ABSTRACT: Adaptation of the ChIP-on-chip protocol, to calculate genomic transcription rates in S. cerevisiae. Keywords: ChIP-chip Overall design: There are 3 different experimental conditions: -Yeast cell growing exponentially in YPD. -Yeast cell stopped after 2 h of changing them to YPGal. -Yeast cell growing exponentially after 14.5 h of changing them to YPGal. We have used 3 different IP protocols: -total RNA pol II using a Myc tagged RNA pol (RPB1-Myc) -RNA pol II CTD using the Ab 8WG16 (Covance) -RNA pol II CTD Phosphorylated on Ser5 (David Bentley\'s lab) There are 3 independent biological replicates of each experiment.
Project description:Adaptation of the ChIP-on-chip protocol, to calculate genomic transcription rates in S. cerevisiae. Keywords: ChIP-chip There are 3 different experimental conditions: -Yeast cell growing exponentially in YPD. -Yeast cell stopped after 2 h of changing them to YPGal. -Yeast cell growing exponentially after 14.5 h of changing them to YPGal. We have used 3 different IP protocols: -total RNApol II using a Myc tagged RNApol (RPB1-Myc) -RNApol II CTD using the Ab 8WG16 (Covance) -RNApol II CTD Phosphorilated on Ser5 (David Bentley\'s lab) There are 3 independent biological replicates of each experiment.
Project description:RPCC (RNA pol II ChIP-on-chip) experiments with different mutants that affect to the accumulation of non active RNA pol II along the yeast genome. Keywords: ChIP-chip Overall design: There are 4 different strains: rap1-sil (without the silencing domain), RAP1 (both from Graham I.R. et al 1999) and tpk1 & tpk2 mutants (Euroscarf). The IP was done using an Ab against the RNApol II CTD (8WG16 Covance).There are 3 independent biological replicates of Rap1 and rap1-sil experiments and 2 for the tpk1 & tpk2 experiments.
Project description:RPCC (RNA pol II ChIP-on-chip) experiments with different mutants that affect to the accumulation of non active RNA pol II along the yeast genome. Keywords: ChIP-chip There are 4 different strains: rap1-sil (without the silencing domain), RAP1 (both from Graham I.R. et al 1999) and tpk1 & tpk2 mutants (Euroscarf). The IP was done using an Ab against the RNApol II CTD (8WG16 Covance).There are 3 independent biological replicates of Rap1 and rap1-sil experiments and 2 for the tpk1 & tpk2 experiments.
Project description:The carboxy-terminal domain (CTD) of RNA polymerase II (Pol II) consists of heptad repeats with the consensus motif Y1-S2-P3-T4-S5-P6-S7. Dynamic phosphorylation of the CTD coordinates Pol II progression through the transcription cycle. Monoclonal antibodies have been used to study in vivo the potentially phosphorylated CTD amino acids (Y1, S2, T4, S5 and S7). However, the epitopes detected by antibodies can be masked by proteins or modifications at neighbouring sites. Therefore, the effectiveness of antibodies in western blot or ChIP analysis reflects the number of accessible CTD phosphorylation marks, but not the total number of phosphorylations. Most importantly, CTD phospho-specific antibodies do not provide any heptad - (location) specific information of CTD phosphorylation. Due to these limitations, the principles and patterns of CTD phosphorylation remained elusive. Here, we use genetic and mass spectrometric approaches to directly detect and map phosphosites along the entire CTD. We confirm phosphorylation of CTD residues Y1, S2, T4, S5 and S7 in mammalian and yeast cells. Although specific phosphorylation signatures dominate, adjacent CTD repeats can be differently phosphorylated, leading to a high variation of coexisting phosphosites in mono- and di-heptad CTD repeats. Inhibition of CDK9 kinase specifically reduces S2 phosphorylation levels within the CTD.
Project description:To investigate the chromatin transcription cycle, we determined genome-wide occupancy profiles for RNA polymerase (Pol) II, its phosphorylated forms, and transcription factors in growing yeast. ChIP-chip was performed to identify the genomic binding locations for Rpb3, TFIIB, Tfg1, Kin28, Cet1, Spt4, Spt5, Spt6, Elf1, Spn1, Bur1, Ctk1, Paf1, Spt16, Pcf11, and Rpb1 phosphorylated at serine 2, 5, and 7 residues of the CTD, respectively.
