RTF1 and SPT6 stimulate transcription elongation by two distinct mechanisms
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA669878 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12844206_1.fastq.gz | Fastqsanger.gz | |||
| SRR12844206_2.fastq.gz | Fastqsanger.gz | |||
| SRR12844207_1.fastq.gz | Fastqsanger.gz | |||
| SRR12844207_2.fastq.gz | Fastqsanger.gz | |||
| SRR12844208_1.fastq.gz | Fastqsanger.gz |
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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:The Paf1 complex (Paf1C) is a conserved transcription elongation factor that regulates transcription elongation efficiency, facilitates co-transcriptional histone modifications, and impacts molecular processes linked to RNA synthesis, such as polyA site selection. Coupling of the activities of Paf1C to transcription elongation requires its association with RNA polymerase II (Pol II). Mutational studies in yeast identified Paf1C subunits Cdc73 and Rtf1 as important mediators of Paf1C association with Pol II on active genes. While the interaction between Rtf1 and the general elongation factor Spt5 is relatively well-understood, the interactions involving Cdc73 have not been fully elucidated. Using a site-specific protein cross-linking strategy in yeast cells, we identified direct interactions between Cdc73 and two components of the Pol II elongation complex, the elongation factor Spt6 and the largest subunit of Pol II. Both of these interactions require the tandem SH2 domain of Spt6. We also show that Cdc73 and Spt6 can interact in vitro and that rapid depletion of Spt6 dissociates Paf1 from chromatin, altering patterns of Paf1C-dependent histone modifications genome-wide. These results reveal interactions between Cdc73 and the Pol II elongation complex and identify Spt6 as a key factor contributing to the occupancy of Paf1C at active genes in Saccharomyces cerevisiae.
2023-02-24 | GSE201436 | GEO
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: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.
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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.
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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.
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Project description:During heart development, an evolutionarily conserved network of cardiac transcription factors collaborate to define the precise timing and location of cardiac progenitor specification. Accumulating evidence suggests that cardiac progenitor specification is subject to transcriptional control beyond the level of transcription initiation. The PAF1C component Rtf1 is a multifunctional transcription regulatory protein that modulates pausing and elongation of RNA Pol II, as well as histone epigenetic modifications. By transient knockdown and CRISPR mutagenesis, we found that Rtf1 is essential for cardiogenesis and that without Rtf1 activity, cardiac progenitors arrest in an immature state. This role in early cardiogenesis was evolutionarily conserved between fish and mammals. We also found that Rtf1's Plus3 domain, which confers interaction with the pausing/elongation factor Spt5, was required for Rtf1's ability to support cardiac progenitor formation, while other regions of the protein were dispensable. We examined the occupancy of RNA Pol II at cardiac genes in rtf1 morphants using ChIP-seq and found that Pol II signals at the TSS of genes was reduced, suggesting a reduction in transcriptional pausing. Intriguingly, pharmacological or morpholino antisense reduction of pause release in rtf1 morphants and mutants restored the formation of cardiac cells and improved Pol II occupancy at the TSS of key cardiac genes. Our findings highlight the crucial role that transcriptional pausing plays in promoting normal levels of gene expression in a cardiac developmental context.
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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.
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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.
2022-08-09 | PXD033771 | Pride
Project description:The histone chaperone Spt6 is involved in promoting elongation of RNA polymerase II (RNAPII), maintaining chromatin structure, regulating co-transcriptional histone modifications, and controlling mRNA processing. These diverse functions of Spt6 are partly mediated through its interactions with RNAPII and other factors in the transcription elongation complex. In this study, we used mass spectrometry to characterize the differences in RNAPII interacting factors between wild-type cells and those depleted for Spt6, leading to the identification of proteins that depend on Spt6 for their interaction with RNAPII. In all, eight samples were processed - four genotypes (1. SPT6, RPB3-untagged; 2. SPT6, RPB3-tagged; 3. spt6-1004, RPB3-untagged; 4. spt6-1004; RPB3-tagged) in biological duplicates.
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