Ontology highlight
ABSTRACT:
PROVIDER: PRJNA507630 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8263710_1.fastq.gz | Fastqsanger.gz | |||
| SRR8263710_2.fastq.gz | Fastqsanger.gz | |||
| SRR8263711_1.fastq.gz | Fastqsanger.gz | |||
| SRR8263711_2.fastq.gz | Fastqsanger.gz | |||
| SRR8263706_1.fastq.gz | Fastqsanger.gz |
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Project description:Promoter-proximal RNA polymerase II (Pol II) pausing is implicated in the regulation of gene transcription. However, the mechanisms of pausing including its dynamics during transcriptional responses remain to be fully understood. We performed global analysis of short capped RNAs and Pol II Chromatin Immunoprecipitation sequencing in MCF-7 breast cancer cells to map Pol II pausing across the genome, and used permanganate footprinting to specifically follow pausing during transcriptional activation of several genes involved in the Epithelial to Mesenchymal Transition (EMT). We find that the gene for EMT master regulator Snail (SNAI1), but not Slug (SNAI2), shows evidence of Pol II pausing before activation. Transcriptional activation of the paused SNAI1 gene is accompanied by a further increase in Pol II pausing signal whereas activation of non-paused SNAI2 gene results in the acquisition of a typical pausing signature. The increase in pausing signal reflects increased transcription initiation without changes in Pol II pausing. Activation of the heat shock HSP70 gene involves pausing release that speeds up Pol II turnover, but does not change pausing location. We suggest that Pol II pausing is retained during transcriptional activation and can further undergo regulated release in a signal-specific manner. Untreated MCF-7 cells were analyzed for the distribution of Pol II using ChIP-sequencing with Anti-Pol II N-20 antibody (two independent biological replicates, A, B), and for the distribution of paused RNA polymerase II by sequencing of short capped RNAs (scRNAs) prepared from nuclei (three independent biological replicates, 1-3). All samples were sequenced on a MiSeq instrument in paired-end format
2015-03-27 | E-GEOD-67041 | biostudies-arrayexpress
Project description:Many mammalian genes are occupied by paused RNA polymerase II (pol II) at promoter-proximal regions on both sides of transcription start sites (TSSs). However, the consequences of pol II pausing on gene expression and cell biology are not fully understood. Here we report that genetic ablation of the b subunit of mouse negative elongation factor (Nelf-b), a key pol II-pausing factor, results in slower progression at multiple cell cycle stages and increased apoptosis. Consistently, a whole-genome analysis indicates that growth and cell death-related genes are highly enriched among the direct target genes of Nelf-b. In particular, Nelf-b deletion increases pol II density in the promoter-distal region of stress response genes and their overall expression levels in the absence of any external stress signals. In addition, our work also reveals a previously unappreciated role of Nelf-b role in curbing TSS-upstream transcription of many mammalian genes. We suggest that Nelf-mediated pol II pausing balances the cellular needs for growth/survival and stress response by preventing excessive basal transcription of stress-induced genes. Examination of Nelf-b bindings sites on chromosomes in mouse embryonic fibroblasts
2011-08-17 | E-GEOD-24113 | biostudies-arrayexpress
Project description:Transcription regulation occurs frequently through promoter-associated pausing of RNA polymerase II (Pol II). We developed a Precision nuclear Run-On and sequencing assay (PRO-seq) to map the genome-wide distribution of transcriptionally-engaged Pol II at base-pair resolution. Pol II accumulates immediately downstream of promoters, at intron-exon junctions that are efficiently used for splicing, and over 3' poly-adenylation sites. Focused analyses of promoters reveal that pausing is not fixed relative to initiation sites nor is it specified directly by the position of a particular core promoter element or the first nucleosome. Core promoter elements function beyond initiation, and when optimally positioned they act collectively to dictate the position and strength of pausing . We test this ‘Complex Interaction’ model with insertional mutagenesis of the Drosophila Hsp70 core promoter. Identification of RNA polymerase active sites in Drosophila S2 cell line using PRO-seq method. Identification of transcription initiation sites in Drosophila S2 cell line using PRO-cap method. Identification of changes in RNA polymerase active sites on transgenic Hsp70 promoters upon disruption of DNA sequence elements in 3 transgenic fly lines using PRO-seq method.
