Mediator and Pol II form diffraction-sized condensates dependent on active transcription in living stem cells
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA474953 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7275200.fastq.gz | Fastqsanger.gz | |||
| SRR7275210.fastq.gz | Fastqsanger.gz | |||
| SRR7275201.fastq.gz | Fastqsanger.gz | |||
| SRR7275202.fastq.gz | Fastqsanger.gz | |||
| SRR7275203.fastq.gz | Fastqsanger.gz |
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Project description:Gene activation is thought to involve a multistep process whereby transcription factors bind to distal enhancer sites and recruit the Mediator complex which contacts the pre-initiating RNA Polymerase II (Pol II) complex assembled at the start site of the gene. The interaction of Mediator and Pol II has yet to be observed in the nucleus of living cells and the dynamics of this interaction are not yet elucidated. Here we use quantitative live cell super-resolution and light sheet imaging to study the organization and dynamics of endogenous Mediator and Pol II directly in living mouse embryonic stem cells. In addition to forming transient clusters with average lifetimes of 11.1 (± 0.9) s, and 12.1 (± 1.4) s respectively, Mediator and Pol II also form large and stable clusters in stem cells (~15 stable clusters per cell). The large and stable Mediator and Pol II clusters gradually disappear within hours after induction of stem cell differentiation. Mediator and Pol II colocalize in the large clusters. Inhibition of Brd4 bromodomains necessary for enhancer association eliminates both Mediator and Pol II stable clusters, and inhibition of transcription elongation selectively eliminates stable Pol II but not stable Mediator clusters. Tracking of Mediator and Pol II stable clusters suggests they are chromatin associated and they coalesce upon contact, a property associated with phase separated droplets. We conclude that Mediator and Pol II associate in diffraction-sized condensates with a defined lifetime dependent on active transcription in living stem cells.
2018-07-22 | GSE115436 | GEO
Project description:The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex, and a transition to an elongation complex. The large subunit of Pol II contains an intrinsically disordered C-terminal domain that is phosphorylated by cyclin-dependent kinases during the transition from initiation to elongation, thus influencing the interaction of the C-terminal domain with different components of the initiation or the RNA-splicing apparatus. Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain. Both the transcription-initiation machinery and the splicing machinery can form phase-separated condensates that contain large numbers of component molecules: hundreds of molecules of Pol II and mediator are concentrated in condensates at super-enhancers, and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites. Here we investigate whether the phosphorylation of the Pol II C-terminal domain regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing. We find that the hypophosphorylated C-terminal domain of Pol II is incorporated into mediator condensates and that phosphorylation by regulatory cyclin-dependent kinases reduces this incorporation. We also find that the hyperphosphorylated C-terminal domain is preferentially incorporated into condensates that are formed by splicing factors. These results suggest that phosphorylation of the Pol II C-terminal domain drives an exchange from condensates that are involved in transcription initiation to those that are involved in RNA processing, and implicates phosphorylation as a mechanism that regulates condensate preference.
2019-06-17 | GSE120656 | GEO
Project description:lncRNA DIGIT and BRD3 protein form phase-separated condensates to regulate endoderm differentiation
2020-07-08 | GSE149032 | GEO
Project description:The gene expression programs that define each cell’s identity are controlled by master transcription factors (TFs) that bind cell-type specific enhancers, as well as signaling factors, which bring extracellular stimuli to these enhancers. Recent studies have revealed that master TFs form phase-separated condensates with the Mediator coactivator at super-enhancers. Here we present evidence that signaling factors for the WNT, TGF-β and JAK/STAT pathways employ their intrinsically disordered regions (IDRs) to enter and concentrate in Mediator condensates at super-enhancers. We show that the WNT coactivator β-catenin interacts both with components of condensates and DNA binding factors to selectively occupy super-enhancer associated genes. We propose that the cell-type specificity of the response to signaling is mediated, in part, by the IDRs of the signaling factors, which cause these factors to partition into condensates established by the master TFs and Mediator at genes with prominent roles in cell identity.
2020-01-31 | GSE134387 | GEO
Project description:Recurrent cancer-causing fusions of NUP98 produce higher-order assemblies known as condensates. How NUP98 oncofusion-driven condensates activate oncogenes remainspoorly understood. Here, we investigate NUP98-PHF23, a leukemogenic chimera of the FG-repeat region of NUP98 and the H3K4me3-binding PHD finger of PHF23. Our integrated analyses using mutagenesis, proteomics, genomics, and condensate reconstitution demonstrate that the PHD finger targets condensates to H3K4me3-demarcated developmental genes and the FG repeats determine condensate composition and gene activation. The FG repeats are necessary to form condensates that partition a specific set of transcriptional regulators, notably the MLL family of H3K4 methylation-writing enzymes and BRD4. The FG repeats are sufficient, when tethered to the genome, to partition these transcriptional regulators and activate genes. NUP98-PHF23 assembles chromatin-bound condensates that partition multiple positive regulators, initiating a feed-forward loop of reading-and-writing active histone modifications. This network of interactions enforces an open chromatin landscape at proto-oncogenes, thereby driving cancerous transcriptional programs.
