CDK7/TFIIH regulates SETD1A/B activity
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Dylan J Taatjes William M. Old
PROVIDER: MSV000081069 | MassIVE | Wed May 10 07:39:00 BST 2017
SECONDARY ACCESSION(S): PXD006485
REPOSITORIES: MassIVE
Similar Datasets
Project description:We previously identified PCIF1 (Phosphorylated CTD Interacting Factor 1) as a novel phosphorylated C-terminal domain (CTD) of RNA polymerase II. We also recently identified PCIF1 as a new cap-specific adenine N6 methyltransferase (CAPAM) responsible for creating N6, 2’-O-dimethyladenosine (m6Am) at the 5’-end of mRNAs. However, it remains unclear how PCIF1 regulates gene expression. To identify genes whose expression levels are affected by PCIF1 knockdown in human cultured cells, we performed gene expression profiling by microarray analysis.
2023-09-30 | GSE156768 | GEO
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.
2023-02-22 | E-MTAB-11506 | biostudies-arrayexpress
Project description:The RNA polymerase II (RNApII) C-terminal domain (CTD)-interacting domain (CID) proteins are involved in two distinct termination pathways and recognize different phosphorylated forms of CTD. To investigate the role of differential CTDM-^VCID interactions in the choice of termination pathway, we altered the CTD-binding specificity of Nrd1 by domain swapping. ChIP-chip was performed to examine the effect of Nrd1 CID swapping on genome-wide RNA polymerase II (Rpb3 antibody, Neoclone) occupancy. Nrd1 with the CID from Rtt103 (Nrd1[CID-Rtt103]; strain YSB2445) causes read-through transcription at many genes, but can trigger termination where multiple Nrd1/Nab3-binding sites and serine 2 phosphorylated CTD co-exist.
2013-12-04 | E-MTAB-1626 | biostudies-arrayexpress
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:he RNA polymerase II carboxy-terminal domain (CTD) consists of conserved repeats of the consensus sequence Y1-S2-P3-T4-S5-P6-S7, which can be phosphorylated to influence distinct stages of the transcription cycle, including RNA processing. Although affinity purification coupled with mass spectrometry has defined CTD-associated proteins, phospho-dependent CTD interactions have remained largely elusive. Proximity-dependent biotinylation (PDB) provides an alternative approach to identify protein-protein associations in the native cellular environment. Here we present a PDB-based map of the fission yeast RNAPII CTD in live cells. The proteomic screen identified known CTD-associated proteins, but also captured new and unexpected CTD proximal proteins. We also used PDB to identify phospho-dependent CTD interactions by using a mutant in which Ser2 was replaced by alanine in every repeat of the fission yeast CTD. Surprisingly, CTD-mediated biotinylation of most 3’ end processing factors was not affected in the S2A mutant, consistent with RNA-seq and ChIP-seq analysis indicating that CTD Ser2 phosphorylation is not required for 3’ end processing and transcription termination. Conversely, we found that CTD Ser2 phosphorylation is critical for the association between RNAPII and the histone methyltransferase Set2 during transcription elongation. We show that loss of CTD Ser2 phosphorylation disables the Set2-Clr6(II) axis, resulting in a global increase in cryptic antisense transcription that correlates with elevated levels of histone acetylation in gene bodies. Our findings reveal that the fundamental role of CTD Ser2 phosphorylation is to establish a chromatin-based repressive state that prevents cryptic intragenic transcription initiation.
2024-03-28 | GSE249235 | GEO
Project description:The RNA polymerase II carboxy-terminal domain (CTD) consists of conserved repeats of the consensus sequence Y1-S2-P3-T4-S5-P6-S7, which can be phosphorylated to influence distinct stages of the transcription cycle, including RNA processing. Although affinity purification coupled with mass spectrometry has defined CTD-associated proteins, phospho-dependent CTD interactions have remained largely elusive. Proximity-dependent biotinylation (PDB) provides an alternative approach to identify protein-protein associations in the native cellular environment. Here we present a PDB-based map of the fission yeast RNAPII CTD in live cells. The proteomic screen identified known CTD-associated proteins, but also captured new and unexpected CTD proximal proteins. We also used PDB to identify phospho-dependent CTD interactions by using a mutant in which Ser2 was replaced by alanine in every repeat of the fission yeast CTD. Surprisingly, CTD-mediated biotinylation of most 3’ end processing factors was not affected in the S2A mutant, consistent with RNA-seq and ChIP-seq analysis indicating that CTD Ser2 phosphorylation is not required for 3’ end processing and transcription termination. Conversely, we found that CTD Ser2 phosphorylation is critical for the association between RNAPII and the histone methyltransferase Set2 during transcription elongation. We show that loss of CTD Ser2 phosphorylation disables the Set2-Clr6(II) axis, resulting in a global increase in cryptic antisense transcription that correlates with elevated levels of histone acetylation in gene bodies. Our findings reveal that the fundamental role of CTD Ser2 phosphorylation is to establish a chromatin-based repressive state that prevents cryptic intragenic transcription initiation.
2024-03-28 | GSE249234 | GEO
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.
2016-01-25 | PXD003159 | Pride
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.
2012-06-29 | E-MTAB-1060 | biostudies-arrayexpress
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.
2017-02-14 | GSE93342 | 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.
2014-09-25 | GSE61713 | GEO