Targeted protein degradation reveals direct roles of SPT6 in POL2 elongation and termination
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA681218 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13159400.fastq.gz | Fastqsanger.gz | |||
| SRR13159401.fastq.gz | Fastqsanger.gz | |||
| SRR13159402.fastq.gz | Fastqsanger.gz | |||
| SRR13159403.fastq.gz | Fastqsanger.gz | |||
| SRR13159404.fastq.gz | Fastqsanger.gz |
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Project description:SPT6 is both, an important histone chaperone and POL2 elongation factor but its primary role on transcription in mammalian cells remains open, as no acute depletion system is available. We used targeted protein degradation to rapidly deplete SPT6 and analyzed defects in POL2 behavior by a multi-omics approach and estimated POL2 processivity, elongation rates and termination and compared it to gene transcription. Our data indicate that SPT6 is a crucial factor for POL2 elongation. Unexpectedly, SPT6 has also a vital role in POL2 termination, as acute depletion induces POL2 read through at most protein coding genes. In contrast, acute depletion did not induce spurious intragenic initiation, while this behavior can be induced by long-term depletion of SPT6 and can therefore be attributed to its function as a histone chaperone. In conclusion, targeted protein degradation of SPT6 allows the kinetic discrimination of its function as a histone chaperone and POL2 elongation factor.
2021-06-23 | GSE162264 | GEO
Project description:SPT6 is a histone chaperone that tightly binds RNA polymerase II (POL2) during transcription elongation. However, its primary role in POL2 transcription is uncertain. We used targeted protein degradation to rapidly deplete SPT6 in human cells and analyzed defects in POL2 behavior by a multi-omics approach and mathematical modeling. Our data indicate that SPT6 is a crucial factor for POL2 processivity and is therefore required for the productive transcription of protein-coding genes. Unexpectedly, SPT6 also has a vital role in POL2 termination, as acute depletion induced readthrough transcription for thousands of genes. Long-term depletion of SPT6 induced cryptic intragenic transcription, as observed earlier in yeast. However, this phenotype was not observed upon acute SPT6 depletion and therefore can be attributed to accumulated epigenetic perturbations in the absence of SPT6. In conclusion, targeted protein degradation of SPT6 allowed the temporal discrimination of its function as an epigenetic safeguard and POL2 elongation factor.
2021-06-28 | PXD025243 | Pride
Project description:The conserved transcription elongation factor Spt6 makes several contacts with the RNA Polymerase II (RNAPII) complex, including a high-affinity interaction between the Spt6 tandem SH2 domain (Spt6-tSH2) and phosphorylated residues in Rpb1 in a region between the catalytic core and the heptad repeats of its C-terminal domain (CTD). This interaction contributes to the global occupancy of Spt6 within transcription units, suggesting that it has a general role in tethering Spt6 to the elongation complex. However, we show here that disrupting this binding caused increases in some transcripts, revealing specific functional roles in regulating the expression of subsets of genes. These included loci whose regulation involves differential transcription start site selection, early termination of transcription, or efficient restoration of chromatin integrity after transcription. Loss of this interaction also caused a defect in splicing, and apparent pausing of RNAPII progression in regions requiring more complex processing of excised introns. The results support a global role for the Spt6-tSH2:Rpb1 interaction as one of several means of stabilizing the association of Spt6 with RNAPII, but they also reveal local functions at specific sites, especially those where dynamic decisions regarding initiation or termination are made, or where changes in the configuration of associated factors occur. We therefore propose that the Spt6-tSH2:Rpb1 interaction can provide a conduit for communication between RNAPII and the elongation factor function of Spt6, or with other factors associated with the Rpb1 CTD, supporting appropriate elongation through challenging templates and efficient co-transcriptional processing.
