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Regulation of Mediator-dependent transcriptional activation of Pol II(G) through TFII

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE33128 | GEO | 2012/01/13

SECONDARY ACCESSION(S): PRJNA149351

REPOSITORIES: GEO

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Regulation of Mediator-dependent transcriptional activation of Pol II(G) through TFII

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

Functional association of Gdown1 with RNA Polymerase II in Human

Project description:Most human genes are loaded with promoter proximally paused RNA polymerase II (RNAP II) molecules that are poised for release into productive elongation by P-TEFb. Gdown1, a protein that renders RNAP II responsive to mediator, is involved in RNAP II elongation control. During in vitro transcription assays Gdown1 specifically blocked elongation stimulation by TFIIF, inhibited the termination activity of TTF2, and influenced pausing factors NELF and DSIF, but did not affect the function of TFIIS or the mRNA capping enzyme. Without P-TEFb, Gdown1 led to the production of stably paused polymerases in the presence of nuclear extract. ChIP-Seq data demonstrated that Gdown1 mapped over essentially all poised polymerases across the human genome. Gdown1 increases the stability of poised polymerases while maintaining their responsiveness to P-TEFb, and mediator overcomes a Gdown1-mediated block of initiation by allowing TFIIF function. 7 ChIP-Seq data for Pol II and Gdown1 in human HeLa cell. See associated publication.

2012-01-13 | E-GEOD-32442 | biostudies-arrayexpress

Whole Cell Extract

Project description:Most human genes are loaded with promoter proximally paused RNA polymerase II (RNAP II) molecules that are poised for release into productive elongation by P-TEFb. Gdown1, a protein that renders RNAP II responsive to mediator, is involved in RNAP II elongation control. During in vitro transcription assays Gdown1 specifically blocked elongation stimulation by TFIIF, inhibited the termination activity of TTF2, and influenced pausing factors NELF and DSIF, but did not affect the function of TFIIS or the mRNA capping enzyme. Without P-TEFb, Gdown1 led to the production of stably paused polymerases in the presence of nuclear extract. ChIP-Seq data demonstrated that Gdown1 mapped over essentially all poised polymerases across the human genome. Gdown1 increases the stability of poised polymerases while maintaining their responsiveness to P-TEFb, and mediator overcomes a Gdown1-mediated block of initiation by allowing TFIIF function. 1 whole cell extract sample in human HeLa cell. See associated publication.

2012-02-21 | E-GEOD-35882 | biostudies-arrayexpress

Whole Cell Extract

2012-02-21 | GSE35882 | GEO

Functional association of Gdown1 with RNA Polymerase II in Human

2012-01-13 | GSE32442 | GEO

Characterization of GFP-Gdown1 interactors by IP and LC-MS/MS

Project description:Approximately half of purified mammalian RNA polymerase II (Pol II) is associated with a tightly interacting sub-stoichiometric subunit, Gdown1. Previous studies have established that Gdown1 inhibits transcription initiation through competitive interactions with general transcription factors and blocks the Pol II termination activity of transcription termination factor 2 (TTF2). However, the biological functions of Gdown1 remain poorly understood. Here, we utilized genetic, microscopic, and multi-omics approaches to functionally characterize Gdown1 in three human cell lines. Acute depletion of Gdown1 caused minimal direct effects on transcription. We show that Gdown1 resides predominantly in the cytoplasm of interphase cells, shuttles between the cytoplasm and nucleus, and is regulated by nuclear export. Gdown1 enters the nucleus at the onset of mitosis. Consistently, genetic ablation of Gdown1 is associated with partial de-repression of mitotic transcription, and Gdown1 KO cells present with evidence of aberrant mitoses coupled to p53 pathway activation. Evidence is presented demonstrating that Gdown1 modulates the combined functions of purified productive elongation factors PAF1C, RTF1, SPT6, DSIF, and P-TEFb in vitro. Collectively, our findings support a model wherein the Pol II-regulatory function of Gdown1 occurs during mitosis and is required for stably maintaining a pattern of gene expression across cell divisions. To characterize potential interactors of Gdown1 in the cytoplasm, an eGFP-TEV-Gdown1 HAP1 cell line was developed. Cytosolic extracts were prepared, GFP-Trap Agarose (Chromotek) was utilized to pull down Gdown1 and associated factors, and associated proteins were characterized by LC-MS/MS.

