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Interactome of chromatin-associated RNA Polymerase II from U2OS cells

ABSTRACT: To determine the interactome of RNA Polymerase II (RNAPII), HA-tagged RPB3 was stably expressed in U2OS cells. The cells were harvested followed by the native stepwise isolation of chromatin-associated RNAPII complexes. HA-RPB3 was immunoprecipitated and interacting proteins analyzed by quantitative mass spectrometry. U2OS cells expressing no HA-tagged protein were used for comparison.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Pranjali Bhandare

LAB HEAD: Elmar Wolf

PROVIDER: PXD054701 | Pride | 2024-12-20

REPOSITORIES: Pride

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			Action	DRS
	2016-10-28_Fusion_8716.raw	Raw
	2016-10-28_Fusion_8717.raw	Raw
	2016-10-28_Fusion_8718.raw	Raw
	2016-10-28_Fusion_8719.raw	Raw
	2016-10-28_Fusion_8720.raw	Raw

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Publications

Phenotypic screens identify SCAF1 as critical activator of RNAPII elongation and global transcription.

Bhandare Pranjali P Narain Ashwin A Hofstetter Julia J Rummel Teresa T Wenzel Julia J Schülein-Völk Christina C Lamer Stephanie S Eilers Ursula U Schlosser Andreas A Eilers Martin M Erhard Florian F Wolf Elmar E

Nucleic acids research 20241219

Transcripts produced by RNA polymerase II (RNAPII) are fundamental for cellular responses to environmental changes. It is therefore no surprise that there exist multiple avenues for the regulation of this process. To explore the regulation mediated by RNAPII-interacting proteins, we used a small interfering RNA (siRNA)-based screen to systematically evaluate their influence on RNA synthesis. We identified several proteins that strongly affected RNAPII activity. We evaluated one of the top hits, ...[more]

PMID: 39698826

Similar Datasets

RNA Polymerase II interactome in presence or absence of SCAF1 in U2OS-SCAF1-AID cells

Project description:Transcripts produced by RNA polymerase II (RNAPII) are fundamental for cellular responses to environmental changes. It is therefore no surprise that there exist multiple avenues for the regulation of this process. To explore the regulation mediated by RNAPII-interacting proteins, we used an siRNA-based screen to systematically evaluate their influence on RNA synthesis. We identified several proteins that strongly affected RNAPII activity. We evaluated one of the top hits, SCAF1, using an auxin-inducible degradation system and sequencing approaches. In agreement with our screen results, acute depletion of SCAF1 decreased RNA synthesis, and showed an increase of Serine-2 phosphorylated-RNAPII (pS2-RNAPII). We found that the accumulation of pS2-RNAPII within the gene body occurred at GC-rich regions and was indicative of stalled RNAPII complexes. The accumulation of stalled RNAPII complexes was accompanied by reduced recruitment of initiating RNAPII, explaining the observed global decrease in transcriptional output. Furthermore, upon SCAF1 depletion, RNAPII complexes showed increased association with components of the proteasomal-degradation machinery. We concluded that in cells lacking SCAF1, RNAPII undergoes a rather interrupted passage, resulting in intervention by the proteasomal-degradation machinery to clear stalled RNAPII. While cells survive the compromised transcription caused by absence of SCAF1, further inhibition of proteasomal-degradation machinery, is synthetically lethal.

2024-12-20 | PXD054675 | Pride

Hypoxia-induced alterations of G2 checkpoint regulators

Project description:Hypoxia promotes an aggressive tumor phenotype with increased genomic instability, partially due to downregulation of DNA repair pathways. However, in addition to DNA repair, genome stability is also controlled by cell cycle checkpoints. An important issue is therefore whether hypoxia also can alter the DNA damage cell cycle checkpoints. Here, we show that hypoxia (24h 0.2% O2) alters the expression of several G2 checkpoint regulators, as examined by microarray gene expression analysis and immunoblotting of U2OS cells. While some of the changes reflected hypoxia-induced inhibition of cell cycle progression, flow cytometric bar-coding analysis of individual cells showed that the levels of several G2 checkpoint regulators were reduced in G2 phase cells after hypoxic exposure, in particular cyclin B1. These effects were accompanied by decreased Cyclin dependent kinase (CDK) activity in G2 phase cells after hypoxia. Furthermore, cells pre-exposed to hypoxia showed a longer G2 checkpoint arrest upon treatment with ionizing radiation. Similar results were found following other hypoxic conditions (~0.03 % O2 20h and 0.2% O2 72h). These results demonstrate that the DNA damage G2 checkpoint can be altered as a consequence of hypoxia, and we propose that such alterations may influence the genome stability of hypoxic tumors. We measured gene expression changes in U2OS cells after treatment with 0.2% hypoxia for 24 hours. The data were used in the exploration of hyopxia induced alterations in the G2 checkpoint.

2016-01-04 | E-GEOD-70530 | biostudies-arrayexpress

Proximity biotinylation of CDK1 associated proteins in U2OS cells

Project description:BirA ligase tagged CDK1 and BirA alone were expressed in U2OS cells. Proximal proteins to CDK1 were labelled by incubating cells with Biotin for 24 hours and biotinylated proteins extracted using Streptavidin pulldown.

