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Targeted protein degradation reveals direct roles of SPT6 in POL2 elongation and termination

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE162264 | GEO | 2021/06/23

REPOSITORIES: GEO

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RNA Polymerase II interactome in presence or absence of SPT6 in U2OS cells

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

Targeted protein degradation reveals direct roles of SPT6 in POL2 elongation and termination

Project description:Targeted protein degradation reveals direct roles of SPT6 in POL2 elongation and termination

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Analysis of RNA polymerase II interacting proteins in wild-type and spt6-1004 yeast cells.

2021-12-05 | PXD027708 | Pride

The histone chaperone Spt6 is required for normal recruitment of the capping enzyme Abd1 to transcribed regions

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The Elongation Factor and Histone Chaperone Spt6 Maintains ESC Pluripotency by Controlling Super-Enhancers and Counteracting Polycomb Repressive Complex 2

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The interaction between the Spt6-tSH2 domain and Rpb1 affects multiple functions of RNA Polymerase II [MNase-seq]

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 of the Rpb1 subunit in the linker between the catalytic core and the C-terminal domain (CTD) heptad repeats. This interaction contributes to generic localization of Spt6, but we show here that it also has gene-specific roles. Disrupting the interface affected transcription start site selection at a subset of genes whose expression is regulated by this choice, and this was accompanied by changes in a distinct pattern of Spt6 accumulation at these sites. Splicing efficiency was also diminished, as was apparent progression through introns that encode snoRNAs. Chromatin-mediated repression was impaired, and a distinct role in maintaining +1 nucleosomes was identified, especially at ribosomal protein genes. The Spt6-tSH2:Rpb1 interface therefore has both genome-wide functions and local roles at subsets of genes where dynamic decisions regarding initiation, transcript processing, or termination are made. We propose that the interaction modulates the availability or activity of the core elongation and histone chaperone functions of Spt6, contributing to coordination between RNAPII and its accessory factors as varying local conditions call for dynamic responses.

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Spt6 association with RNAPII directs mRNA turnover during transcription

Project description:Spt6 is an essential histone chaperone that mediates nucleosome reassembly during gene transcription. Spt6 interacts with elongating RNA polymerase II (RNAPII) via a tandem Src2 homology (tSH2) domain, but it is not known whether this particular interaction is required for the nucleosome reassembly activity of Spt6. Here, we show that Spt6 recruitment to genes and its nucleosome reassembly functions are largely independent of association with RNAPII. Instead, the Spt6-RNAPII association is required for post-transcriptional mRNA turnover. Mechanistically, association of Spt6 with RNAPII couples the Ccr4-Not complex to the transcribed regions of genes, which we show regulates the timely deadenylation and degradation of a broad range of mRNAs including those required for cell cycle progression. Thus, our findings reveal an unexpected control mechanism for mRNA turnover facilitated by a histone chaperone during transcription.

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Essential histone chaperones collaborate to regulate transcription and chromatin integrity

Project description:Histone chaperones are critical for controlling chromatin integrity during transcription, DNA replication, and DNA repair. We have discovered that the physical interaction between two essential histone chaperones, Spt6 and Spn1/Iws1, is required for transcriptional accuracy and nucleosome organization. To understand this requirement, we have isolated suppressors of an spt6 mutation that disrupts the Spt6-Spn1 interaction. Several suppressors are in a third essential histone chaperone, FACT, while another suppressor is in the transcription elongation factor Spt5/DSIF. The FACT suppressors weaken FACT-nucleosome interactions and bypass the requirement for Spn1, possibly by restoring a necessary balance between Spt6 and FACT on chromatin. In contrast, the Spt5 suppressor modulates Spt6 function in a Spn1-dependent manner. Despite these distinct mechanisms, both suppressors alleviate the nucleosome organization defects caused by disruption of the Spt6-Spn1 interaction. Taken together, we have uncovered a network in which histone chaperones and other elongation factors coordinate transcriptional integrity and chromatin structure.

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