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RNA polymerase II pausing contributes to maintain chromatin organization in erythrocytes [RNA-seq]

ABSTRACT: Chicken erythrocytes are nucleated cells often referred to as transcriptionally inactive, although the epigenetic changes and chromatin remodeling that mediate transcriptional repression and the extent of gene silencing during avian terminal erythroid differentiation are not fully understood. Here we characterized the changes in gene expression, chromatin accessibility, genome organization, and chromatin nuclear disposition during the terminal stages of erythropoiesis in chicken and found a complex chromatin reorganization at different genomic scales. We identified a robust decrease in transcription in erythrocytes. Nevertheless, a set of genes maintains their expression in erythrocytes, including genes involved in RNA pol II promoter-proximal pausing. Erythrocytes exhibit an inverted nuclear architecture and reposition euchromatin towards the nuclear periphery together with the paused RNA polymerase. In erythrocytes, chromatin domains are partially lost genome-wide except at mini domains retained around paused promoters. Our results suggest that promoter-proximal pausing of the RNA pol II participates in the transcriptional regulation of the erythroid genome and highlight the role of RNA polymerase in the maintenance of local chromatin organization.

ORGANISM(S): Gallus gallus

PROVIDER: GSE206193 | GEO | 2023/06/08

REPOSITORIES: GEO

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RNA polymerase II pausing contributes to maintain chromatin organization in erythrocytes [Hi-C]

Project description:Chicken erythrocytes are nucleated cells often referred to as transcriptionally inactive, although the epigenetic changes and chromatin remodeling that mediate transcriptional repression and the extent of gene silencing during avian terminal erythroid differentiation are not fully understood. Here we characterized the changes in gene expression, chromatin accessibility, genome organization, and chromatin nuclear disposition during the terminal stages of erythropoiesis in chicken and found a complex chromatin reorganization at different genomic scales. We identified a robust decrease in transcription in erythrocytes. Nevertheless, a set of genes maintains their expression in erythrocytes, including genes involved in RNA pol II promoter-proximal pausing. Erythrocytes exhibit an inverted nuclear architecture and reposition euchromatin towards the nuclear periphery together with the paused RNA polymerase. In erythrocytes, chromatin domains are partially lost genome-wide except at mini domains retained around paused promoters. Our results suggest that promoter-proximal pausing of the RNA pol II participates in the transcriptional regulation of the erythroid genome and highlight the role of RNA polymerase in the maintenance of local chromatin organization.

2023-06-08 | GSE206192 | GEO

RNA polymerase II pausing contributes to maintain chromatin organization in erythrocytes [ATAC-seq]

2023-06-08 | GSE206191 | GEO

RNA polymerase II pausing contributes to maintain chromatin organization in erythrocytes [ChIP-Seq]

2023-06-08 | GSE228694 | GEO

PAF1, a molecular regulator of promoter-proximal pausing by RNA Polymerase II

Project description:The control of promoter-proximal pausing and the release of RNA polymerase II (RNA Pol II) is a widely used mechanism for regulating gene expression in metazoans, especially for genes that respond to environmental and developmental cues. Here, we identify Pol II associated Factor 1 (PAF1) as a major regulator of promoter-proximal pausing. Knockdown of PAF1 leads to increased release of paused Pol II into gene bodies at thousands of genes. Genes with the highest levels of paused Pol II exhibit the largest redistribution of Pol II from the promoter-proximal region into the gene body in the absence of PAF1. PAF1 depletion results in increased nascent transcription and increased levels of phosphorylation of Pol II’s c-terminal domain on serine 2 (Ser2P). These changes can be explained by the recruitment of the Ser2P kinase Super Elongation Complex (SEC) effecting increased release of paused Pol II into productive elongation, thus establishing a novel function for PAF1 as a major regulator of pausing in metazoans. ChIP-seq of Pol II of different forms, SEC subunits, PAFc subunits and H2Bub in human cell lines targeted by PAF1 or scramble shRNA. ChIP-seq of total Pol II in HCT116 cells targeted by BRE1A or scramble shRNA. ChIP-seq of total Pol II in S2 cells targeted by Paf1 or LacZ RNAi. Total RNA-seq, nascent RNA-seq and GRO-seq in HCT116 cells targeted by PAF1 or scramble shRNA.

