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Distinctive Interactomes of RNA polymerase II phosphorylation during different stages of transcription

ABSTRACT: We have discovered distinctive protein interactomes for Ser2 and Ser5 phosphorylation of the C-terminal domain (CTD) of RNA Pol II. Calcium Homeostasis Endoplasmic reticulum (CHERP) was identified in our pulldown to directly interact with Ser2 phosphomarks on the CTD of Pol II. As Pol II is a chromatin-associated protein, we sought to identify genomic regions that CHERP, a Pol II binding protein, occupy through ChIP-seq analysis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE226908 | GEO | 2023/08/17

REPOSITORIES: GEO

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Distinctive Interactomes of RNA polymerase II phosphorylation during different stages of transcription

Project description:During eukaryotic transcription, RNA polymerase II undergoes dynamic post-translational modification on the C-terminal domain (CTD) of the largest subunit , generating a sophisticated PTM landscape for the spatiotemporal recruitment to transcriptional regulators. To delineate the protein interactomes recruited to Pol II at different stages of transcription, we in vitro reconstructed phosphorylation patterns of the CTD at Ser5 and Ser2 positions, the hallmark phosphorylation at the initation and productive elongation stages of transcription, respectively. Distinctive protein interactomes indicates different proteins are recruited to RNA polymerase II at different stages of transcription by the phosphorylation of Ser2 and Ser5 of the CTD heptads. Calcium Homeostasis Endoplasmic Reticulum Protein (CHERP) specifically binds to the Ser2 of the heptad. The loss of the interaction between CHERP and Pol II results in broad alternative splicing events. Our method points to a new method to distinguish the PTM codes that coordinate the transcription process.

2023-02-06 | MSV000091213 | MassIVE

Distinctive Interactomes of RNA polymerase II phosphorylation during different stages of transcription

Project description:To investigate the whole-transcriptome effects of Calcium Homeostasis Endoplasmic Reticulum Protein (CHERP)/SCAF6 which specifically binds to the Ser2 of C-terminal domain of RNA Polymerase II, we established HEK293 cell line in which SCAF6 gene was knocked down by shRNA. We then performed gene expression and alternative splicing profiling using data obtained from RNA-seq of 2 cell lines (HEK293 Control vs HEK293 SCAF6 KD).

2023-08-17 | GSE221328 | GEO

CDK12 globally stimulates RNA polymerase II transcription elongation and carboxyl-terminal domain phosphorylation

Project description:Cyclin-dependent kinase 12 (CDK12) phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) but its roles in transcription beyond the expression of DNA damage response genes remain unclear. Here, we have used TT-seq and mNET-seq to monitor the direct effects of rapid CDK12 inhibition on transcription activity and CTD phosphorylation in human cells. CDK12 inhibition causes a genome-wide defect in transcription elongation and a global reduction of CTD Ser2 and Ser5 phosphorylation. The elongation defect is explained by the loss of the elongation factors LEO1 and CDC73, part of PAF1 complex, and SPT6 from the newly-elongating pol II. Our results indicate that CDK12 is a general activator of pol II transcription elongation and indicate that it targets both Ser2 and Ser5 residues of the pol II CTD.

2020-06-18 | GSE133156 | GEO

CDK12 globally stimulates RNA polymerase II transcription elongation and carboxyl-terminal domain phosphorylation

2020-06-03 | GSE115290 | GEO

Budding yeast RNA polymerase II CTD phosphorylation and transcription termination factor localization

2010-09-15 | GSE23960 | GEO

HDACs and Phosphorylated Pol II CTD recruit Spt6 for cotranscriptional histone reassembly

Project description:Spt6 is a multifunctional histone chaperone involved in the maintenance of chromatin structure during elongation by RNA polymerase II (Pol II). Spt6 has a tandem SH2 (tSH2) domain within its C-terminus that recognizes Pol II CTD peptides phosphorylated on Ser2, Ser5 or Try1 in vitro. Deleting the tSH2 domain, however, only has a partial effect on Spt6 occupancy in vivo, suggesting that more complex mechanisms are involved in the Spt6 recruitment. Our results show that the Ser2 kinases Bur1 and Ctk1, but not the Ser5 kinase Kin28, cooperate in recruiting Spt6, genome-wide. Interestingly, the Ser2 kinases promote the association of Spt6 in early transcribed regions and not toward the 3' end of genes, where phosphorylated Ser2 reaches its maximum level. Additionally, our results uncover an unexpected role for histone deacetylases (Rpd3 and Hos2) in promoting Spt6 interaction with elongating Pol II. Finally, our data suggest that phosphorylation of the Pol II CTD on Tyr1 promotes the association of Spt6 with the 3' end of transcribed genes, independently of Ser2 phosphorylation. Collectively, our results show that a complex network of interactions, involving the Spt6 tSH2 domain, CTD phosphorylation and histone deacetylases, coordinate the recruitment of Spt6 to transcribed genes in vivo.

