Nuclear export restricts Gdown1 to a mitotic function
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA772715 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR16482000_1.fastq.gz | Fastqsanger.gz | |||
| SRR16482000_2.fastq.gz | Fastqsanger.gz | |||
| SRR16482001_1.fastq.gz | Fastqsanger.gz | |||
| SRR16482001_2.fastq.gz | Fastqsanger.gz | |||
| SRR16482002_1.fastq.gz | Fastqsanger.gz |
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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
Project description: Not available
| S-EPMC8887472 | biostudies-literature
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
Project description:Dysregulated transcription and aberrant cell cycle progression represent core cancer hallmarks, yet not many transcriptional regulators are known to simultaneously govern both pathways as potent tumor suppressors. GDOWN1 is a Pol II-interacting factor mainly sequestered in the cytoplasm in somatic cells, and its nuclear biological functions remain largely undefined. We previously generated a nuclear-localized GDOWN1 mutant (nGDOWN1(10M)) that strongly represses transcription and cell proliferation. Here, we systematically dissect the underlying molecular mechanisms and validate its broad-spectrum anticancer potential. Combining genetic manipulation, multi-omics profiling and multiple functional assays, we demonstrate that nGDOWN1(10M) disturbs Pol II CTD phosphorylation homeostasis and triggers promoter-proximal premature termination, causing global repression of nascent transcription. Terminated Pol II complexes undergo cytoplasmic translocation alongside associated the CTD phosphatases PP2A and PP1, leading to massive cytoplasmic accumulation of these phosphatases. Concurrently, TP53 and CDKN1A are targeted by nGDOWN1(10M) and transcriptionally upregulated. The above dual regulatory circuit synergistically activates RB family tumor suppressors: p53-p21 signaling inhibits cyclin-dependent kinases, while cytoplasmic PP2A/PP1 enhance cellular phosphatase activity to dephosphorylate and stabilize RB and p130. Activated RB and p130 form functional RB and DREAM complexes and inhibit the transcription of their target genes to induce robust cell cycle arrest. Notably, nGDOWN1(10M) exerts profound antiproliferative effects across cancer cell lines, mouse xenograft models and patient-derived tumor organoids, independent of p53 status. Collectively, this work uncovers a novel interconnected regulatory cascade triggered by nuclear translocation of GDOWN1: it remodels Pol II phosphorylation landscape and induces global transcriptional repression, which further drives cytoplasmic kinase-phosphatase coordination to activate master cell cycle suppressors and ultimately arrest cell cycle progression. Our results illuminate a coordinated molecular circuit governing two central cancer hallmarks and establish nuclear GDOWN1 as a promising therapeutic candidate for both p53-proficient and -deficient malignancies.
2026-08-10 | GSE336704 | GEO
Project description:Dysregulated transcription and aberrant cell cycle progression represent core cancer hallmarks, yet not many transcriptional regulators are known to simultaneously govern both pathways as potent tumor suppressors. GDOWN1 is a Pol II-interacting factor mainly sequestered in the cytoplasm in somatic cells, and its nuclear biological functions remain largely undefined. We previously generated a nuclear-localized GDOWN1 mutant (nGDOWN1(10M)) that strongly represses transcription and cell proliferation. Here, we systematically dissect the underlying molecular mechanisms and validate its broad-spectrum anticancer potential. Combining genetic manipulation, multi-omics profiling and multiple functional assays, we demonstrate that nGDOWN1(10M) disturbs Pol II CTD phosphorylation homeostasis and triggers promoter-proximal premature termination, causing global repression of nascent transcription. Terminated Pol II complexes undergo cytoplasmic translocation alongside associated the CTD phosphatases PP2A and PP1, leading to massive cytoplasmic accumulation of these phosphatases. Concurrently, TP53 and CDKN1A are targeted by nGDOWN1(10M) and transcriptionally upregulated. The above dual regulatory circuit synergistically activates RB family tumor suppressors: p53-p21 signaling inhibits cyclin-dependent kinases, while cytoplasmic PP2A/PP1 enhance cellular phosphatase activity to dephosphorylate and stabilize RB and p130. Activated RB and p130 form functional RB and DREAM complexes and inhibit the transcription of their target genes to induce robust cell cycle arrest. Notably, nGDOWN1(10M) exerts profound antiproliferative effects across cancer cell lines, mouse xenograft models and patient-derived tumor organoids, independent of p53 status. Collectively, this work uncovers a novel interconnected regulatory cascade triggered by nuclear translocation of GDOWN1: it remodels Pol II phosphorylation landscape and induces global transcriptional repression, which further drives cytoplasmic kinase-phosphatase coordination to activate master cell cycle suppressors and ultimately arrest cell cycle progression. Our results illuminate a coordinated molecular circuit governing two central cancer hallmarks and establish nuclear GDOWN1 as a promising therapeutic candidate for both p53-proficient and -deficient malignancies.
