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Transcription machinery clustering by Integrator synchronizes histone gene expression

ABSTRACT: Numerous components of the transcription machinery, including RNA polymerase II (Pol II), accumulate in high-concentration clusters at gene promoters. Whether these clusters assemble on demand during transcription or constitute regulatory units remains unclear. Examining the Drosophila histone locus—where histone genes normally transcribe exclusively during S phase—we unexpectedly found large clusters containing non-chromatin-bound Pol II and elongation factors outside of S phase. When transcription activates during S phase, P-TEFb drives an accelerated turnover of the cluster components. Dispersion of clusters through depletion of the Integrator complex endonuclease module causes histone transcription to have a diminished S-phase peak and instead occur ectopically throughout the cell cycle. We propose that clusters constitute gate-keeping hubs that maintain a poised machinery pool, restricting gene activation to defined temporal windows.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE334998 | GEO | 2026/06/10

REPOSITORIES: GEO

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			Action	DRS
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	GSE334998_DMSO_PAF1_bg_corrected.bw	Other
	GSE334998_DMSO_Pol2_bg_corrected.bw	Other
	GSE334998_DMSO_Spt5_bg_corrected.bw	Other
	GSE334998_FP_PAF1_bg_corrected.bw	Other

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Phase separation of RNA-binding protein promotes polymerase binding and transcription

Project description:An RNA-involved phase-separation model has been proposed for transcription control. Yet, the molecular links that connect RNA to the transcription machinery remain missing. Here we find RNA-binding proteins (RBPs) constitute half of the chromatin proteome in embryonic stem cells (ESCs), and some are colocalized with RNA polymerase (Pol) II at promoters and enhancers. Biochemical analyses of representative RBPs show that the paraspeckle protein PSPC1 inhibits the RNA-induced premature release of Pol II, and makes use of RNA as multivalent molecules to enhance the formation of transcription condensates and subsequent phosphorylation and release of Pol II. This synergistic interplay enhances polymerase engagement and activity via the RNA-binding and phase-separation activities of PSPC1. In ESCs, auxin-induced acute degradation of PSPC1 leads to genome-wide defects in Pol II binding and nascent transcription. We propose that promoter-associated RNAs and their binding proteins synergize the phase separation of polymerase condensates to promote active transcription.

2020-05-12 | GSE150399 | GEO

DSIF factor Spt5 coordinates transcription, maturation and exoribonucleolysis of RNA polymerase II transcripts

Project description:The timely termination of RNA polymerase II (Pol II) transcription is essential for recycling of this enzyme and preventing transcriptional interference. While 5'-3' exonuclease Xrn2 is known to degrade nascent RNA after poly(A) site cleavage to promote Pol II release, its molecular mechanism remains not fully understood. Spt5 is one of highly conserved transcription factors that controls and regulates multiple aspects of Pol II transcription. Here, using mass spectrometry analysis of immunoprecipitated Flag-tagged Spt5-WT and Spt5-mutant complexes from fission yeast cells, we show that Xrn2 is highly enriched in these samples. Through in vitro BS3 crosslinking experiments with purified components, we further demonstrated that Xrn2 associates with transcription machinery and we probed interfaces between different subunits of transcription machinery

2024-11-22 | PXD058168 | Pride

The SPOC proteins DIDO3 and PHF3 co-regulate gene expression and neuronal differentiation - 3' RNA-seq

Project description:Transcription is regulated by a multitude of activators and repressors, which bind to the RNA polymerase II (Pol II) machinery and modulate its progression. Death-inducer obliterator (DIDO) and PHD finger protein 3 (PHF3) are paralogue proteins that regulate transcription elongation by docking onto phosphorylated serine-2 in the C-terminal domain (CTD) of Pol II through their SPOC domains. Here we show that DIDO3 and PHF3 form a complex that bridges the Pol II elongation machinery with chromatin and RNA processing factors, and tethers Pol II in a phase-separated microenvironment. Their SPOC domains and C-terminal intrinsically disordered regions are critical for transcription regulation. The dataset contains 3' Sequencing of WT, PHF3 KO, and PHF3 dSPOC mutant HEK293 cell line. Cytoplasmic and nuclear fractions were profiled separately.