2015-08-13 | E-GEOD-70408 | biostudies-arrayexpress

PAF1, a molecular regulator of promoter-proximal pausing by RNA Polymerase II

2015-08-13 | GSE70408 | GEO

RNA Polymerase II Pausing Can Be Retained or Acquired During Activation of Genes Involved in the Epithelial to Mesenchymal Transition

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

RNA Polymerase II-associated factor 1 regulates the release and phosphorylation of paused RNA Polymerase II

Project description:Release of promoter-proximal paused RNA polymerase II (Pol II) during early elongation is a critical step in transcriptional regulation in metazoan cells. Paused Pol II release is thought to require the kinase activity of cyclin-dependent kinase 9 (CDK9) for the phosphorylation of DRB sensitivity-inducing factor, negative elongation factor, and C-terminal domain (CTD) serine-2 of Pol II. We found that Pol II-associated factor 1 (PAF1) is a critical regulator of paused Pol II release, that positive transcription elongation factor b (P-TEFb) directly regulates the initial recruitment of PAF1 complex (PAF1C) to genes, and that the subsequent recruitment of CDK12 is dependent on PAF1C. These findings reveal cooperativity among P-TEFb, PAF1C, and CDK12 in pausing release and Pol II CTD phosphorylation. Comparison of the chromatin occupancy of [1] PAF1, CDC73, LEO1, CTR9, total Pol II, and CTD serine 2-phosphorylated Pol II by ChIP-seq in THP1 cells; [2] PAF1, Pol II, Pol II (ser-5p), CDK12, and CDK9 by ChIP-seq in control and PAF1 knockdown cells; [3] LEO1 and Pol II by ChIP-seq in control and flavopiridol treated THP1 cells.

2015-12-11 | E-GEOD-62171 | biostudies-arrayexpress

GAGA Factor maintains promoters in nucleosome-free conformation and allows promoter-proximal pausing [MNase]

Project description:Promoter-proximal pausing of RNA polymerase II (Pol II) is a widespread in higher eukaryotes. Previous studies have shown that GAF is enriched at paused genes, but the role of GAF in pausing has not been well characterized on a genome-wide level. To investigate the role of GAF in pausing, we RNAi-depleted GAF from Drosophila S2 cells, and examined the effects on promoter-proximal polymerase. We confirmed the importance of GAF for pausing on the classic pause model gene Hsp70. To determine the dependence of pausing on GAF genome-wide, we assayed the levels of transcriptionally-engaged polymerase genome-wide using GRO-seq in control and GAF-RNAi cells. We found that promoter-proximal polymerase was significantly reduced on a subset of paused genes with GAF-bound promoters. There is a dramatic change in nucleosome distribution at genes with reduction in pausing upon GAF depletion and intergenic GAF binding sites in GAF knock-down, suggesting that GAF allows the establishment of pausing at these genes by directing nucleosome displacement off of the promoter. In addition, the insulator factor BEAF, BEAF-interacting protein Chriz, and transcription M1BP enrichment on unaffected genes suggests that redundant transcription factors or insulators protect other GAF-bound paused genes from GAF knock-down effects. Three biological replicates of MNase digested chromatin from LacZ-RNAi and GAGA factor-RNAi cells.

2015-04-04 | E-GEOD-58956 | biostudies-arrayexpress

A Unified View of the Sequence and Functional Organization of the Human RNA Polymerase II Promoter

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

RNA Polymerase II-associated factor 1 regulates the release and phosphorylation of paused RNA Polymerase II

2015-12-11 | GSE62171 | GEO

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