2026-08-10 | GSE336705 | GEO
Project description:Dysregulated transcription and aberrant cell cycle progression represent core cancer hallmarks, yet not many transcriptional regulators are known to simultaneously govern both pathways as potent tumor suppressors. GDOWN1 is a Pol II-interacting factor mainly sequestered in the cytoplasm in somatic cells, and its nuclear biological functions remain largely undefined. We previously generated a nuclear-localized GDOWN1 mutant (nGDOWN1(10M)) that strongly represses transcription and cell proliferation. Here, we systematically dissect the underlying molecular mechanisms and validate its broad-spectrum anticancer potential. Combining genetic manipulation, multi-omics profiling and multiple functional assays, we demonstrate that nGDOWN1(10M) disturbs Pol II CTD phosphorylation homeostasis and triggers promoter-proximal premature termination, causing global repression of nascent transcription. Terminated Pol II complexes undergo cytoplasmic translocation alongside associated the CTD phosphatases PP2A and PP1, leading to massive cytoplasmic accumulation of these phosphatases. Concurrently, TP53 and CDKN1A are targeted by nGDOWN1(10M) and transcriptionally upregulated. The above dual regulatory circuit synergistically activates RB family tumor suppressors: p53-p21 signaling inhibits cyclin-dependent kinases, while cytoplasmic PP2A/PP1 enhance cellular phosphatase activity to dephosphorylate and stabilize RB and p130. Activated RB and p130 form functional RB and DREAM complexes and inhibit the transcription of their target genes to induce robust cell cycle arrest. Notably, nGDOWN1(10M) exerts profound antiproliferative effects across cancer cell lines, mouse xenograft models and patient-derived tumor organoids, independent of p53 status. Collectively, this work uncovers a novel interconnected regulatory cascade triggered by nuclear translocation of GDOWN1: it remodels Pol II phosphorylation landscape and induces global transcriptional repression, which further drives cytoplasmic kinase-phosphatase coordination to activate master cell cycle suppressors and ultimately arrest cell cycle progression. Our results illuminate a coordinated molecular circuit governing two central cancer hallmarks and establish nuclear GDOWN1 as a promising therapeutic candidate for both p53-proficient and -deficient malignancies.
2026-08-10 | GSE336707 | GEO
Project description:Dysregulated transcription and aberrant cell cycle progression represent core cancer hallmarks, yet not many transcriptional regulators are known to simultaneously govern both pathways as potent tumor suppressors. GDOWN1 is a Pol II-interacting factor mainly sequestered in the cytoplasm in somatic cells, and its nuclear biological functions remain largely undefined. We previously generated a nuclear-localized GDOWN1 mutant (nGDOWN1(10M)) that strongly represses transcription and cell proliferation. Here, we systematically dissect the underlying molecular mechanisms and validate its broad-spectrum anticancer potential. Combining genetic manipulation, multi-omics profiling and multiple functional assays, we demonstrate that nGDOWN1(10M) disturbs Pol II CTD phosphorylation homeostasis and triggers promoter-proximal premature termination, causing global repression of nascent transcription. Terminated Pol II complexes undergo cytoplasmic translocation alongside associated the CTD phosphatases PP2A and PP1, leading to massive cytoplasmic accumulation of these phosphatases. Concurrently, TP53 and CDKN1A are targeted by nGDOWN1(10M) and transcriptionally upregulated. The above dual regulatory circuit synergistically activates RB family tumor suppressors: p53-p21 signaling inhibits cyclin-dependent kinases, while cytoplasmic PP2A/PP1 enhance cellular phosphatase activity to dephosphorylate and stabilize RB and p130. Activated RB and p130 form functional RB and DREAM complexes and inhibit the transcription of their target genes to induce robust cell cycle arrest. Notably, nGDOWN1(10M) exerts profound antiproliferative effects across cancer cell lines, mouse xenograft models and patient-derived tumor organoids, independent of p53 status. Collectively, this work uncovers a novel interconnected regulatory cascade triggered by nuclear translocation of GDOWN1: it remodels Pol II phosphorylation landscape and induces global transcriptional repression, which further drives cytoplasmic kinase-phosphatase coordination to activate master cell cycle suppressors and ultimately arrest cell cycle progression. Our results illuminate a coordinated molecular circuit governing two central cancer hallmarks and establish nuclear GDOWN1 as a promising therapeutic candidate for both p53-proficient and -deficient malignancies.
2026-08-10 | GSE336706 | GEO
Project description:Nuclear export of circular RNA
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Project description:Nuclear export of circular RNA
| PRJNA941274 | ENA
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