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Overlapping and distinctfunctions of SPT6, PNUTS and PCF11 in regulating transcription termination [ChIP-Seq]

ABSTRACT: The histone chaperone and transcription elongation factor SPT6, integral to RNA Polymerase II (RNAPII) activity, also plays a crucial role in regulating transcription termination. In an attempt to identify the pathways employed by SPT6 in this regulation, we found that while SPT6 and its closest partner IWS1 interact and co-localize with RNAPII, their functions diverge significantly at gene termination sites. Our data revealed that SPT6 depletion, unlike IWS1, results in extensive readthrough transcription, indicating that SPT6 independently regulates transcription termination. Further analysis identified that the cleavage and polyadenylation factor PCF11 and the phosphatase regulatory protein PNUTS collaborate with SPT6 in this process. These findings suggest that SPT6 may facilitate transcription termination by recruiting PNUTS and PCF11 to RNAPII. Additionally, SPT6 and PNUTS jointly restrict promoter upstream transcripts (PROMPTs), whereas PCF11 presence is essential for their activation in the absence of SPT6. This study elucidates the distinct and overlapping functions of SPT6, PCF11, and PNUTS in transcription termination, highlighting the pivotal role of SPT6 in ensuring proper transcription termination at the 5’ and 3’ ends of genes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE272847 | GEO | 2025/03/14

REPOSITORIES: GEO

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Overlapping and distinctfunctions of SPT6, PNUTS and PCF11 in regulating transcription termination [RNA-Seq]

Project description:The histone chaperone and transcription elongation factor SPT6, integral to RNA Polymerase II (RNAPII) activity, also plays a crucial role in regulating transcription termination. In an attempt to identify the pathways employed by SPT6 in this regulation, we found that while SPT6 and its closest partner IWS1 interact and co-localize with RNAPII, their functions diverge significantly at gene termination sites. Our data revealed that SPT6 depletion, unlike IWS1, results in extensive readthrough transcription, indicating that SPT6 independently regulates transcription termination. Further analysis identified that the cleavage and polyadenylation factor PCF11 and the phosphatase regulatory protein PNUTS collaborate with SPT6 in this process. These findings suggest that SPT6 may facilitate transcription termination by recruiting PNUTS and PCF11 to RNAPII. Additionally, SPT6 and PNUTS jointly restrict promoter upstream transcripts (PROMPTs), whereas PCF11 presence is essential for their activation in the absence of SPT6. This study elucidates the distinct and overlapping functions of SPT6, PCF11, and PNUTS in transcription termination, highlighting the pivotal role of SPT6 in ensuring proper transcription termination at the 5’ and 3’ ends of genes.

2025-03-14 | GSE272848 | GEO

Overlapping and distinctfunctions of SPT6, PNUTS and PCF11 in regulating transcription termination [RNA-Seq]

Project description:Overlapping and distinctfunctions of SPT6, PNUTS and PCF11 in regulating transcription termination [RNA-Seq]

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Overlapping and distinctfunctions of SPT6, PNUTS and PCF11 in regulating transcription termination [ChIP-Seq]

Project description:Overlapping and distinctfunctions of SPT6, PNUTS and PCF11 in regulating transcription termination [ChIP-Seq]

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Spt6 is required for heterochromatic silencing in the fission yeast Schizosaccharomyces pombe

Project description:Spt6 is a conserved factor, critically required for several transcription and chromatin related processes. We now show that Spt6 and its binding partner, Iws1, are required for heterochromatic silencing in Schizosaccharomyces pombe. Our studies demonstrate that Spt6 is required for silencing of all heterochromatic loci and that an spt6 mutant has an unusual combination of heterochromatic phenotypes compared to previously studied silencing mutants. Unexpectedly, we find normal nucleosome positioning over heterochromatin and normal levels of histone H3K9 dimethylation. However, we also find greatly reduced levels of H3K9 trimethylation, elevated levels of H3K14 acetylation, and reduced recruitment of several silencing factors. Our evidence suggests that Spt6 plays a role at both the transcriptional and post-transcriptional levels; in an spt6 mutant, RNA polymerase II (RNAPII) occupancy at the pericentric regions is only modestly increased, while production of small interfering RNAs (siRNAs) is lost. Taken together, our results suggest that Spt6 is required for multiple steps in heterochromatic silencing by controlling chromatin, transcriptional, and post-transcriptional processes.

