5 is more recruited to its target genes in cells expressing slow Pol II than those expressing fast Pol II. In cells expressing slow Pol II chromatin associated THOC5 interacts with CDK12 (a protein that modulates mRNA elongation rates), RNA helicases DDX5, DDX17, and THOC6. Importantly these interactions were not observed in cells expressing fast Pol II. 3’ cleavage of 50% of THOC5 target genes is also regulated by CDK12 and THOC6. The CDK12/THOC5 interaction promotes CDK12 recruitment to R-loops in a THOC6-dependent manner. These data demonstrate that THOC5/THOC6 play a part in transcription elongation. Given the role of THOC5 in primitive cell survival its phosphorylation by agonists, oxidative stress and oncogenes the pathway we identified has relevance in the physiology and pathology of stem cells.

2022-12-16 | GSE173374 | GEO

NDF Is a Transcription Factor that Stimulates Elongation by RNA Polymerase II

Project description:RNA polymerase II (Pol II) elongation is a critical step in gene expression. Here we find that NDF, which was identified as a bilaterian nucleosome-destabilizing factor, is also a Pol II transcription factor that stimulates elongation with plain DNA templates in the absence of nucleosomes. NDF binds directly to Pol II and enhances elongation by a different mechanism than does transcription factor TFIIS. Moreover, yeast Pdp3, which is related to NDF, binds to Pol II and stimulates elongation. Thus, NDF is a Pol II-binding transcription elongation factor that is localized over gene bodies and is conserved from yeast to humans.

2022-03-10 | GSE185464 | GEO

Paf1C regulates RNA polymerase II progression by modulating elongation rate

Project description:Elongation factor Paf1C regulates several stages of the RNA polymerase II (Pol II) transcription cycle, although it is unclear how it modulates Pol II distribution and progression in mammalian cells. We found that conditional ablation of Paf1 resulted in the accumulation of unphosphorylated and Ser5 phosphorylated Pol II around promoter proximal regions and within the first 20-30 kb of gene bodies, respectively. Paf1 ablation did not impact the recruitment of other key elongation factors, namely, Spt5, Spt6, and the FACT complex, suggesting that Paf1 function may be mechanistically distinguishable from each of these factors. Moreover, loss of Paf1 triggered an increase in TSS-proximal nucleosome occupancy, which could impose a considerable barrier to Pol II elongation past TSS-proximal regions. Remarkably, accumulation of Ser5P in the first 20-30 kb coincided with reductions in histone H2B ubiquitylation within this region. Furthermore, we show that nascent RNA species accumulate within this window, suggesting a mechanism whereby Paf1 loss leads to aberrant, prematurely terminated transcripts and diminution of full-length transcripts. Importantly, we found that loss of Paf1 results in Pol II elongation rate defects with significant rate compression. Our findings suggest that Paf1C is critical for modulating Pol II elongation rates by functioning beyond the pause-release step as an "accelerator" over specific early gene body regions.

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