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Total RNA Pol2 ChIP-seq in mES cells treated with flavopiridol (Cdk9 inhibitor) after DSIF and NELF knockdown

ABSTRACT: Recent studies have found that promoter-proximal pausing occurs at most Pol2-regulated genes. DSIF and NELF function as negative elongation factors and promote Pol2 pausing. P-TEFb, whose enzymatic activity lies in the kinase Cdk9, positively regulates the transition into productive elongation by phosphorylating subunits in DSIF, NELF and Pol2. To gain insights into the interplay between these factors in regulating transcriptional pause release, we tested if knockdown of either pausing factor could bypass P-TEFb function. We find that P-TEFb function is still required for transcriptional elongation after DSIF or NELF knockdown. mES cells were infected with a shRNA hairpin to knockdown Spt5, Spt4, NelfE or control. The resulting cells were then treated with flavopiridol (1uM for 60 minutes). DNA was enriched by chromatin immunoprecipitation (ChIP) and analyzed by Solexa sequencing. A sample of whole cell extract was sequenced and used as the background to determine enrichment. ChIP was performed using an antibody against total RNA Pol2 (Rpb1 N-terminus, Santa Cruz sc-899).

ORGANISM(S): Mus musculus

SUBMITTER: Richard Young

PROVIDER: E-GEOD-26658 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Total RNA Pol2 ChIP-seq in mES cells treated with flavopiridol (Cdk9 inhibitor) after DSIF and NELF knockdown

Project description:Recent studies have found that promoter-proximal pausing occurs at most Pol2-regulated genes. DSIF and NELF function as negative elongation factors and promote Pol2 pausing. P-TEFb, whose enzymatic activity lies in the kinase Cdk9, positively regulates the transition into productive elongation by phosphorylating subunits in DSIF, NELF and Pol2. To gain insights into the interplay between these factors in regulating transcriptional pause release, we tested if knockdown of either pausing factor could bypass P-TEFb function. We find that P-TEFb function is still required for transcriptional elongation after DSIF or NELF knockdown.

2011-01-19 | GSE26658 | GEO

Genome-wide map of RNA polymerase II, NELF, and DSIF in HeLa cells

Project description:Most metazoan promoters have RNA polymerase II (Pol II) paused slightly downstream of the transcription start site by NELF and DSIF. This pausing keeps these promoters available for rapid induction by P-TEFb, whose activity causes NELF to dissociate and Pol II and DSIF to elongate on the gene. ChIP-Seq data was generated for Pol II, NELF, and DSIF in HeLa cells and used to look at pausing downstream of unannotated promoters. 3x ChIP-Seq (Pol II, NELF, DSIF) in HeLa cells

2014-02-01 | E-GEOD-53008 | biostudies-arrayexpress

Whole Cell Extract

Project description:Most human genes are loaded with promoter proximally paused RNA polymerase II (RNAP II) molecules that are poised for release into productive elongation by P-TEFb. Gdown1, a protein that renders RNAP II responsive to mediator, is involved in RNAP II elongation control. During in vitro transcription assays Gdown1 specifically blocked elongation stimulation by TFIIF, inhibited the termination activity of TTF2, and influenced pausing factors NELF and DSIF, but did not affect the function of TFIIS or the mRNA capping enzyme. Without P-TEFb, Gdown1 led to the production of stably paused polymerases in the presence of nuclear extract. ChIP-Seq data demonstrated that Gdown1 mapped over essentially all poised polymerases across the human genome. Gdown1 increases the stability of poised polymerases while maintaining their responsiveness to P-TEFb, and mediator overcomes a Gdown1-mediated block of initiation by allowing TFIIF function. 1 whole cell extract sample in human HeLa cell. See associated publication.

2012-02-21 | E-GEOD-35882 | biostudies-arrayexpress

Functional association of Gdown1 with RNA Polymerase II in Human

Project description:Most human genes are loaded with promoter proximally paused RNA polymerase II (RNAP II) molecules that are poised for release into productive elongation by P-TEFb. Gdown1, a protein that renders RNAP II responsive to mediator, is involved in RNAP II elongation control. During in vitro transcription assays Gdown1 specifically blocked elongation stimulation by TFIIF, inhibited the termination activity of TTF2, and influenced pausing factors NELF and DSIF, but did not affect the function of TFIIS or the mRNA capping enzyme. Without P-TEFb, Gdown1 led to the production of stably paused polymerases in the presence of nuclear extract. ChIP-Seq data demonstrated that Gdown1 mapped over essentially all poised polymerases across the human genome. Gdown1 increases the stability of poised polymerases while maintaining their responsiveness to P-TEFb, and mediator overcomes a Gdown1-mediated block of initiation by allowing TFIIF function. 7 ChIP-Seq data for Pol II and Gdown1 in human HeLa cell. See associated publication.

