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Transcription Pausing Regulates Mouse Embryonic Stem Cell Differentiation [GRO-seq]

ABSTRACT: The pluripotency of embryonic stem cells (ESCs) relies on appropriate responsiveness to developmental cues. Promoter-proximal pausing of RNA polymerase II (Pol II) has been suggested to play a role in keeping genes poised for future activation. To identify the role of Pol II pausing in regulating ESC pluripotency, we have generated mouse ESCs carrying a mutation in the pause-inducing factor SPT5. Consistent with previous in vitro studies showing the pausing deficiency of this mutant SPT5, our genomic analysis reveals genome-wide reduction of paused Pol II in mutant mESCs. Furthermore, we find that genes differentially regulated by mutant SPT5 correlates with distinct chromatin states. Functionally, this pausing-deficient SPT5 disrupts ESC differentiation without affecting self-renewal. Thus, our study uncovers an important role of Pol II pausing in regulating ESC differentiation and also suggests that Pol II pausing can both positively and negatively influence transcription depending on the local chromatin environment.

ORGANISM(S): Mus musculus

PROVIDER: GSE99747 | GEO | 2017/12/08

SECONDARY ACCESSION(S): PRJNA389560

REPOSITORIES: GEO

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Transcription Pausing Regulates Mouse Embryonic Stem Cell Differentiation [ATAC-seq]

Project description:The pluripotency of embryonic stem cells (ESCs) relies on appropriate responsiveness to developmental cues. Promoter-proximal pausing of RNA polymerase II (Pol II) has been suggested to play a role in keeping genes poised for future activation. To identify the role of Pol II pausing in regulating ESC pluripotency, we have generated mouse ESCs carrying a mutation in the pause-inducing factor SPT5. Consistent with previous in vitro studies showing the pausing deficiency of this mutant SPT5, our genomic analysis reveals genome-wide reduction of paused Pol II in mutant mESCs. Furthermore, we find that genes differentially regulated by mutant SPT5 correlates with distinct chromatin states. Functionally, this pausing-deficient SPT5 disrupts ESC differentiation without affecting self-renewal. Thus, our study uncovers an important role of Pol II pausing in regulating ESC differentiation and also suggests that Pol II pausing can both positively and negatively influence transcription depending on the local chromatin environment.

2017-12-08 | GSE99746 | GEO

Pausing of RNA polymerase II regulates mammalian developmental potential

Project description:The remarkable capacity for pluripotency and self-renewal in embryonic stem cells (ESCs) requires a finely-tuned transcriptional circuitry wherein the genes and pathways that initiate differentiation are suppressed, but poised to respond rapidly to developmental signals. To elucidate transcriptional control in mouse ESCs in the naïve, ground state, we defined the distribution of engaged RNA polymerase II (Pol II) at high-resolution. We find that promoter-proximal pausing of Pol II is widespread at genes encoding signaling molecules that control ESC proliferation, differentiation and metabolism, but is not enriched at developmental control genes. Accordingly, ablation of the primary pause-inducing factor, NELF, causes proliferation defects, early embryonic lethality and dysregulation of critical ESC signaling pathways. Moreover, loss of NELF in ESCs causes resistance to cell fate commitment through dramatic attenuation of FGF/ERK activity. This work thus uncovers an unanticipated role for NELF-mediated pausing in establishing the responsiveness of ESCs to developmental cues.

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The Polycomb group mutant esc leads to augmented the levels of paused Pol II in the Drosophila embryo.

Project description:The role of Polycomb group (PcG) was studied on RNA Polymerase II (Pol II) pausing phenomenon. Wild type and Polycomb mutant (esc) embryos were used for ChIP-Seq experiments using Pol II and histone methylation antibodies. Enhanced Pol II occupancy was observed for thousands of genes in esc mutant embryos, including genes not known to be bound by PRC2 or having H3K27me3 associated. Most of these genes exhibit a concomitant reduction in H3K27me3 and increase in H3K4me3 at promoter-proximal regions. Silent genes lacking promoter-associated paused Pol II in wild-type embryos are converted into âpoisedâ genes with paused Pol II in esc mutants. We suggest that this conversion of silent genes into poised genes might render differentiated cell types susceptible to switches in identity in PcG mutants. ChIP-Seq data was generated for antibodies against Pol-II, H3K4me3, H3K27me3 in both wild type and esc mutant Drosophila embryos (0-2hr, 8-12hr, and 18-24hr embryos). In addition GRO-Seq data was generated for 18-24hr esc mutant embryos.

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Genome wide localization of Activation induced cytidine deaminase (AID), Spt5, and RNA Polymerase II (PolII) in mouse

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Regulation of RNA polymerase II processivity by Spt5 is restricted to a narrow window in elongation

Project description:Spt5 is a highly conserved RNA polymerase II (Pol II)-associated pausing and elongation factor. However, its impact on global elongation and Pol II processivity in mammalian cells has not been clarified. Here, we show that depleting Spt5 in mouse embryonic fibroblasts (MEFs) does not cause global elongation defects or decreased elongation rates. Instead, in the absence of Spt5, a fraction of Pol II molecules are dislodged during elongation thus decreasing the number of Pol II complexes that complete the transcription cycle. Most strikingly, this decrease is restricted to a narrow window between 15-20 kb from the promoter, a distance which coincides with the stage where accelerating Pol II attains maximum elongation speed and processivity. Consequently, long genes show a greater dependency on Spt5 for optimal elongation efficiency and overall gene expression than short genes. We propose that an important role of Spt5 in mammalian elongation is to promote the processivity of those Pol II complexes that are transitioning towards maximum elongation speed 15-20 kb from the promoter.

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RNA Polymerase II pausing temporally coordinates cell cycle progression and erythroid differentiation [RNA-seq]

Project description:The controlled release of promoter-proximal RNA polymerase II (Pol II) pausing into productive elongation is a major step in gene regulation. Yet, functionally analyzing Pol II pausing is difficult because the factors that regulate pause release are generally essential. Here, we identified heterozygous loss-of-function mutations in SUPT5H, encoding SPT5, in individuals with β-thalassemia trait unlinked to the β-globin locus. Recapitulating the pathogenic mutations in human erythroid cells or acute treatment with transcription inhibitors replicates the patient phenotype of altered globin gene expression. Furthermore, in SUPT5H-edited cells, Pol II pause release was globally disrupted. We observed dynamic patterns of pausing at cell cycle and erythroid differentiation genes at the transition from progenitors to precursors during erythropoiesis. At this same transition, in SUPT5H-edited cells, we observed delayed differentiation and altered cell cycle kinetics, as well as a delay in the onset of gene expression programs. Therefore, hindering pause release perturbs cell cycle and delays differentiation at key stages of erythropoiesis, revealing a role for Pol II pausing in the temporal coordination between the cell cycle and differentiation.

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