2025-01-30 | GSE280397 | GEO
Project description:Pol II phosphorylation regulates a switch between transcriptional and splicing condensates
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Project description:Pol II(G) is a novel form of RNA polymerase II that contains a tightly associated Gdown1 polypeptide. Whereas Pol II suffices for robust activator-dependent transcription in the absence of Mediator, Pol II(G) is highly dependent upon Mediator in a biochemically defined in vitro system. However, the mechanism(s) whereby Gdown1 alters the coactivator-dependence of Pol II is unknown. Here we show that Gdown1 competes with TFIIF for binding to the RPB1 and RPB5 subunits of Pol II in vitro, resulting in inhibition of a critical function of TFIIF in facilitating PIC assembly. The apparent inability of Pol II(G) to associate with TFIIF suggests that Gdown1 must be removed from Pol II for transcription initiation. However, Mediator can actually help Pol II(G) bind to the promoter prior to subsequent Mediator functions. Complementary cell-based analyses reveal that Pol II(G) is recruited to promoter regions of subsets of actively transcribed genes, where Pol II(G) appears to modulate transcription. Our findings indicate that differential regulation of subsets of genes by Pol II and Pol II(G) occurs through the regulation of TFIIF and consequent alterations in Mediator requirements. Genome-wide localization of PolII and Gdown1 in human IMR90 fibroblast cells using ChIP-seq
2012-01-13 | E-GEOD-33128 | biostudies-arrayexpress
Project description:We investigated the role of the nucleolar protein Treacle in organizing and regulating the nucleolus in human cells. Our results support Treacle’s ability to form liquid-like phase condensates through electrostatic interactions among molecules. The formation of these biomolecular condensates is crucial for segregating nucleolar fibrillar centers from the dense fibrillar component and ensuring high levels of rRNA gene transcription and accurate rRNA processing. Both the central and C-terminal domains of Treacle are required to form liquid-like condensates. The initiation of phase separation is attributed to the C-terminal domain. The central domain is characterized by repeated stretches of alternatively charged amino-acid residues and is vital for condensate stability. Overexpression of mutant forms of Treacle that cannot form liquid-like phase condensates compromises the assembly of fibrillar centers, suppressing rRNA gene transcription and disrupting rRNA processing. These mutant forms also fail to recruit DNA topoisomerase II binding protein 1 (TOPBP1), suppressing the DNA damage response in the nucleolus.
2025-03-26 | GSE292850 | GEO
Project description:Recurrent cancer-causing fusions of NUP98 produce higher-order assemblies known as condensates. How NUP98 oncofusion-driven condensates activate oncogenes remains poorly understood. Here, we investigate NUP98-PHF23, a leukemogenic chimera of the disordered FG-repeats-rich region of NUP98 and the H3K4me3/2-binding PHD finger domain of PHF23. Our integrated analyses using mutagenesis, proteomics, genomics, and condensate reconstitution demonstrate that the PHD domain targets condensates to H3K4me3/2-demarcated developmental genes while the FG repeats determine condensate composition and gene activation. The FG repeats are necessary to form condensates that partition a specific set of transcriptional regulators, notably the KMT2/MLL family of H3K4 methyltransferases, histone acetyltransferases and BRD4. The FG repeats are sufficient to partition these transcriptional regulators and activate a reporter when tethered to a locus. NUP98-PHF23 assembles the chromatin-bound condensates that partition multiple positive regulators, initiating a feed-forward loop of reading-and-writing active histone modifications. This network of interactions enforces an open chromatin landscape at proto-oncogenes, thereby driving cancerous transcriptional programs.
2024-12-31 | PXD056948 | Pride
Project description:Pol II(G) is a novel form of RNA polymerase II that contains a tightly associated Gdown1 polypeptide. Whereas Pol II suffices for robust activator-dependent transcription in the absence of Mediator, Pol II(G) is highly dependent upon Mediator in a biochemically defined in vitro system. However, the mechanism(s) whereby Gdown1 alters the coactivator-dependence of Pol II is unknown. Here we show that Gdown1 competes with TFIIF for binding to the RPB1 and RPB5 subunits of Pol II in vitro, resulting in inhibition of a critical function of TFIIF in facilitating PIC assembly. The apparent inability of Pol II(G) to associate with TFIIF suggests that Gdown1 must be removed from Pol II for transcription initiation. However, Mediator can actually help Pol II(G) bind to the promoter prior to subsequent Mediator functions. Complementary cell-based analyses reveal that Pol II(G) is recruited to promoter regions of subsets of actively transcribed genes, where Pol II(G) appears to modulate transcription. Our findings indicate that differential regulation of subsets of genes by Pol II and Pol II(G) occurs through the regulation of TFIIF and consequent alterations in Mediator requirements.
2012-01-13 | GSE33128 | GEO