2021-05-12 | PXD024796 | Pride

Ubiquitin-dependent turnover of MYC promotes loading of the PAF complex on RNA Polymerase II to drive transcriptional elongation (ChIP-seq)

Project description:The MYC transcription factor is an unstable protein and its turnover is controlled by the ubiquitin system. Ubiquitination enhances MYC-dependent transactivation, but the underlying mechanism remains unresolved. Here we show that proteasomal turnover of MYC is dispensable for recruitment of RNA polymerase II (RNAPII), but is required to promote transcriptional elongation at MYC target genes. Degradation of MYC stimulates histone acetylation and recruitment of BRD4 and P-TEFb to target promoters, leading to phosphorylation of RNAPII CTD and the release of elongating RNAPII. In the absence of degradation, the RNA polymerase II-associated factor (PAF) complex associates with MYC via interaction of its CDC73 subunit with a conserved domain in the amino-terminus of MYC ("MYC box I"), suggesting that a MYC/PAF complex is an intermediate in transcriptional activation. Since histone acetylation depends on a second highly conserved domain in MYCs amino-terminus ("MYC box II"), we propose that both domains co-operate to transfer elongation factors onto paused RNAPII. ChIP-Seq experiments for MYC-HA (HA-IP) and RNAPII (total,Ser2p,Ser5p) performed in IMEC primary breast epithelial cells. Input-samples were sequenced as controls. The following antibodies were used: HA (Abcam; ab 9110)/ total RNAPII (Santa Cruz; sc-899x)/ Ser2p RNAPII (Abcam; ab 5095)/ Ser5p RNAPII (Covance; MMS-128P)

2016-01-04 | E-GEOD-70001 | biostudies-arrayexpress

Gene expression of osteosarcoma cell (U2OS, MG63) lines relative to normal human osteoblasts (HOB)

Project description:Gain or loss of genes and deregulation of gene expression can result in cumulative and progressive disruptions of normal cellular functions. Cancer-specific changes in gene expression play an essential role in cancer occurrence, and ultimately lead to cancer-related self-sufficiency in growth signals, insensitivity to growth inhibitory signals, evasion of apoptosis, limitless replicative potential, angiogenesis, and metastasis. We aimed to analyse such changes in gene expression related to osteosarcoma. Keywords: Comparative We performed genome-wide comparison of gene expression and identified genes that are differentialy expressed in osteosarcoma (U2OS, MG63) cell lines relative to normal human osteoblasts (HOB)

2009-05-01 | E-GEOD-11414 | biostudies-arrayexpress

Investigation of the arsenic regulated proteome of PML bodies

Project description:A proteomic analysis of PML bodies purified from U2OS cells expressing YFP-PMLV either treated or not with 1uM arsenic for 2 hours, in comparison with parallel purifications from wt-U2OS cells treated identically.

2023-01-05 | PXD030330 | Pride

Effects of sumoylation-impairing mutation of TFIIF subunit Tfg1 on genome-wide RNA polymerase II (RNAPII) levels in budding yeast

Project description:Many transcription-related proteins, including components of the RNA polymerase II (RNAPII) general transcription factors (GTFs), have been shown through proteomics studies to be modifiable by SUMO conjugation. However, we determined that the large subunit of TFIIF, Tfg1, is likely the major target of sumoylation among GTFs in budding yeast. The primary site of sumoylation on Tfg1 is Lys residues 60 or 61, which lies in a region of the protein that is proximal to the Rpb2 subunit of RNAPII when the GTF-RNAPII preinitiation complex (PIC) assembles at promoters. As TFIIF and RNAPII are highly functionally and physically linked, we examined whether blocking Tfg1 sumoylation through Lys-to-Arg mutations could affect the recruitment or association of RNAPII with chromatin. RNAPII ChIP-seq was performed in strains expressing wild-type (Tfg1-HA) or sumoylation-deficient (Tfg1-K60,61R-HA) forms of Tfg1. Indeed, nearly half of all protein-coding genes showed significantly reduced RNAPII levels in the mutant strain. These data suggest that sumoylation of TFIIF plays an important role in regulating the dynamics of RNAPII association with genes.

2021-09-21 | GSE167426 | GEO

Transcription profiling by array of human osteosarcoma U2OS cell line conditionally expressing HA-tagged PRDM5

Project description:PRDM5 is a recently identified member of the PRDM family of proteins, which functions as a transcriptional repressor by recruiting histone methyltransferase G9A to DNA, and behaves as a putative tumor suppressor in different types of cancer. We investigated PRDM5 function by identifying its target genes in U2OS cells at different time points after expression of PRDM5 protein. Experiment Overall Design: We analyzed gene expression profiles of a U2OS cell line conditionally expressing HA-tagged PRDM5 (U2OS-PRDM5), where PRDM5 is under the transcriptional control of a doxycycline-inducible promoter. Expression of PRDM5 protein is detectable after 8 hours of induction with 2ug/ml doxycycline, and increases steadily up to 48 hours, after which cells begin to undergo apoptosis. U2OS cells containing the empty cloning vector (U2OS-pSG213) were used as controls for each condition.

2009-01-31 | E-GEOD-10580 | biostudies-arrayexpress

Repression of pervasive transcription is the primary role of RNA polymerase II CTD serine 2 phosphorylation in fission yeast

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.

2024-05-06 | PXD047023 | Pride

Protein interactions with HA-tagged cytoplasmic dynein-2 DATASET2

Project description:To identify those proteins interacting with the cytoplasmic dynein-2 using mammalian cell lines stably expressing HA-tagged intermediate chain subunits, WDR34 (DYNC2I2) or WDR60 (DYNC2I1).

2023-01-24 | PXD031152 | Pride

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