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TOX4 Facilitates Promoter-Proximal Pausing and C-Terminal Domain Dephosphorylation of RNA Polymerase II in Human Cells

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE164277 | GEO | 2022/03/29

REPOSITORIES: GEO

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Distinct Cdk9-phosphatase switches act on elongation factor Spt5 at the beginning and end of the RNA polymerase II transcription cycle

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2020-08-04 | GSE138548 | GEO

Control of RNA pol II speed by PNUTS-PP1 and Spt5 dephosphorylation facilitates termination by a “sitting duck torpedo” mechanism

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2019-10-30 | GSE134198 | GEO

Tox4 is an evolutionarily conserved regulator of elongation and recycling of RNA polymerase II (TTchem-seq)

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

Tox4 is an evolutionarily conserved regulator of elongation and recycling of RNA polymerase II (CUT&Tag)

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Tox4 is an evolutionarily conserved regulator of elongation and recycling of RNA polymerase II (scRNA-seq)

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Tox4 is an evolutionarily conserved regulator of elongation and recycling of RNA polymerase II (ATAC-seq)

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Tox4 is an evolutionarily conserved regulator of elongation and recycling of RNA polymerase II (RNA-seq)

2023-09-08 | GSE190038 | GEO

A Cdk9-PP1 switch regulates the elongation-termination transition of RNA polymerase II

Project description:The end of the RNA polymerase II (Pol II) transcription cycle is strictly regulated to ensure proper mRNA maturation and prevent interference between neighboring genes. Pol II slowing downstream of the cleavage and polyadenylation signal (CPS) leads to recruitment of cleavage and polyadenylation factors and termination, but how this chain of events is initiated remains unclear. In a chemical-genetic screen we identified protein phosphatase 1 (PP1) isoforms as substrates of human positive transcription elongation factor b (P-TEFb), the cyclin-dependent kinase 9 (Cdk9)-cyclin T1 complex. Here we show that Cdk9 and PP1 govern phosphorylation of the conserved transcription factor Spt5 in the fission yeast Schizosaccharomyces pombe. Cdk9 phosphorylates both Spt5 and a negative regulatory site on the PP1 isoform Dis2. Sites phosphorylated by Cdk9 in the Spt5 carboxy-terminal domain (CTD) are dephosphorylated by Dis2 in vitro, and Cdk9 inhibition in vivo leads to rapid Spt5 dephosphorylation that is retarded by concurrent Dis2 inactivation. Chromatin immunoprecipitation and sequencing (ChIP-seq) analysis indicates that Spt5 is dephosphorylated as transcription complexes traverse the CPS, prior to or concomitant with Pol II pausing. A Dis2-inactivating mutation stabilizes Spt5 phosphorylation (pSpt5) on chromatin, promotes transcription beyond the normal termination zone detected by precision run-on transcription and sequencing (PRO-seq), and is suppressed by ablation of Cdk9 target sites in Spt5. These results support a model whereby the transition of Pol II from elongation to termination is regulated by opposing activities of Cdk9 and Dis2 towards their common substrate Spt5—a bistable switch analogous to a Cdk1-PP1 module that controls exit from mitosis.

2018-06-07 | GSE102590 | GEO

Transcriptional elongation requires DNA break-induced signaling

Project description:We have previously shown that RNA polymerase II (Pol II) pause release and transcriptional elongation involve phosphorylation of the factor TRIM28 by the DNA damage response (DDR) kinases ATM and DNA-PK. Here, we report a significant role for DNA breaks and DDR signaling in the mechanisms of transcriptional elongation in stimulus-inducible genes in humans. Our data show the enrichment of TRIM28 and Î³H2AX on serum-induced genes and the important function of DNA-PK for Pol II pause release and transcriptional activation-coupled DDR signaling on these genes. Î³H2AX accumulation decreases when P-TEFb is inhibited, confirming that DDR signaling results from transcriptional elongation. In addition, transcriptional elongation-coupled DDR signaling involves topoisomerase II because inhibiting this enzyme interferes with Pol II pause release and Î³H2AX accumulation. Our findings propose that DDR signaling is required for effective Pol II pause release and transcriptional elongation through a novel mechanism involving TRIM28, DNA-PK, and topoisomerase II 42 samples in total. IP targets were gammaH2ax, s2-pol-II, pol-II, pTRIM28, DNA-pk, topo-IIB. Experimental conditions included DMSO treatment (control), pTEFb, topoII-i, dnapk-i. Matched non-specific IP samples used for control in peak calling.

2015-11-20 | E-GEOD-75170 | biostudies-arrayexpress

RNA polymerase II termination involves CTD tyrosine dephosphorylation by CPF subunit Glc7

Project description:At the 3'-ends of genes, RNA polymerase (Pol) II is dephosphorylated at tyrosine 1 residues of its C-terminal domain (CTD), resulting in recruitment of transcription termination factors. We show that the multisubunit cleavage and polyadenylation factor (CPF) is a Pol II CTD phosphatase and its Glc7 subunit is required for Tyr1 dephosphorylation at the poly-adenylation site and Pol II termination in vivo. ChIP-chip was performed to examine the effect of Glc7 nuclear depletion on genome-wide Pol II occupancy [using ?-Rpb3 (1Y26, cat. no. W0012, neoclone) antibody] and CTD tyrosine 1 phosphorylation levels [using ?-TyrY1P (3D12, D. Eick) antibody].

2013-12-16 | E-MTAB-1528 | biostudies-arrayexpress

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