2013-02-01 | E-GEOD-42117 | biostudies-arrayexpress
Project description:Activation induced cytidine deaminase (AID) initiates antibody gene diversification by creating U:G mismatches. However, AID is not specific for antibody genes. Off-target lesions can activate oncogenes or cause chromosome translocations. Despite its importance in these key transactions little is known about how AID finds its target sites. To examine AID regulation we performed an shRNA screen. We found that Spt5, a factor associated with paused RNA polymerase II (Pol II) and single stranded DNA (ssDNA), is required for class switch recombination (CSR). Spt5 interacts with AID, it is required for the association of AID and Pol II, and for AID recruitment to switch regions. ChIP-seq experiments reveal that Spt5 co-localizes with AID and stalled Pol II. Further, Spt5 accumulation at sites of Pol II pausing is predictive of AID-induced mutation. Our data suggest that Spt5 acts as an adaptor, which brings AID to Pol II on target DNA. PolII, AID, and Spt5 were immunoprecipitated from in vitro activated B cells. For Spt5 and AID, two different antibodies were used in separate, biological replicates
2010-10-05 | E-GEOD-24178 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE31485: CTCF promotes RNA pol II pausing and links DNA methylation to alternative splicing [ChIP-Seq] GSE31486: CTCF promotes RNA pol II pausing and links DNA methylation to alternative splicing [RNA-Seq] Refer to individual Series
2011-10-05 | E-GEOD-31278 | biostudies-arrayexpress
Project description:DNA faces continuous threats from various endogenous and exogenous stresses. When damaged, DNA requires repair to enable proper cell functioning. Cells have evolved multiple repair pathways to maintain genome stability. Transcription mediated by RNA polymerase II (RNA Pol II), which is conventionally linked with gene expression, plays an integral role in DNA repair. Transcription occurring at sites of DNA damage facilitates repair by generating functional transcripts, recruiting DNA repair factors, or modulating chromatin architecture. Conversely, the shutdown of transcription in nearby genes is crucial to ensure effective DNA repair. UV-induced DNA lesions have been shown to lead to a genome-wide suppression of transcription through RNA Pol II ubiquitylation and subsequent degradation. However, the global transcriptional response to double-strand breaks remains unclear.In this study, we employed mNET-Seq analysis to examine nascent transcripts bound to the CTD Y1P, S2P and S5P of RNA Pol II. Our data reveal a global transcriptional shutdown, as evidenced by a greater number of downregulated protein coding genes in response to irradiation. Interestingly, we observed a significant increase in Y1P antisense transcripts near promoters, known as PROMPTs, which are associated with the downregulated protein coding genes upon irradiation. Furthermore, an increased promoter pausing index correlated with elevated expression of PROMPTs in response to double strand breaks induction. Motif enrichment analysis uncovered binding sites for the Polycomb repressive complex 2 (PRC2) in upregulated PROMPTs. Using proximity ligation assays, we demonstrated an increased proximity between Y1P RNA Pol II and PRC2 upon irradiation. Finally, we also report that inhibition of Y1P or depletion of PRC2 results in the loss of transcriptional repression of protein coding genes, associated with PROMPTs.
2024-02-13 | GSE244353 | GEO
Project description:During transcription, DNA supercoiling generated by the advance of RNA polymerase II (Pol II) is resolved by DNA topoisomerases, enzymes that bind chromatin and produce transient breaks to relax DNA. Recently, this idea of mere facilitators of transcription progression is changing, as topoisomerases are being assigned new functions in regulating the expression of specific genes. In fact, mammalian type II topoisomerases, both the [Symbol] (TOP2A) and [Symbol] (TOP2B) paralogs, are enriched at promoter regions, where they have been proposed to trigger transcription through the generation of DNA double-strand breaks (DSBs). However, this is difficult to reconcile with the intrinsic catalytic properties of TOP2 and the high risk of genome instability that continuous production and repair of DSBs implies. Here, we show that TOP2A enforces promoter-proximal pausing of Pol II by removing transcription-associated negative DNA supercoiling. Interestingly, this topological balance and its disruption is essential for the transcriptional control of Immediate Early Genes (IEGs) and their typical bursting behaviour in response to stimulus. We therefore uncover a novel layer of transcriptional regulation that relies on canonical functions of TOP2A that are independent of aberrant DSB formation, providing a topological framework for the control of promoter-proximal pausing and the tight regulation of IEGs.
2021-04-14 | GSE141798 | GEO
Project description:To expand our understanding of the interplay of human RNA polymerase II (Pol II) promoter elements, promoter-proximal pausing, local chromatin structure, and co-transcriptional RNA processing, the 5′ and 3′ ends of nascent, capped transcripts and the locations of nearby nucleosomes were accurately identified. High-quality visualization tools revealed sequence elements defining the core promoter, sites of pausing, and nucleosome positioning across over 177,000 transcription start regions (TSRs). We found that cap methylation begins as transcripts attain a length of about 30 nt. A nuclear run-off assay was developed that utilizes the unique properties of the DNA fragmentation factor (DFF) to demonstrate the relative location of nucleosomes and paused Pol II. Our data support the conclusion that human Pol II promoters are functional TFIID binding sites with built-in downstream information directing ubiquitous promoter proximal pausing and the downstream nucleosome location.
2020-04-29 | GSE139237 | GEO
Project description:Work from our mouse models showed that COBRA1, the B subunit of NELF, could facilitate R-loop (DNA:RNA hybrid) accumulation in the BRCA1-deficient mouse mammary epithelium. Since NELF could promoter RNA PolII pausing at promoter proximal regions, we asked whether COBRA1-mediated RNA Pol II pausing could contribute to accumulated R-loops. To answer this question, we performed chromatin immunoprecipitation-sequencing (ChIP-seq) for RNA Pol II, as well as two NELF subunits, NELF-A and NELF-B (COBRA1) in mouse primary mammary epithelium cells. To locate R-loops in the same cell population, we performed DNA:RNA immunoprecipitation-sequencing (DRIP-seq) with or without treatment of RNase H, a nuclease that specifically degrades RNA in the R-loop structure. Our sequencing results showed that RNA Pol II, NELF and R-loops are all enriched at transcription start sites (TSSs). Notably, genes with TSS R-loop accumulation are significantly enriched for COBRA1 binding. Taken together, the genomic data lent additional support to the notion that COBRA1-mediated RNAPII pausing contributes to R-loop dynamics in mammary epithelium.
2021-03-30 | GSE67702 | GEO
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