2022-02-14 | PXD029425 | Pride

Nuclear export restricts Gdown1 to a mitotic function

Project description:Approximately half of purified mammalian RNA polymerase II (Pol II) is associated with a tightly interacting sub-stoichiometric subunit called Gdown1, encoded by the POLR2M gene. Previous in vitro and structural studies have established that Gdown1 potently inhibits transcription initiation through competitive interactions with general transcription factors and blocks the Pol II termination activity of transcription termination factor 2 (TTF2). However, the biological functions of Gdown1 remain poorly understood. Here, we utilized genetic, microscopic, and multi-omics approaches to functionally characterize Gdown1 in multiple human cell lines. Remarkably, acute depletion of Gdown1 led to minimal direct effects on gene expression and transcription. We show that Gdown1 resides predominantly in the cytoplasm of interphase cells, shuttles between the cytoplasm and nucleus, and is directly regulated by nuclear export. Gdown1 enters the nucleus at the onset of mitosis prior to complete breakdown of the nuclear envelope, whereupon association with Pol II becomes possible. Consistently, genetic ablation of Gdown1 is associated with partial de-repression of mitotic transcription, and Gdown1 KO cells present with a growth defect and evidence of aberrant mitoses coupled to p53 pathway activation. Evidence is presented demonstrating that Gdown1 association with Pol II elongation complexes in vitro modulates the combined functions of purified productive elongation factors PAF1C, RTF1, SPT6, DSIF, and P-TEFb. Collectively, our findings support a model wherein the Pol II-regulatory function of Gdown1 occurs during mitosis and is required for genome integrity.

2022-01-03 | GSE186133 | GEO

Activation of GPCR ligand binding genes revealed through degradation of RNA polymerase II

Project description:RNA polymerase II drives mRNA gene expression, yet our understanding of global Pol II repression is limited. Using auxin-inducible degron, we degraded Pol II's RPB1 subunit in mESCs, resulting in global repression, yet certain genes exhibited increased RNA levels post-degradation. These genes are associated with GPCR ligand binding and are characterized by being less paused and comprising polycomb-bound short genes. RPB1 degradation globally increased KDM6B binding, which was insufficient to explain specific gene activation. In contrast, RPB2 degradation repressed nearly all genes, accompanied by decreased H3K9me3 and SUV39H1 occupancy. We observed a specific increase in serine 2 phosphorylated Pol II at Pol II degradation-upregulated genes, indicating their resistance to degradation. Additionally, a-amanitin or UV treatment resulted in RPB1 degradation and global gene repression, unveiling subsets of upregulated genes. Our findings highlight the activation of signaling genes as a potential mechanism for cells to counter global transcription shutdown during stress.

2024-05-28 | PXD048023 |

Functional link between TFIIB and Mediator

Project description:In this study, we generate genomic maps of Mediator, Pol II, TBP, TFIIH, TFIIA, TFIIB, TFIIE, TFIIF, by ChIP coupled to next generation sequencing technology (ChIP-seq), in wild type strains from Saccharomyces cerevisiae and in a mutant for the Mediator essential subunit Med10

2017-01-24 | E-MTAB-4607 | biostudies-arrayexpress

The RNA Polymerase II factor RPAP1 is critical for Mediator-driven transcription and cell identity

Project description:The RNA Polymerase II Associated Protein 1 (RPAP1) is conserved across metazoa and critical for stem cell differentiation in plants, however, very little is known about its mechanism of action or its role in mammalian cells. Here, we report that RPAP1 is essential for the expression of lineage specifying factors and for viability. Accordingly, inhibition of RPAP1 triggers somatic cell de-differentiation and facilitates reprogramming into pluripotent stem cells. Conversely, interfering with RPAP1 in ES cells severely impairs their differentiation capacity. Mechanistically, we show that RPAP1 is essential for the interaction between Pol II and Mediator, as well as for the recruitment of important regulators, such as the Mediator-specific Pol II factor POLR2M/Gdown1 and the CTD phosphatase RPAP2. In agreement, depletion of RPAP1 disturbs the loading of Pol II and Pol II Ser5 phosphorylation levels and impairs expression of super-enhancer-driven genes. We conclude that Mediator-RPAP1-Pol II is an ancient module, conserved from plants to mammals, critical for establishing and maintaining cell identity.

2018-01-10 | PXD007114 | Pride

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