Project description:The protein Seb1 from fission yeast contains a conserved CTD-interacting domain (CID) with which it can bind to phosphorylated forms of the Pol II C-terminal domain (CTD) during active transcription. It mainly interacts with Ser2P-CTD but also with Ser5P-CTD. Here, we show the recruitment profile of the protein to chromatin using ChIP-Seq which is mediated mainly via binding to the Pol II-CTD. In addition, it can also interact with nascent RNA via its RNA recognition motif (RRM) domain. Overall design: Two inputs and two immunoprecipitated Seb1-TAP samples were used to perform ChIP-seq. An untagged control (called WT) is also provided.
Project description:ChIP-chip was performed to identify the genomic binding locations for the termination factors Nrd1, and Rtt103, and for RNA polymerase (Pol) II phosphorylated at the tyrosine 1 and threonine 4 position of its C-terminal domain (CTD). In different phases of the transcription cycle, Pol II recruits different factors via its CTD, which consists of heptapeptide repeats with the sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Here we show that the CTD of transcribing yeast Pol II is phosphorylated at Tyr1, and that this impairs recruitment of termination factors. Tyr1 phosphorylation levels rise downstream of the transcription start site (TSS), and decrease before the polyadenylation (pA) site. Tyr1-phosphorylated gene bodies are depleted of CTD-binding termination factors Nrd1, Pcf11, and Rtt103. Tyr1 phosphorylation blocks CTD binding by these termination factors, but stimulates binding of elongation factor Spt6. These results show that CTD modifications can not only stimulate but also block factor recruitment, and lead to an extended CTD code for transcription cycle coordination.
Project description:Using pol II mutants in human cells we found that slow transcription repositioned specific co-transcriptionally deposited chromatin modifications; H3K36me3 shifted within genes toward 5’ ends and H3K4me2 extended further upstream of start sites. Slow transcription also evoked a hyperphosphorylation of CTD Ser2 residues at 5’ ends of genes that is conserved in yeast. We propose a “dwell-time in the target zone” model to explain the effects of transcriptional dynamics on establishment of co-transcriptionally deposited protein modifications. Promoter-proximal Ser2 phosphorylation is associated with longer pol II dwell time at start sites and reduced transcriptional polarity due to strongly enhanced divergent antisense transcription at promoters. Overall design: The effect of transcription elongation rate on histone H3K36me3, H3K4me2 and pol II CTD phosphorylation was analyzed by ChIP-seq in isogenic human HEK293 cell lines that inducibly express a-amanitin resistant mutants of the RNA polymerase II large subunit with slow elongation rates. Anti-pol II total nascent RNA sequencing (tNET-seq) was developed to assay transcription by WT and slow pol II. Slow pol II mutants in S. cerevisiae were also assayed for pol II CTD Ser2 phosphorylation.
Project description:Transcription controls splicing and other gene regulatory processes, yet mechanisms remain obscure due to our fragmented knowledge of the molecular connections between the dynamically phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD) and regulatory factors. By systematically isolating phosphorylation states of the CTD heptapeptide repeat (Y1S2P3T4S5P6S7), we identify hundreds of protein factors that are differentially enriched, revealing unappreciated connections between the Pol II CTD and co-transcriptional processes. These data uncover a novel role for threonine-4 in 3’ end processing through controlling the transition between cleavage and termination. Furthermore, serine-5 phosphorylation seeds spliceosomal assembly immediately downstream of 3’ splice sites through a direct interaction with spliceosomal subcomplex, U1. Strikingly, threonine-4 phosphorylation also impacts splicing through serving as a mark of spliceosomal release and ensuring efficient post-transcriptional splicing genome-wide. Thus, comprehensive Pol II interactomes identify the complex and functional connections between transcription machinery and other gene regulatory complexes. Overall design: NET-seq of WT, rai1, rtt103 and Pol II CTD threonine-4 mutants. Nascent RNA-seq of WT and Pol II CTD threonine-4 mutant. RNA-seq of WT and Pol II CTD threonine-4 mutants. ChIP-nexus analysis of phospho-Ser5 and phospho-Thr4 of the Pol II CTD and ChIP-nexus of splicing factors.
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.