Differential recruitment of Ser2/5 phosphorylated RPB1, a component of the elongating RNAPII, in Plasmoidium falciparum parasite with disrupted PfNot1.1 (PF3D7_1103800).
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ABSTRACT:
PROVIDER: PRJNA596454 | ENA |
REPOSITORIES: ENA
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Project description:The study aims to investigate the role of PfNOT1.1 in transcription in the malaria parasite, P. falciparum. To achieve this goal, the recruitment of RPB1 was assessed by chromatin immunoprecipitatiion and microarray technology The endogenous PfNot1.1 was disrupted. DNA bound by Ser2/5-P RPB1 (ChIP DNA) was immunoprecipitated by Ser2/5-P RPB1 antibody in the PfNot1.1 knockout line and the wildtype line at six timepoints across one parasite's intraerythrocytic life cycle. ChIP DNA and Input genomic DNA were analyzed by microarray.
2021-08-31 | GSE142276 | GEO
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. We examined the genome-wide distribution (using ChIP-chip) of Spt6. Spt6 occupancy was also assayed in mutants for CTD Serine 2 and Serine 5 kinases and in mutants for histone deacetylases. ChIPs were performed with a Myc-tagged version of Spt6. Most ChIPs (in Cy5) were hybridyzed against a control ChIP sample from an isogenic non-tagged strain (in Cy3). In the ChIP experiments with the spt6-202del mutant, non immunoprecipitated DNA (input) was used as the control. In addition to Spt6 ChIPs, the project includes RNAPII (Rpb3) ChIP-chip datasets, where an anti-Rpb3 antibody was used to ChIP RNAPII and non immunoprecipitated DNA (input) was used as the control. All ChIP-chip experiments were done in duplicates. Each microarray was normalized using the Lima Loess and replicates were combined using a weighted average method as previously described (Pokholok et al., 2005).
2014-09-25 | E-GEOD-61713 | biostudies-arrayexpress
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.
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Project description:We performed ChIP-Seq on MV4;11 WT, CDK12AS/NULL, CDK13AS/AS, #1CDK12AS/NULL;CDK13AS/NULL and #2CDK12AS/NULL;CDK13AS/AS MV4;11 clones treated with 1-NM-PP1 or vehicle for 4 hours to determine the location of total Pol II and phosphorylated Ser2 of Pol II occupancy
2020-04-29 | GSE141365 | GEO
Project description:We report calibrated transcriptome of rpb1-CTD-S2A and WT of S. pombe cells. Phosphorylation of the RNA polymerase II (Pol II) C-terminal domain on heptad Y1S2P3T4S5P6S7 coordinates key events during transcription and when its deregulation leads to defects in transcription and RNA processing. Here we report that alanine substitution of all Ser2 in CTD result in increased antisense transcription.
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Project description:The conserved transcription elongation factor Spt6 makes several contacts with the RNA Polymerase II (RNAPII) complex, including a high-affinity interaction between the Spt6 tandem SH2 domain (Spt6-tSH2) and phosphorylated residues of the Rpb1 subunit in a region linking the catalytic core and the heptad repeats at the C-terminal domain (CTD). This interaction contributes to generic localization of Spt6, but we show here that it also has gene-specific roles. Disrupting the interface affected transcription start site selection at a subset of genes whose expression is regulated by this choice, and this was accompanied by changes in the distinct pattern of Spt6 occupancy at these sites. Splicing efficiency was also impacted, as was apparent progression through introns that encode snoRNAs. Expression of genes that are particularly sensitive to efficient restoration of chromatin after transcription were affected, and a distinct role in maintaining +1 nucleosomes was identified, especially at ribosomal protein genes. The Spt6-tSH2:Rpb1 interface therefore has both genome-wide functions and local roles at subsets of genes where dynamic decisions regarding initiation, transcript processing, or termination are made. We propose that the Spt6-tSH2:Rpb1 interaction participates in coordinating appropriate responses to the varying local conditions encountered RNAPII at each gene, perhaps by modulating the elongation rate to allow reconfiguration of accessory factor activity or availability.
2021-12-20 | GSE184954 | GEO
Project description:Transcription is a major contributor to genome instability.A main cause of transcription-associated instability relies on the capacity of transcription to stall replication. Such genome instability is increased in RNAPII mutants. ChIP-chips performed in asynchronous cultures showed an increase of the Rrm3 binding signal all over the genome in rpb1-1 compared to wild-type. ChIP-chip studies were perfomed with antibody against Flag-tagged Rrm3 protein in both wild-type and rpb1-1 cells.
2014-12-04 | E-GEOD-55184 | biostudies-arrayexpress
Project description:The conserved transcription elongation factor Spt6 makes several contacts with the RNA Polymerase II (RNAPII) complex, including a high-affinity interaction between the Spt6 tandem SH2 domain (Spt6-tSH2) and phosphorylated residues of the Rpb1 subunit in the linker between the catalytic core and the C-terminal domain (CTD) heptad repeats. This interaction contributes to generic localization of Spt6, but we show here that it also has gene-specific roles. Disrupting the interface affected transcription start site selection at a subset of genes whose expression is regulated by this choice, and this was accompanied by changes in a distinct pattern of Spt6 accumulation at these sites. Splicing efficiency was also diminished, as was apparent progression through introns that encode snoRNAs. Chromatin-mediated repression was impaired, and a distinct role in maintaining +1 nucleosomes was identified, especially at ribosomal protein genes. The Spt6-tSH2:Rpb1 interface therefore has both genome-wide functions and local roles at subsets of genes where dynamic decisions regarding initiation, transcript processing, or termination are made. We propose that the interaction modulates the availability or activity of the core elongation and histone chaperone functions of Spt6, contributing to coordination between RNAPII and its accessory factors as varying local conditions call for dynamic responses.
2021-12-20 | GSE189831 | GEO
Project description:We investigated whether RNA Pol II mediated transcription was altered in Carm1 KO compared to control KO tumor cells. We examined p-Ser2 CTD Pol II relative to total CTD Pol II using mammalian native elongating transcript sequencing (mNET-Seq) (Nojima et al., 2016). Inactivation of the Carm1 gene substantially increased the normalized read density of p-Ser2 CTD Pol II tags relative total Pol II tags. These data provide evidence for altered transcriptional regulation in Carm1 deficient cells.
2022-01-31 | GSE148905 | GEO
Project description:Transcription is a major contributor to genome instability.A main cause of transcription-associated instability relies on the capacity of transcription to stall replication. Such genome instability is increased in RNAPII mutants. ChIP-chips performed in asynchronous cultures showed an increase of the Rrm3 binding signal all over the genome in rpb1-1 compared to wild-type.
2014-12-04 | GSE55184 | GEO