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Rate of elongation by RNA polymerase II is influenced by specific gene features and histone modifications

ABSTRACT: 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-β-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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE55534 | GEO | 2014/04/01

SECONDARY ACCESSION(S): PRJNA239893

REPOSITORIES: GEO

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Rate of elongation by RNA polymerase II is influenced by specific gene features and histone modifications

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

Genome-wide miRNA turnover profile in various cell types after transcription inhibition

Project description:We measured miRNA turnover rates in 8 mammalian cell types post transcription inhibitors ActinomycinD (ActD) or 5,6-Dichlorobenzimidazole 1-β-D-ribofuranoside (DRB) treatment with miRNA expression profiling, generating 218 expression profiles on 528 endogenous miRNAs.

2015-02-05 | GSE46877 | GEO

Pre-mRNA Splicing is Facilitated by an Optimal RNA Polymerase II Elongation Rate

Project description:Transcription and pre-mRNA alternative splicing was analyzed by in isogenic human HEK293 cell lines that inducibly express a-amanitin resistant mutants of the RNA polymerase II large subunit with slow and fast elongation rates. Alternative splicing was analyzed by, RNA-seq and RASL-seq of polyA+ RNA from a-amanitin treated cells; transcription elongation rate was analyzed by BrUTP-labelled GRO-seq of a-amanitin treated cells at time points after release from a DRB (5,6-dichloro-1-bold beta-D-ribofuranosylbenzimidazole) block.

2014-11-25 | E-GEOD-63375 | biostudies-arrayexpress

Genome-wide miRNA turnover profile in various cell types after transcription inhibition

Project description:We measured miRNA turnover rates in 8 mammalian cell types post transcription inhibitors ActinomycinD (ActD) or 5,6-Dichlorobenzimidazole 1-M-NM-2-D-ribofuranoside (DRB) treatment with miRNA expression profiling, generating 218 expression profiles on 528 endogenous miRNAs. Cells were plated in six-well plates at around 200,000 cells/well. A media change was performed one day after plating. ActD and DRB treatment were started after confluency (around two days after plating) for 0, 2, 4, 6, 12, and 24 hours post treatment with biological duplicate (ActD treatment) or biological triplicate (DRB treatment). The concentration of ActD and DRB for different cell lines were titrated to achieve rapid c-myc down-regulation after 2 hours of ActD and DRB treatment. After treatment at indicated time points, cells were washed with PBS once and lysed in TriZol reagent immediately (Invitrogen). RNA extraction was performed following the manufacturerM-bM-^@M-^Ys protocol. miRNA profiling was performed using a Luminex bead-based method.

2015-02-05 | E-GEOD-46877 | biostudies-arrayexpress

Chemical Perturbation of CDK9 and RUVBL2 Reveals Insights on Regulation of the Circadian Phase

Project description:We report the CRY1 ChIPseq data. Mice were intraperitoneally (I.P.) injected with DRB at CT02 (CT: circadian time). DRB (5,6-dichlorobenzimidazone-1-β-D-ribofuranoside) is a well-known inhibitor of the transcription factor CDK9 which has crucial roles in the RNA polymerase II transcriptional pausing. The mouse liver was collected at 0, 2 and 4hr after treatment. Then, the samples were processed as we previously reported (Koike et al, 2012, Science). This study provides the evidence that DRB regulates the CRY1 binding dynamics.

2021-11-01 | GSE107187 | GEO

DRB-PRO-Seq for measuring Pol II transcriptional elongation rate alteration up on CDK12 and CDK13 inhibition by THZ531

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

DRB-PRO-Seq for measuring Pol II transcriptional elongation rate alterations upon inhibition of analog sensitive CDK12 and/or CDK13

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.

2020-04-29 | GSE141373 | GEO

ChIP-seq of HA-METTL14 in HepG2 cells

Project description:The METTL3-METTL14 heterodimer is the core component of the N6-methyltransferase complex (MTC) that catalyzes methylation of adenosine residues at the N(6) position of RNA. As the non-catalytic subunit of the MTC, METTL14 functions as the RNA-binding scaffold that recognizes the RNA substrate. We found METTL14 could interacte with chromatin through recognition of H3K36me3. ChIP-seq with HA-tagged METTL14 using an HA antibody identified 300 peaks in HepG2 cells, among which 53% peaks were located in the gene body and only 3% peaks were located in the promoter. The binding sites of METTL14 on chromatin correlate with that of H3K36me3.To investigat whether this interaction is Pol II dependent, we treated cells with a Pol II inhibitor 5,6-Dichlorobenzimidazole 1-β-D-ribofuranoside (DRB) for a short time. Interestingly, we found the interaction of METTL14 with chromatin in gene body region was not affected by DRB treatment .

2019-02-21 | GSE121943 | GEO

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)

2014-05-09 | E-GEOD-57116 | biostudies-arrayexpress

Widespread backtracking by RNA pol II is a major effector of gene activation, 5’ pause release, termination and transcription elongation rate

Project description:In addition to phosphodiester bond formation, RNA polymerase II has an RNA endonuclease activity, stimulated by TFIIS, which rescues complexes that have arrested and backtracked. We identified backtracking sites using mNET-seq in human cells expressing dominant-negative TFIIS (TFIISDN) that inhibits RNA cleavage and stabilizes backtracked complexes. Backtracking is most frequent within 2 kb of start sites, consistent with slow elongation early in transcription, and in 3’ flanking regions where termination is enhanced by TFIISDN. Inhibition of the rescue from backtracked pol II by TFIISDN impaired the escape from 5’ pause sites, the completion of long transcripts, and activation of stress-inducible genes. TFIISDN slowed elongation rates genome-wide by half as measured by sequencing nascent transcripts pulse labeled with Bromouridine (Bru-seq) and by anti-pol II ChIP-seq at time points after release of a DRB block. These results suggest that rescue of backtracked pol II by TFIIS is a major stimulus of elongation.

2018-11-30 | GSE120201 | GEO

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