2014-09-25 | GSE61713 | GEO

Threonine-4 of Mammalian RNA Polymerase II CTD is Targeted by Polo-like Kinase-3 and Required for Transcriptional Elongation

Project description:Eukaryotic RNA polymerase II (Pol II) has evolved an array of heptad repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 at the carboxy-terminal domain (CTD) of the large subunit (Rpb1). Differential phosphorylation of Ser2, Ser5, and Ser7 in the 5’ and 3’ regions of genes coordinates the binding of transcription and RNA processing factors to the initiating and elongating polymerase complexes. Here, we report phosphorylation of Thr4 by Polo-like-kinase-3 in mammalian cells. ChIPseq analyses indicate an increase of Thr4-P levels in the 3’ region of genes occurring subsequently to an increase of Ser2-P levels. A Thr4/Ala mutant of Pol II displays a lethal phenotype. This mutant reveals a global defect in RNA elongation, while initiation is largely unaffected. Since Thr4 replacement mutants are viable in yeast we conclude that this amino acid has evolved an essential function(s) in the CTD of Pol II for gene transcription in mammalian cells.

2012-06-04 | GSE37519 | GEO

ChIP-chip in RNA Pol II mutants

Project description:The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) orchestrates dynamic recruitment of specific cellular machines during different stages of transcription. Signature phosphorylation patterns of Y1S2P3T4S5P6S7 heptapeptide repeats of the CTD engage specific “readers.” While phospho-Ser5 and phospho-Ser2 marks are ubiquitous, phospho-Thr4 is reported to only impact specific genes. Here, we investigate the genome-wide occupancy of Pol II, phospho-Thr4, and key reader Rtt103 in WT and CTD-mutant strains of S. cerevisiae.

2017-05-02 | GSE95418 | GEO

Budding yeast RNA polymerase II CTD phosphorylation and transcription termination factor localization

Project description:Whole genome analysis of total RNA pol II, Ser2-, Ser5- and Ser7-phosphorylated RNA pol II, in WT and mutants of the C-terminal domain (CTD) kinases Ctk1 and Kin28, and localization of the termination factors Pcf11, Nrd1 and Rat1. ChIP-chip using ligation-mediated PCR-amplified material hybridized to NimbleGen 385K arrays (50mers, median probe spacing 32 bp, cat. No. C4214-00-01).

2010-09-15 | E-GEOD-23960 | biostudies-arrayexpress

Role of RNA polymerase II "pausing" in Drosophila development

Project description:According to previous studies, during Drosophila embryogenesis, RNA polymerase II is recruited to promoters at developmental stages preceding the stages of active transcription of genes. This work is aimed at exploring whether this mechanism is used during Drosophila metamorphosis. We performed ChIP-Seq analysis using antibodies to various modifications of RNA polymerase II (total, Pol II CTD Ser5P and Pol II CTD Ser2P), as well as to subunits of NELF, DSIF, PAF complexes and Brd4/Fs(1)h that control transcription elongation. We found that like in mid-embryogenesis during metamorphosis, promoters bind RNA polymerase II in the "paused" state preparing for activation at later stages of development. During mid-embryogenesis, RNA polymerase II in "pause" is phosphorylated at Ser5 and Ser2 of Rpb1 CTD and binds NELF, DSIF, and PAF complexes, but not Brd4/Fs(1)h. During metamorphosis, the "paused" RNA polymerase II complex includes Brd4/Fs(1)h in addition to NELF, DSIF, and PAF. The RNA polymerase II in this complex is phosphorylated at Ser5 at Rpb1 CTD, but not at Ser2.

2022-09-10 | GSE210971 | GEO

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