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

ORGANISM(S): Homo sapiens

PROVIDER: GSE136024 | GEO | 2019/10/01

REPOSITORIES: GEO

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

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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.

2019-10-01 | GSE136022 | 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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Selected Spt5-Pol II inhibitors effectively relieve Huntington’s disease neuropathology and slow down its progression in mice [INCPMPM-17698]

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-16068]

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Transcription Pausing Regulates Mouse Embryonic Stem Cell Differentiation [GRO-seq]

Project description:The pluripotency of embryonic stem cells (ESCs) relies on appropriate responsiveness to developmental cues. Promoter-proximal pausing of RNA polymerase II (Pol II) has been suggested to play a role in keeping genes poised for future activation. To identify the role of Pol II pausing in regulating ESC pluripotency, we have generated mouse ESCs carrying a mutation in the pause-inducing factor SPT5. Consistent with previous in vitro studies showing the pausing deficiency of this mutant SPT5, our genomic analysis reveals genome-wide reduction of paused Pol II in mutant mESCs. Furthermore, we find that genes differentially regulated by mutant SPT5 correlates with distinct chromatin states. Functionally, this pausing-deficient SPT5 disrupts ESC differentiation without affecting self-renewal. Thus, our study uncovers an important role of Pol II pausing in regulating ESC differentiation and also suggests that Pol II pausing can both positively and negatively influence transcription depending on the local chromatin environment.

2017-12-08 | GSE99747 | 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

SPT5 licenses transition into productive transcription elongation by regulating promoter-proximal Pol II stability

Project description:An acute protein depletion system coupled with proteomic and genomic analyses reveal how SPT5 regulates promoter-proximal Pol II stability in eukaryotic cells, linking transcriptional initiation to productive elongation.

2021-02-04 | MSV000086806 | MassIVE

SPT5 stabilizes promoter-proximal RNA polymerase II prior to release into gene bodies

Project description:The regulation of gene expression by RNA polymerase II (Pol II) is a multistep process requiring the concerted action of diverse transcription factors. Very little is known about in vivo function of transcription factor SPT5 as its deletion results in loss of viability. To circumvent this issue and define in vivo mechanism of action for SPT5, we employed acute degradation of SPT5 and studied its consequence on transcription. We find that SPT5 loss triggers degradation of the core Pol II subunit RPB1, a process which we show to be evolutionarily conserved from yeast to human. This RPB1 degradation requires the E3 ubiquitin ligase Cullin 3, the unfoldase VCP/p97 and a novel form of CDK9 kinase complex. SPT5 specifically stabilizes Pol II at promoter proximal regions, permitting Pol II release from promoters to gene bodies. Our findings provide mechanistic insight into the in vivo function of SPT5 in stabilization of promoter-proximal Pol II prior to release into gene bodies for safeguarding accurate gene expression.

2021-08-31 | GSE168827 | GEO

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.

2022-10-01 | GSE202749 | GEO

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