DRB-PRO-Seq for measuring Pol II transcriptional elongation rate alterations upon inhibition of analog sensitive CDK12 and/or CDK13
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE141373 | GEO | 2020/04/29
REPOSITORIES: GEO
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Project description:We performed PRO-Seq to measure the Pol II elongation rate by measruing the distance of Pol II travels at viarous time points after relased from DRB induced pausing. We compared DMSO/DRB or THZ531/DRB co-treated sample to assess the effect of CDK12 and CDK13 inhibtion by THZ531 on transcriptional elongation rate.
2020-04-29 | GSE141370 | GEO
Project description:CDK9 is a critical kinase required for the productive transcription of protein-coding genes by RNA polymerase II (pol II) in higher eukaryotes. Phosphorylation of targets including the elongation factor SPT5 and the carboxyl-terminal domain (CTD) of RNA pol II allow the polymerase to pass an early elongation checkpoint (EEC), which is encountered soon after initiation. In addition to halting RNA polymerase II at the EEC, CDK9 inhibition also causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, suggesting that CDK9 and PP2A, working together, regulate the coupling of elongation and transcription termination to RNA maturation. Our phosphoproteomic analyses, using either DRB or an analog-sensitive CDK9 cell line confirm the splicing factor SF3B1 as an additional key target of this kinase. CDK9 inhibition causes loss of interaction of splicing and export factors with SF3B1, suggesting that CDK9 also helps to co-ordinates coupling of splicing and export to transcription.
2022-08-22 | PXD026720 | Pride
Project description:To investigate the question that whether Rpb9 affects elongation velocity, thereby indirectly affecting splicing the regulation, we performed time-course GRO-seq after DRB release. In this assay, DRB was first used to block transcription elongation, which caused Pol II to accumulate at TSSs. Upon DRB wash-off, we performed GRO-seq at 0, 10, 20, and 30min to determine the movement of Pol II wave toward gene 3’ end.Based on the hidden Markov model established earlier, we detected a marked reduction of Pol II re-initiation upon DRB removal in response to Rpb9 knockdown (KD) in HeLa cells. This is consistent with the role of Rpb9 in transcription initiation. By determining the mean elongation rate between 10 and 20min and between 20 and 30m, we found accelerated Pol II elongation, as reported earlier, and interestingly, Rpb9 knockdown appeared to have slightly decreased, rather increased, the rate of transcription. We divided all impacted splicing events 4 equal bins and then asked if a higher degree of induced exon skipping tends to occur on genes with faster Pol II movement, finding that this is not the case. Conversely, we divided Rpb9 KD-altered Pol II rate into 4 equal binds and asked whether splicing shows any correlation to Pol II Rpb9 KD-altered Pol II elongation rate, and again, we detected no correlation. We conclude from these data that Rpb9 KD has some minor impact on the Pol II elongation rate in vivo, and importantly, whatever measurable changes in Rpb9 KD-induced Pol II elongation rate are not linked to changes in Rpb9 KD-induced co-transcriptional splicing.
2023-12-25 | GSE221839 | GEO
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
Project description:Urinary tract infections (UTIs) constitute a highly relevant model of microbial adaptation, in which the contrasting effects of pathogens and commensals on host tissues are clearly displayed. While virulent Escherichia coli cause severe, potentially life-threatening disease by breaking the inertia of the mucosal barrier and infecting the kidneys, the most common outcome of bacteriuria is an asymptomatic carrier state resembling commensalism at other mucosal sites. It remains unclear if the lack of destructive inflammation merely reflects low virulence or if carrier strains actively inhibit disease associated responses in the host. To address this question, we examined the effects of asymptomatic bacterial carriage on host gene expression. The asymptomatic strain E. coli 83972 caused reduction in Pol II phosphorylation in the nuclei of human kidney epithelial A498 cells. To specifically address if Pol II inhibition alters the response to infection, A498 cells were pretreated with 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB). This adenosine analogue has been proposed to specifically and reversibly inhibit Pol II transcription without directly affecting other cellular functions. A498 cultered cells were infected with E. coli 83972 or DRB for 4 hours. The culture medium with DMSO was used as a background control. A498 cells were infected with E. coli 83972 or DRB for 4 h. Isolated RNA was subjected to whole genome transcriptome analysis.
2013-02-23 | E-GEOD-43886 | biostudies-arrayexpress
Project description:To examine the effects of Tet3-inactivation on genome-wide transcriptional initiation, we determined the binding profiles of Pol II pSer5, a specific form of Pol II that marks transcriptional initiation sites, by ChIP-seq after DRB-induced block of transcriptional elongation.
2022-09-20 | GSE166815 | GEO
Project description:The rate of transcription elongation plays important roles in the timing of expression of full-length transcripts as well as for the regulation of alternative splicing. In this study we coupled Bru-Seq technology with 5,6-dichlorobenzimidazole 1-M-NM-2-D-ribofuranoside (DRB) to estimate the elongation rates of over 2,000 individual genes in human cells. This technique, BruDRB-Seq, revealed gene-specific differences in elongation rates with a median rate of around 1.5 kb/min. We found that genes with fast elongation rates showed higher densities of H3K79m2 and H4K20me1 marks compared to slower elongating genes. Furthermore, fast elongation rates had a positive correlation with gene length, low complexity DNA sequence and distance from nearest active transcription unit. Features that negatively correlated with elongation rate included exon density and the number of LINE sequences in the gene. The BruDRB-Seq technique offers new opportunities to interrogate mechanisms of regulation of transcription elongation. Measurement of RNA Pol II elogation rate. Normal fibroblasts (HF1 and TM), Cockayne syndrome group B fibroblasts, K562 and MCF-7 cells were exposed to DRB for 60 minutes, after which a washout was performed. Nascent RNA was labeled using bromouridine for 10 minutes immediately after the washout. The genomic region extending from actice Trancription Start Sites was used to determine the gene's elongation rate. Please note that the nf_0h_3* samples are duplicated sample records of GSM1062445 and GSM1062446, for the convenient retrieval of the complete raw data from SRA.
2014-04-01 | E-GEOD-55534 | biostudies-arrayexpress
Project description:Recent studies reveal a striking phenomenon that RNA Polymerase II (Pol II) appears to travel on gene body in an accelerated fashion, but the mechanism has remained unknown. We performed synchronized transcription coupled with deep sequencing, observing an inverse relationship between initial rate and acceleration in different cell types. We directly tested several correlative events and detected a positive contribution of the splicing commitment factor SRSF2 to Pol II acceleration, suggesting a functional benefit of co-transcriptional pre-mRNA splicing in transcription elongation. Unexpectedly, we found that perturbation of Pol II Ser2 phosphorylation had little impact on Pol II elongation or acceleration. While H3K79me2 has been positively correlated with Pol II elongation, we showed that reduction of this histone modification event actually accelerated Pol II elongation. Together, these data suggest a combined effect of gradual gain-of-competence and gradual lost-of-epigenetic barriers as the mechanism for accelerated Pol II elongation. DRB time-course releasing assay under functional perturbation
2015-12-31 | E-GEOD-66021 | biostudies-arrayexpress
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
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-41324 | biostudies-arrayexpress