Genome-wide RNA Pol II-CTD occupancy in wild type and mutant worms
Ontology highlight
ABSTRACT:
ORGANISM(S): Caenorhabditis elegans
SUBMITTER: Xiang-Dong Fu
PROVIDER: E-GEOD-68203 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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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
Project description:At Schizosaccharomyces pombe centromeres, heterochromatin formation is required for de novo incorporation of the histone H3 variant CENP-A/Cnp1, which in turn directs kinetochore assembly and ultimately chromosome segregation during mitosis. Noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (Pol II) directs heterochromatin formation via the RNAi machinery, but also through RNAiindependent RNA processing factors. Control of centromeric ncRNA transcription is therefore a key factor for proper centromere function. We here use transcriptional profiling, gene inactivation experiments, and chromatin immunoprecipitation analyses to demonstrate that the Mediator complex directs ncRNA transcription and regulates centromeric heterochromatin formation in fission yeast. Mediator co-localizes with Pol II at centromeres and loss of the Mediator subunit Med20 causes a dramatic increase in pericentromeric transcription and desilencing of the core centromere. As a consequence, heterochromatin formation is impaired both via the RNAi dependent and independent pathways, resulting in loss of CENP-A/Cnp1 from the core centromere, defect kinetochore function, and a severe chromosome segregation defect. Interestingly, the increased centromeric transcription observed in med20Δ appears to directly block CENP-A/Cnp1 incorporation and inhibition of Pol II transcription can suppress the observed phenotypes. Our data thus identify Mediator as a crucial regulator of ncRNA transcription at fission yeast centromeres and add another crucial layer of regulation to centromere function. 3 samples examined: wild type chromatin incubated with beads as the non antibody control, wild type chromatin incubated with RNA Polymerase II CTD domain antibody and Protein G beads, and TAP-Med7 cells chromatin incubated with IgG beads.
2012-11-06 | E-GEOD-35524 | biostudies-arrayexpress
Project description:The heptarepeats of the C-terminal domain of Pol II are extensively modified throughout the transcription cycle. The CTD coordinates RNA synthesis and processing by recruiting transcription regulation factors as well as RNA capping, splicing and 3’end processing factors. The SPOC domain of PHF3 was recently identified as a new CTD reader domain specifically binding to phosphorylated Serine-2 residues in adjacent CTD repeats. Here, we establish the SPOC domains of the human proteins DIDO, SHARP and RBM15 as phosphoserine binding modules that can act as CTD readers but also recognize other phosphorylated binding partners. We report the crystal structure of SHARP (SPEN) SPOC-CTD and identify the molecular determinants for its specific binding to phosphorylated Serine-5. PHF3 and DIDO SPOC domains preferentially interact with the Pol II elongation complex, while RBM15 and SHARP SPOC domains engage with the m6A writer and reader proteins. Our findings establish the SPOC domain as a major interface between the transcription machinery and regulators of transcription and co-transcriptional processes. Here we include ChIP seq data from SHARP and PHF3 with and without the SPOC domain.
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Project description:RNA Polymerase II transcribes protein-coding and many non-coding RNA genes in eukaryotes. The largest subunit of RNA Polymerase II, Rpb1, contains a hepta-peptide repeat on its C-terminal tail with three potential phosphorylation sites (Serine 2, Serine 5 and Serine 7). Mammalian Rpb1 contains 52 repeats. The phosphorylation events are catalyzed by specific protein kinases where the phosphorylation of specific residues is coupled to the transcription cycle. For example, the Cdk7 subunit of TFIIH phosphorylates both Serine 5 and Serine 7 during intiation and the Cdk9 subunit of P-TEFb phosphorylates Serine 2 during the transition into productive elongation. The dataset presented here is the genome-wide distribution of RNA Pol II with Serine 7 of the CTD phosphorylated in murine embryonic stem cells. This data, in addition to phospho-specific datasets generated in the same cell type in Rahl et al. Cell 2010 and Seila et al. Science 2008, represents the genome-wide distribution of multiple RNA Pol II isoforms in murine embryonic stem cells: total Pol II, hypophosphorylated CTD Pol II, Serine 2 phosphorylated CTD Pol II, Serine 5 phosphorylated CTD Pol II and Serine 7 phosphorylated CTD Pol II. An antibody specific to RNA Pol II Serine 7 phosphorylated CTD (gift of Dirk Eick; Chapman et al. Science 2008) was used to enrich for DNA fragments associated with this Pol II isoform in murine embryonic stem cells. DNA was purified and prepared for Illumina/Solexa sequencing following their standard protocol. This is a single dataset but together with datasets from Rahl et al. Cell 2010 and Seila et al. Science 2008, these datasets represent the genome-wide distribution of multiple RNA Pol II isoforms in murine embryonic stem cells: total Pol II, hypophosphorylated CTD Pol II, Serine 2 phosphorylated CTD Pol II, Serine 5 phosphorylated CTD Pol II and Serine 7 phosphorylated CTD Pol II.