2023-10-31 | E-MTAB-12757 | biostudies-arrayexpress

Mediator and Pol II form diffraction-sized condensates dependent on active transcription in living stem cells

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

Pol II phosphorylation regulates a switch between transcriptional and splicing condensates

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

The SPOC proteins DIDO3 and PHF3 co-regulate gene expression and neuronal differentiation - RNAseq

Project description:Transcription is regulated by a multitude of activators and repressors, which bind to the RNA polymerase II (Pol II) machinery and modulate its progression. Death-inducer obliterator (DIDO) and PHD finger protein 3 (PHF3) are paralogue proteins that regulate transcription elongation by docking onto phosphorylated serine-2 in the C-terminal domain (CTD) of Pol II through their SPOC domains. Here we show that DIDO3 and PHF3 form a complex that bridges the Pol II elongation machinery with chromatin and RNA processing factors, and tethers Pol II in a phase-separated microenvironment. Their SPOC domains and C-terminal intrinsically disordered regions are critical for transcription regulation. PHF3 and DIDO exert cooperative and antagonistic effects on the expression of neuronal genes and are both essential for neuronal differentiation. The dataset contains RNAseq of HEK293 cells with various perturbations of PHF3 and DIDO1 genes.

2023-11-16 | E-MTAB-12782 | biostudies-arrayexpress

Chromatin remodeling by antisense Pol II primes efficient Pol III transcription

Project description:The packaging of the genetic material into chromatin imposes the remodeling of this barrier to allow efficient transcription. RNA polymerase II activity is associated with several histone modification complexes that enforce remodeling. How RNA polymerase III (Pol III) counteracts the inhibitory effect of chromatin is unknown. We report here that antisense RNA Polymerase II (Pol II) transcription is critical to prime and maintain nucleosome depletion at Pol III loci and allow efficient Pol III recruitment upon re-initiation of growth from stationary phase. Antisense Pol II is recruited by the Pcr1 transcription factor, which affects local histone occupancy through the associated SAGA complex and a Pol II phospho-S2 CTD / Mst2 pathway. These data expand the central role of Pol II in gene expression beyond mRNA synthesis.

2022-12-24 | GSE193552 | GEO

RNA polymerase II pausing downstream of core histone genes is different from genes producing polyadenylated transcripts

Project description:Recent genome-wide chromatin immunoprecipitation coupled high throughput sequencing (ChIP-seq) analyses performed in various eukaryotic organisms, analysed RNA Polymerase II (Pol II) pausing around the transcription start sites of genes. In this study we have further investigated genome-wide binding of Pol II downstream of the 3' end of the annotated genes (EAGs) by ChIP-seq in human cells. At almost all expressed genes we observed Pol II occupancy downstream of the EAGs suggesting that Pol II pausing 3' from the transcription units is a rather common phenomenon. Downstream of EAGs Pol II transcripts can also be detected by global run-on and sequencing, suggesting the presence of functionally active Pol II. Based on Pol II occupancy downstream of EAGs we could distinguish distinct clusters of Pol II pause patterns. On core histone genes, coding for non-polyadenylated transcripts, Pol II occupancy is quickly dropping after the EAG. In contrast, on genes, whose transcripts undergo polyA tail addition [poly(A)+], Pol II occupancy downstream of the EAGs can be detected up to 4-6 kb. Inhibition of polyadenylation significantly increased Pol II occupancy downstream of EAGs at poly(A)+ genes, but not at the EAGs of core histone genes. The differential genome-wide Pol II occupancy profiles 3' of the EAGs have also been confirmed in mouse embryonic stem (mES) cells, indicating that Pol II pauses genome-wide downstream of the EAGs in mammalian cells. Moreover, in mES cells the sharp drop of Pol II signal at the EAG of core histone genes seems to be independent of the phosphorylation status of the C-terminal domain of the large subunit of Pol II. Thus, our study uncovers a potential link between different mRNA 3' end processing mechanisms and consequent Pol II transcription termination processes. Examination of RNA Polymerase II in MCF-7 cell line

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