TOX4 Facilitates Promoter-Proximal Pausing and C-Terminal Domain Dephosphorylation of RNA Polymerase II in Human Cells
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ABSTRACT:
PROVIDER: PRJNA689787 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13360800_1.fastq.gz | Fastqsanger.gz | |||
| SRR13360800_2.fastq.gz | Fastqsanger.gz | |||
| SRR13360801_1.fastq.gz | Fastqsanger.gz | |||
| SRR13360801_2.fastq.gz | Fastqsanger.gz | |||
| SRR13360808_1.fastq.gz | Fastqsanger.gz |
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Project description:TOX4 is one of the regulatory proteins of PP1 phosphatases with poorly understood functions. Here we show that chromatin occupancy pattern of TOX4 resembles that of RNA polymerase II (Pol II), and its loss increases cellular level of C-terminal domain (CTD) phosphorylated Pol II but mainly decreases Pol II occupancy on promoters. In addition, elongation rate analyses by 4sUDRB-seq suggest that TOX4 restricts pause release and early elongation but promotes late elongation. Moreover, TT-seq analyses indicate that TOX4 loss mainly decreases transcriptional output. Mechanistically, TOX4 may restrict pause release through facilitating CTD serine 2 and DSIF dephosphorylation, and promote Pol II recycling and reinitiation through facilitating CTD serines 2 and 5 dephosphorylation. Furthermore, among the PP1 phosphatases, TOX4 preferentially binds PP1α and is capable of facilitating Pol II CTD dephosphorylation in vitro. These results lay the foundation for a better understanding of the role of TOX4 in transcriptional regulation.
2022-03-29 | GSE164277 | GEO
Project description:TOX4 is one of the regulatory factors of PP1 phosphatases with poorly understood functions. Here we show that chromatin occupancy pattern of TOX4 resembles that of RNA polymerase II (Pol II), and its loss increases cellular level of C-terminal domain (CTD) phosphorylated Pol II but mainly decreases Pol II occupancy on promoters. In addition, elongation rate analyses by 4sUDRB-seq suggest that TOX4 restricts pause release and early elongation but promotes late elongation. Moreover, TT-seq analyses indicate that TOX4 loss mainly decreases transcriptional output. Mechanistically, TOX4 may restrict pause release through facilitating CTD serine 2 and DSIF dephosphorylation, and promote Pol II recycling and reinitiation through facilitating CTD serines 2 and 5 dephosphorylation. Furthermore, among the PP1 phosphatases, TOX4 preferentially binds PP1α and is capable of facilitating Pol II CTD dephosphorylation in vitro. These results lay the foundation for a better understanding of the role of TOX4 in transcriptional regulation.
| S-EPMC8975821 | biostudies-literature
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.
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Project description:This SuperSeries is composed of the following subset Series: GSE20529: Promoter proximal pausing and its regulation by c-Myc in embryonic stem cells: ChIP-chip GSE20530: Promoter proximal pausing and its regulation by c-Myc in embryonic stem cells: ChIP-Seq Refer to individual Series
2010-04-14 | E-GEOD-20485 | biostudies-arrayexpress
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.
2015-08-13 | GSE70408 | GEO
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
Project description:RNA polymerase II (pol II) transcribes all protein-coding and many non-coding RNAs in the human genome. Pol II transcription initiation is governed by the Pre-Initiation Complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, pol II, and Mediator. After initiation, pol II enzymes typically pause after transcribing less than 100 bases, and paused polymerases represent a common regulatory intermediate. Accordingly, paused pol II has been implicated in enhancer function, development and homeostasis, and diseases ranging from cancer to viral pathogenesis. Precisely how pol II promoter-proximal pausing is enforced and regulated remains unclear; however, protein complexes such as NELF and DSIF increase pausing whereas the activity of CDK9 (P-TEFb complex) correlates with pause release. To address specific mechanistic questions about pol II pausing and its regulation, we reconstituted human pol II promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pol II pausing in vitro, whereas P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pol II pausing, suggesting that pausing is an inherent property of the PIC. In agreement, pol II pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP); moreover, pausing was dependent upon TFIID subunits TAF1 and TAF2. TAF1/2 bind genomic DNA downstream of the pol II initiation site, invoking a “complex interaction” model for pausing. Consistent with this model, PRO-Seq experiments revealed increased transcription upon acute depletion (t=60 min) of TAF1 and TAF2 in human cells, and pol II pausing was disrupted at thousands of genes. Similar results were obtained in TAF1-depleted Drosophila S2 cells. Collectively, these data establish the general transcription factor TFIID as a genome-wide regulator of pol II promoter-proximal pausing.
2020-05-28 | GSE132764 | GEO
Project description:HMG-box protein Tox4 is a regulator of PP1 phosphatases with unknown function in development. Here we show that although pan-hematopoietic Tox4 knockout produces minimal defects, T cell-specific knockout reduces thymic cellularity by 50% and increases cell death. In addition, single cell RNA-seq discovered that Tox4 loss impairs T cell development starting from double positive (DP) blast stage partially due to downregulation of genes critical for cell cycle progression through G2 and M phases. Moreover, Tox4 loss decreases transcriptional output of thousands of genes in DP cells, and mechanistically, Tox4 restricts transcriptional elongation and facilitates transcriptional reinitiation in a dephosphorylation-dependent manner, which is consistent with what we have found in human cells. Furthermore, small effect of Tox4 loss on chromatin accessibility suggest that it mainly regulates transcription in an HMG box independent manner. These results establish an evolutionarily conserved a role of Tox4 in transcriptional elongation and reinitiation.
2023-09-08 | GSE192698 | GEO
Project description:HMG-box protein Tox4 is a regulator of PP1 phosphatases with unknown function in development. Here we show that although pan-hematopoietic Tox4 knockout produces minimal defects, T cell-specific knockout reduces thymic cellularity by 50% and increases cell death. In addition, single cell RNA-seq discovered that Tox4 loss impairs T cell development starting from double positive (DP) blast stage partially due to downregulation of genes critical for cell cycle progression through G2 and M phases. Moreover, Tox4 loss decreases transcriptional output of thousands of genes in DP cells, and mechanistically, Tox4 restricts transcriptional elongation and facilitates transcriptional reinitiation in a dephosphorylation-dependent manner, which is consistent with what we have found in human cells. Furthermore, small effect of Tox4 loss on chromatin accessibility suggest that it mainly regulates transcription in an HMG box independent manner. These results establish an evolutionarily conserved a role of Tox4 in transcriptional elongation and reinitiation.
2023-09-08 | GSE190040 | GEO
Project description:HMG-box protein Tox4 is a regulator of PP1 phosphatases with unknown function in development. Here we show that although pan-hematopoietic Tox4 knockout produces minimal defects, T cell-specific knockout reduces thymic cellularity by 50% and increases cell death. In addition, single cell RNA-seq discovered that Tox4 loss impairs T cell development starting from double positive (DP) blast stage partially due to downregulation of genes critical for cell cycle progression through G2 and M phases. Moreover, Tox4 loss decreases transcriptional output of thousands of genes in DP cells, and mechanistically, Tox4 restricts transcriptional elongation and facilitates transcriptional reinitiation in a dephosphorylation-dependent manner, which is consistent with what we have found in human cells. Furthermore, small effect of Tox4 loss on chromatin accessibility suggest that it mainly regulates transcription in an HMG box independent manner. These results establish an evolutionarily conserved a role of Tox4 in transcriptional elongation and reinitiation.
2023-09-08 | GSE190039 | GEO