2010-05-20 | E-GEOD-21917 | biostudies-arrayexpress
Project description:The rate of RNA polymerase II (pol II) elongation can influence splice site selection in nascent transcripts, yet the extent and physiological relevance of this kinetic coupling between transcription and alternative splicing is not well understood. We performed experiments to perturb pol II elongation and then globally compared alternative splicing patterns with genome-wide pol II occupancy. RNA binding and RNA processing functions were significantly enriched among the genes with pol II elongation inhibition-dependent changes in alternative splicing. Under conditions that interfere with pol II elongation, including cell stress, increased pol II occupancy was detected in the intronic regions flanking the alternative exons in these genes, and these exons generally became more included. A disproportionately high fraction of these exons introduced premature termination codons that elicited nonsense-mediated mRNA decay (NMD), thereby further reducing transcript levels. Our results provide evidence that kinetic coupling between transcription, alternative splicing and NMD affords a rapid mechanism by which cells can respond to changes in growth conditions, including cell stress, to coordinate the levels of RNA processing factors with mRNA levels. To monitor pol II distributions, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) was performed using an anti-pol II antibody (4H8) and cross-linked chromatin preparations from Jurkat cells, treated with or without pol II elongation inhibitor 5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole (DRB) at 10 and 25 ug/ml respectively prior to phorbol 12-myristate 13-acetate (PMA) stimulation, for 5000+ alternative splicing events.
2010-12-09 | E-GEOD-25494 | biostudies-arrayexpress
Project description:We report genome-wide detection of long noncoding RNA (lncRNA) generate by Pol V in transcriptional gene silencing in Arabidopsis thaliana. We further show that most of these transcripts are bound by AGO4. RNA immunoprecipitation followed by high-throughput sequencing (RIP-seq) RNA immunoprecipitation with an anti-AGO4 antibody was performed in one biological replicate and included Col-0, nrpe1, and ago4-1; RNA immunoprecipitation with an anti-NRPE1 (largest subunit of Pol V) was performed in two replicates; replicate 1 included Col-0 and nrpe1, replicated two included Col-0, nrpe1, ago4-1, and idn2-1
2016-10-24 | E-GEOD-70290 | biostudies-arrayexpress
Project description:We have generated single-nucleotide resolution, nascent transcription profiles from HeLa cells by developing Native Elongation Transcript sequencing technology for mammalian chromatin (mNET-seq). Our extensive data sets provide a substantial resource to study mammalian nascent transcript profiles. We reveal unanticipated phosphorylation states for RNA polymerase II C-terminal domain (Pol II CTD) at both gene ends. We also observe that following 5’ splice site cleavage by the spliceosome, upstream exon transcripts are tethered to Pol II CTD phosphorylated on the serine 5 position (S5P) which is accumulated over downstream exons. We further show that depletion of termination factors substantially reduces Pol II pausing at gene ends leading to termination defects. Remarkably termination factors play an additional promoter role by restricting non-productive RNA synthesis and redistributing Pol II CTD S2P to promoters. These data demonstrate that CTD phosphorylation is more dynamic and variably distributed across mammalian transcription units than previously envisaged. To monitor nascent RNA within the mammalian Pol II complex, and its association with different CTD phosphorylation states, we employed mNET-seq methodology on HeLa cells, complemented with direct sequencing of chromatin-bound RNA (ChrRNA-seq). mNET-seq was preformed using the antibodies 8WG16, CMA602, CMA603 and CMA601, which are specific for unphosphorylated CTD, Ser2 phosphorylation, Ser5 phosphorylation and all CTD isoforms, respectively. In another experiment, to evaluate the effect of transcription termination factors in nascent RNA production by Pol II, mNET-seq and complemented with ChrRNA-seq was preformed on HeLa cells transfected with siRNA against PTBP1, CPSF73, CstF64+CstF64tau or Xrn2, and the gene profiles were compared with profiles from HeLa transfected with siRNA for Luciferase generated by the same protocol.
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Project description:RNA polymerase II (Pol II) was immunoprecipitated from Arabidopsis thaliana seedlings, using antibodies that specifically recognize: 1) C’ terminal Domain (CTD), 2) CTD Serine 5 Phosphorylation and 3) CTD Serine 2 Phosphorylation. Proteins that co-immunoprecipitate with different Pol II pools were analysed.
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