2021-12-20 | GSE174427 | GEO
Project description:SPT6 is a histone chaperone that tightly binds RNA polymerase II (RNAPII) during transcription elongation. However, its primary role in transcription is uncertain. We used targeted protein degradation to rapidly deplete SPT6 in human cells and analyzed defects in RNAPII behavior by a multi-omics approach and mathematical modeling. Our data indicate that SPT6 is a crucial factor for RNAPII processivity and is therefore required for the productive transcription of protein-coding genes. Unexpectedly, SPT6 also has a vital role in RNAPII termination, as acute depletion induced readthrough transcription for thousands of genes. Long-term depletion of SPT6 induced cryptic intragenic transcription, as observed earlier in yeast. However, this phenotype was not observed upon acute SPT6 depletion and therefore can be attributed to accumulated epigenetic perturbations in the prolonged absence of SPT6. In conclusion, targeted degradation of SPT6 allowed the temporal discrimination of its function as an epigenetic safeguard and RNAPII elongation factor.
| S-EPMC8354102 | biostudies-literature
Project description:Influence of a C-terminal deletion of the RNA polymerase II elongation factor Spt6 on the transcription of the yeast genome. In parallel the influence of a Spt6 overexpression on gene expression is analyzed.
2009-04-08 | E-MEXP-2123 | biostudies-arrayexpress
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.
2021-06-21 | GSE159633 | GEO
Project description:In adult tissue, stem and progenitor cells must tightly regulate the balance between proliferation and differentiation to sustain homeostasis. It is assumed that the rate-limiting step during the induction of somatic tissue differentiation is the recruitment of RNA polymerase II (Pol II) to promoters. Here we show that ~30% of induced differentiation genes already contain stalled Pol II at the promoters in epidermal stem and progenitor cells which is then released into productive transcription elongation upon differentiation. Central to this process is the histone chaperone, SPT6 which is necessary for the elongation of these differentiation genes. Upon SPT6 depletion there is a loss of human skin differentiation and stratification. Unexpectedly, loss of SPT6 also caused the spontaneous transdifferentiation of epidermal cells into an intestinal phenotype due to the stalled transcription of the master regulator of epidermal fate P63. Our findings suggest that control of transcription elongation through SPT6 plays a prominent role in adult somatic tissue differentiation and the inhibition of alternative cell fate choices.
2020-12-21 | GSE153129 | GEO
Project description:Termination of RNA polymerase II (Pol II) transcription is a key step, that is important for 3’end formation of functional mRNA, mRNA release and Pol II recycling. Even so, this underlying termination mechanism is not yet understood. Here, we demonstrate that the conserved and essential termination factor Seb1 interacts with Pol II near the end of the RNA exit channel and the Rpb4/7 stalk. Furthermore, the Seb1 interaction surface with Pol II largely overlaps with that of the elongation factor Spt5. Notably, Seb1 co-transcriptional recruitment is dependent on Spt5 de-phosphorylation by the conserved PP1 phosphatase Dis2, which also de-phosphorylates threonine 4 within the Pol II heptad repeated C-terminal domain. We propose that Dis2 orchestrates the transition from elongation to termination phase during the transcription cycle by mediating elongation to termination factor exchange and de-phosphorylation of Pol II C-terminal domain.
2018-12-26 | GSE111326 | GEO
Project description:Spt6 coordinates nucleosome dis- and re-assembly, transcriptional elongation and mRNA processing. Here, we report that depleting Spt6 in ESCs reduced expression of pluripotency factors, increased expression of cell lineage-affiliated developmental regulators, and induced cell morphological and biochemical changes indicative of ESC differentiation. Selective down-regulation of pluripotency factors upon Spt6 depletion may be mechanistically explained by its enrichment at ESC super-enhancers where Spt6 controlled H3K27 acetylation and methylation, and super-enhancer RNA transcription. In ESCs, Spt6 interacted with the PRC2 core subunit Suz12 and prevented H3K27me3 accumulation at ESC super-enhancers and associated promoters. Biochemical as well as functional experiments revealed that Spt6 could compete for binding of the PRC2 methyltransferase Ezh2 to Suz12 and reduce PRC2 chromatin engagement. Thus, in addition to serving as histone chaperone and transcription elongation factor, Spt6 counteracts repression by opposing H3K27me3 deposition at critical genomic regulatory regions.
2017-09-01 | GSE103180 | GEO
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.
2022-08-09 | PXD033771 | Pride