2012-01-13 | E-GEOD-32442 | biostudies-arrayexpress

Genome-wide map of RNA polymerase II, NELF, and DSIF in HeLa cells

2014-02-01 | GSE53008 | GEO

Mus musculus

Project description:Total RNA Pol2 ChIP-seq in mES cells treated with flavopiridol (Cdk9 inhibitor) after DSIF and NELF knockdown

| PRJNA136399 | ENA

Role of RNA polymerase II "pausing" in Drosophila development

Project description:According to previous studies, during Drosophila embryogenesis, RNA polymerase II is recruited to promoters at developmental stages preceding the stages of active transcription of genes. This work is aimed at exploring whether this mechanism is used during Drosophila metamorphosis. We performed ChIP-Seq analysis using antibodies to various modifications of RNA polymerase II (total, Pol II CTD Ser5P and Pol II CTD Ser2P), as well as to subunits of NELF, DSIF, PAF complexes and Brd4/Fs(1)h that control transcription elongation. We found that like in mid-embryogenesis during metamorphosis, promoters bind RNA polymerase II in the "paused" state preparing for activation at later stages of development. During mid-embryogenesis, RNA polymerase II in "pause" is phosphorylated at Ser5 and Ser2 of Rpb1 CTD and binds NELF, DSIF, and PAF complexes, but not Brd4/Fs(1)h. During metamorphosis, the "paused" RNA polymerase II complex includes Brd4/Fs(1)h in addition to NELF, DSIF, and PAF. The RNA polymerase II in this complex is phosphorylated at Ser5 at Rpb1 CTD, but not at Ser2.

2022-09-10 | GSE210971 | GEO

The negative elongation factor NELF promotes active transcription of Drosophila ecdysone-dependent genes

Project description:The current work describes molecular mechanism used by the Negative elongation complex (NELF) to control the Pol II pause at genes whose transcription is induced by 20-hydroxyecdysone (20E). According to our data NELF complex is recruited to the promoters and enhancers of 20E-dependent genes. Its presence at the regulatory sites of 20E-dependent genes correlates with the described interaction of its NELF-A subunit with EcR receptor. The NELF complex, consisting of at least three subunits, is formed at the 20E-dependent promoters and participates in their active transcription and maintenance of their repressed state. NELF depletion causes a significant decrease in transcription induced by 20E. We associate this effect with the disruption of Pol II elongation complexes formation. A considerable reduction in the promoter-bound level of Spt5 subunit of DSIF was observed at the 20E-dependent genes upon NELF depletion. We presume that participation in stabilization of the Pol II-DSIF complex can be an important function of NELF with a big impact on transcription of its target genes. Part of our study was to directly link NELF to regulation of 20E-dependent genes in development. We showed presence of NELF at the promoters of 20E-dependent genes during their active transcription both in embryogenesis and metamorphosis. We also demonstrate that 20E-dependent promoters remain in a Pol II paused state at the larva stage, where they are temporarily repressed.

2021-01-10 | GSE156847 | GEO

Identification of candidate genes affecting juvenile wood stiffness in Pinus radiata

Project description:Wood stiffness is the most important wood quality trait of forest trees for structural timber production. We investigated genes differentially transcribed in radiate pine trees with distinct wood stiffness using bulked segregant analysis (BSA) and cDNA microarrays. Transcript accumulation in earlywood (EW) and latewood (LW) of high (HS) and low stiffness (LS) trees in two progeny trials was compared. Radiata pine trees used for microarray experiment were selected from two progeny trials planted at Flynn and Kromelite, Australia. Based on the IML-based MOE measurement, five families with highest and lowest MOE each were selected from each trial, which represented two segregant populations with contrasting wood stiffness. Two individuals from each selected family were further sampled. Developing xylem tissues of selected trees in Flynn trial were sampled in spring (October) and autumn (April), representing earlywood (EW) and latewood (LW) of juvenile aged trees, respectively. Collection of xylem tissues from Kromelite trial was arranged in summer (late November) when latewood (LW) was formed. The xylem tissues were scraped at breast height with a sharp chisel after the bark was removed. In Flynn trial EW and LW tissues were collected from the same sampled trees on opposite sides of the trunk. Transcript accumulation was compared in trees with highest (HS) and lowest stiffness (LS) using xylem samples from Flynn collected in spring (EW) and autumn (LW), as well as Kromelite in summer (LW), respectively. Bulked segregant analysis (BSA) was used for the experiment design. Total RNA samples extracted from the five trees with HS were pooled at equal amount, and compared to the bulked five individuals with LS. This pooling strategy can partly minimize the genetic variation among different genotypes. Dye swaps were applied in each biological replicate.

2011-06-01 | E-GEOD-23020 | biostudies-arrayexpress

Gene-specific mechanisms dictate glucocorticoid receptor-mediated repression of inflammatory response genes in macrophages [RNA-seq]

Project description:The Glucocorticoid Receptor (GR) potently represses macrophage-elicited inflammation, however, the underlying mechanisms remain obscure. Our genome-wide analysis reveals that pro-inflammatory paused genes, activated via global negative elongation factor (NELF) dissociation and RNA Polymerase (Pol)2 release from early elongation arrest, and non-paused genes, induced by de novo Pol2 recruitment, are equally susceptible to acute glucocorticoid repression. Moreover, in both cases the dominant mechanism involves rapid GR tethering to p65 at NF-kB binding sites. Yet, specifically at paused genes, GR activation triggers widespread promoter accumulation of NELF, with myeloid cell-specific NELF deletion conferring glucocorticoid resistance. Conversely, at non-paused genes, GR attenuates the recruitment of p300 and histone acetylation, leading to a failure to assemble BRD4 and Mediator at promoters and enhancers, ultimately blocking Pol2 initiation. Thus, GR displays no preference for a specific pro-inflammatory gene class, however, it effects repression by targeting distinct temporal events and components of transcriptional machinery

2018-02-28 | GSE110243 | GEO

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