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Genome-wide view of the impact of Spt5-Pol II inhibitors (SPIs) on transcription [4sU-seq]

ABSTRACT: We identified the first Spt5-Pol II inhibitors (SPIs). SPIs faithfully reproduced Spt5 knockdown effects on proximal-promoter-pausing, NF-κB activation and the expanded-repeat huntingtin gene in neuronal cells. Using SPIs we identified Spt5 target genes that responded with profoundly diverse kinetics and a novel regulatory element of proximal-promoter-pausing. To validate that the effects of SPIs are at the transcriptional level, cellular RNA was metabolically labeled with 4-thio-uridine (4sU) for 2 hours in the presence of DMSO or SPI-21 in the presence or absence of TNFα. Newly synthesized labeled RNA was then purified and subjected to RNA-seq.

ORGANISM(S): Homo sapiens

PROVIDER: GSE136025 | GEO | 2019/10/01

REPOSITORIES: GEO

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Genome-wide view of the impact of Spt5-Pol II inhibitors (SPIs) on transcription [GRO-Seq]

Project description:We identified the first Spt5-Pol II inhibitors (SPIs). SPIs faithfully reproduced Spt5 knockdown effects on proximal-promoter-pausing, NF-κB activation and the expanded-repeat huntingtin gene in neuronal cells. Using SPIs we identified Spt5 target genes that responded with profoundly diverse kinetics and a novel regulatory element of proximal-promoter-pausing. To obtain a genome-wide view of the impact of SPIs on transcription, we performed Global run-on sequencing (GRO-seq) using nuclei of HeLa cells treated with either DMSO (control) or SPI-21.

2019-10-01 | GSE136024 | GEO

Genome-wide view of the impact of Spt5-Pol II inhibitors (SPIs) on mRNA levels [RNA-Seq 24h]

Project description:We identified the first Spt5-Pol II inhibitors (SPIs). SPIs faithfully reproduced Spt5 knockdown effects on proximal-promoter-pausing, NF-κB activation and the expanded-repeat huntingtin gene in neuronal cells. We determined the global effect of short and long-term SPI-21 treatment by RNA-seq and compared the affected genes between the two treatments. The results revealed that temporary and constitutive Spt5-regulated genes can be distinguished by SPIs.

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Genome-wide view of the impact of Spt5-Pol II inhibitors (SPIs) on mRNA levels [RNA-Seq 2h]

Project description:We identified the first Spt5-Pol II inhibitors (SPIs). SPIs faithfully reproduced Spt5 knockdown effects on proximal-promoter-pausing, NF-κB activation and the expanded-repeat huntingtin gene in neuronal cells. We determined the global effect of short and long-term SPI-21 treatment by RNA-seq and compared the affected genes between the two treatments. The results revealed that temporary and constitutive Spt5-regulated genes can be distinguished by SPIs.

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Selected Spt5-Pol II inhibitors effectively relieve Huntington’s disease neuropathology and slow down its progression in mice [INCPMPM-16068]

Project description:Huntington’s disease (HD) is an incurable inherited disorder caused by repeat expansion in the huntingtin gene (Htt). The mutant protein causes neuronal degeneration leading to severe motor and psychological abnormalities. Selective downregulation of the mutant Htt by targeting Spt4/Spt5 or the Spt5-PolII transcription elongation complexes was proposed as a potential therapy. We report the identification of SPI-24 and SPI-77 small molecule inhibitors of Spt5-PolII that selectively lower mutant Htt. In the BACHD mouse model of HD, direct delivery to the striatum, subcutaneous injection and oral administration of these inhibitors diminished mutant Htt levels, ameliorated mitochondrial dysfunction and restored BDNF expression. Prolonged treatment attenuated motor and anxious-like phenotypes of HD mice and delayed disease deterioration. SPI-24 long-term treatment had no side-effects or global changes in gene expression. Mechanistically, SPIs reduce Spt5 and PolII occupancies of mutant Htt. Thus, SPIs can be an effective therapeutic strategy for HD.

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Selected Spt5-Pol II inhibitors effectively relieve Huntington’s disease neuropathology and slow down its progression in mice [INCPMPM-17698]

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Genome-wide view of the impact of Spt5-Pol II inhibitors (SPIs) on transcription [4sU-seq]

Project description:Genome-wide view of the impact of Spt5-Pol II inhibitors (SPIs) on transcription [4sU-seq]

| PRJNA560967 | ENA

The pausing zone and control of RNA polymerase II elongation by Spt5: implications for the pause-release model

Project description:The pause-release model of transcription proposes that pol II pauses 40-100 bases from the start site resulting in a pile-up that is relieved by subsequent release into productive elongation. Pause release is facilitated by PTEFb phosphorylation of the pol II elongation factor, Spt5. We mapped paused polymerases by eNETseq and found frequent pausing in zones that extend ~0.3-3kb into genes, even when PTEFb is inhibited. The fraction of paused polymerases or “pausiness” declines gradually over several kb, and not abruptly as predicted for a discrete pause release event. Spt5 depletion extends pausing zones suggesting that it promotes maturation of elongation complexes to a low-pausing state. Expression of mutants after Spt5 depletion showed that phosphomimetic substitutions in the Spt5 CTR1 domain diminished pausing throughout genes. In contrast mutants that prevent phosphorylation of the Spt5 RNA-binding domain strengthened pausing. Thus distinct Spt5 phospho-isoforms set the balance between pausing and elongation.

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Genome wide localization of Activation induced cytidine deaminase (AID), Spt5, and RNA Polymerase II (PolII) in mouse

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Distinct Cdk9-phosphatase switches act on elongation factor Spt5 at the beginning and end of the RNA polymerase II transcription cycle

Project description:The Pol II transcription cycle is ordered by CDKs and phosphatases. In fission yeast, Cdk9 phosphorylates carboxy-terminal repeats (CTRs) of Spt5 while inhibiting PP1 during elongation. Transcription past the cleavage and polyadenylation signal (CPS) coincides with PP1-dependent Spt5 dephosphorylation and leads to Pol II pausing with phosphorylated CTD-Ser2 (pSer2). Here we show this switch is conserved in humans: Cdk9 inhibition decreases phosphorylation of both PP1g and Spt5-Thr806 (pThr806), and induces pSer2 upstream of the CPS, whereas PP1 depletion increases pThr806. Moreover, in unperturbed cells, pThr806 is diminished in 3’-paused complexes where pSer2 is maximal. Cdk9 also phosphorylates Spt5 on Ser666, a PP1-refractory site between conserved KOW4 and KOW5 motifs; pSer666 increases upon promoter-proximal pause release and, in contrast to pThr806, persists beyond the CPS. We identify PP4—another target of inhibitory phosphorylation by Cdk9—as a pSer666 phosphatase. PP4 and PP1g are enriched at 5’ and 3’ ends of genes, respectively, consistent with pSer666 and pThr806 distributions. Therefore, distinct Cdk9-phosphatase switches operate on Spt5 at different steps in transcription.

2020-08-04 | GSE138548 | GEO

Genome-wide view of the impact of Spt5-Pol II inhibitors (SPIs) on transcription [GRO-Seq]

Project description:Genome-wide view of the impact of Spt5-Pol II inhibitors (SPIs) on transcription [GRO-Seq]

| PRJNA560966 | ENA