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IWS1 Stimulates Pol II Transcription Elongation In Vivo [ChIP-Seq]

ABSTRACT: Interacts with SPT6 1 (IWS1) has been implicated in various co-transcriptional processes, yet its direct role in RNA Polymerase (Pol) II transcription in vivo remains elusive. In this study, we utilized a rapid depletion system of IWS1 in human cells, followed by multi-omics analysis and biochemical assays, to elucidate its function. We show that IWS1 depletion results in a global decrease of RNA synthesis, but does not affect the chromatin recruitment of the histone methyltransferase SETD2. In vivo and in vitro analyses of nascent transcription and histone modifications revealed that the observed decrease in methylation is a secondary effect of the transcription defect caused by IWS1 depletion. Whereas these results show that IWS1 is essential for maintaining normal RNA Pol II elongation velocity in vivo, and that it can directly stimulate transcription.

ORGANISM(S): Homo sapiens

PROVIDER: GSE276550 | GEO | 2025/07/30

REPOSITORIES: GEO

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IWS1 Stimulates Pol II Transcription Elongation In Vivo

Project description:The protein IWS1 (Interacts with SPT6 1) has been implicated in various transcription-associated processes, but whether it has a direct role in RNA polymerase (Pol) II transcription remains unclear. Here, we establish a rapid depletion system for IWS1 in human cells, and use it to elucidate its function in vivo by multi-omics kinetic analysis. We show that IWS1 depletion results in a global decrease of RNA synthesis in vivo that is due to a decrease in Pol II elongation velocity and in vitro biochemical assays reveal that IWS1 directly stimulates Pol II transcription by lowering the nucleosome entry barrier. Further analyses show that H3K36me3 decreases upon IWS1 depletion although the recruitment of the histone methyltransferase SETD2 to chromatin is unaffected. We conclude that IWS1 has a direct role in Pol II transcription and is essential for maintaining normal RNA elongation, which in turn is required for normal chromatin methylation.

2025-07-30 | PXD056248 | Pride

IWS1 Stimulates Pol II Transcription Elongation In Vivo (revision data)

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IWS1 Stimulates Pol II Transcription Elongation In Vivo [mNET-seq]

Project description:Interacts with SPT6 1 (IWS1) has been implicated in various co-transcriptional processes, yet its direct role in RNA Polymerase (Pol) II transcription in vivo remains elusive. In this study, we utilized a rapid depletion system of IWS1 in human cells, followed by multi-omics analysis and biochemical assays, to elucidate its function. We show that IWS1 depletion results in a global decrease of RNA synthesis, but does not affect the chromatin recruitment of the histone methyltransferase SETD2. In vivo and in vitro analyses of nascent transcription and histone modifications revealed that the observed decrease in methylation is a secondary effect of the transcription defect caused by IWS1 depletion. Whereas these results show that IWS1 is essential for maintaining normal RNA Pol II elongation velocity in vivo, and that it can directly stimulate transcription.

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IWS1 Stimulates Pol II Transcription Elongation In Vivo [RNA-Seq]

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RTF1 and SPT6 stimulate transcription elongation by two distinct mechanisms

Project description:We have previously shown that the RNA polymerase II (Pol II)-DSIF complex associates with the PAF1 complex (PAF), RTF1 and SPT6 to form an activated elongation complex in vitro. Here we investigate the mechanisms that these factors use to stimulate Pol II elongation in vivo. We combine rapid factor depletion from human cells with genome-wide analyses of changes in RNA synthesis and occupancy with engaged Pol II. Whereas depletion of PAF subunits has little effect on transcription in vivo, depletion of RTF1 or SPT6 strongly compromises RNA synthesis, albeit in fundamentally different ways. RTF1 depletion decreases Pol II velocity, whereas SPT6 depletion impairs Pol II progression through nucleosomes. These results show that distinct transcription elongation factors stimulate Pol II velocity and Pol II progression through chromatin in vivo. Our results also provide evidence for two distinct barriers to elongation at the beginning of genes, the promoter-proximal pause site and the +1 nucleosome.

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Project description:In vitro studies identified various factors including P-TEFb, SEC, SPT6, PAF1, DSIF, and NELF functioning at different stages of transcription elongation driven by RNA polymerase II (RNA Pol II). What remains unclear is how these factors cooperatively regulate pause/release and productive elongation in the context of living cells. Using an acute 5 protein-depletion approach, prominent release and a subsequent increase in mature transcripts, whereas long genes fail to yield mature transcripts due to a loss of processivity. Mechanistically, loss of SPT6 results in loss of PAF1 complex (PAF1C) from RNA Pol II, leading to NELF-bound RNA Pol II release into the gene bodies. Furthermore, SPT6 and/or PAF1 depletion impairs heat shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause/release through the recruitment of PAF1C during the early elongation.

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