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PP1/PNUTS phosphatase binds the restrictor complex and stimulates RNA pol II transcription termination

Project description:The restrictor, ZC3H4/WDR82, terminates antisense transcription from bidirectional promoters, but its mechanism is poorly understood. We report that ZC3H4/WDR82 immunoprecipitate with PP1 phosphatase and its nuclear targeting subunit, PNUTS, which binds to WDR82. AlphaFold predicts a complex of PP1/PNUTS with restrictor where both PNUTS and ZC3H4 contact WDR82. A substrate trap, PP1H66K-PNUTS, comprising inactive PP1 fused to the PNUTS C-terminus antagonizes restrictor mediated termination whereas PP1WT-PNUTS has less effect suggesting that phosphatase activity is required for termination. One PP1/PNUTS substrate implicated in termination by restrictor is pol II CTD Ser5-P. PP1H66K-PNUTS induces Ser5-P hyperphosphorylation at 5’ ends presumably by inhibiting dephosphorylation. NET-seq analysis suggests that CTD Ser5 dephosphorylation would promote termination by increasing pol II pausing. Both inhibition of termination and CTD hyperphosphorylation require the WDR82 binding domain of PP1H66K-PNUTS that mediates restrictor binding. In summary, the PP1/PNUTS phosphatase associated with restrictor via WDR82 promotes efficient transcription termination.

2025-04-06 | PXD061598 | Pride

Restrictor synergizes with Symplekin and PNUTS to terminate extragenic transcription

Project description:Transcription termination pathways mitigate the detrimental consequences of unscheduled promiscuous initiation occurring at hundreds of thousands of genomic cis-regulatory elements. The Restrictor complex, composed of the Pol II-interacting protein WDR82 and the RNA-binding protein ZC3H4, suppresses processive transcription at thousands of extragenic sites in mammalian genomes. Restrictor-driven termination does not involve nascent RNA cleavage and its interplay with other termination machineries is unclear. Here we show that efficient termination at Restrictor-controlled extragenic transcription units involves the recruitment of the protein phosphatase 1 (PP1) regulatory subunit PNUTS, a negative regulator of the Spt5 elongation factor, and Symplekin, a protein associated with RNA cleavage complexes but also involved in cleavage-independent and phosphatase-dependent termination of non-coding RNAs in yeast. PNUTS and Symplekin act synergistically with, but independently from Restrictor to dampen processive extragenic transcription. Moreover, the presence of limiting nuclear levels of Symplekin imposes a competition for its recruitment among multiple transcription termination machineries, resulting in mutual regulatory interactions. Hence, by synergizing with Restrictor, Symplekin and PNUTS enable efficient termination of processive, long-range extragenic transcription.

2024-05-23 | PXD043638 | Pride

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

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.

2021-12-05 | PXD027708 | Pride

Pcf11/Spt5 condensates stall RNA polymerase II to facilitate termination and piRNA-guided heterochromatin formation [CUT&Tag]

Project description:The Piwi-interacting RNA (piRNA) pathway plays a crucial role in protecting animal germ cells by repressing transposons. However, the mechanism behind this RNA-mediated epigenetic regulation that leads to heterochromatin formation is not yet fully understood. Through RNA interference screens, we discovered Pcf11 and PNUTS, two conserved termination factors of RNA polymerase II (Pol II), as essential for piRNA-guided silencing. When Pcf11 is artificially tethered to a reporter, it leads to co-transcriptional repression and Pol II stalling, both of which are dependent on an alpha-helical region of Pcf11 capable of forming condensates when triggered by the C-terminal repeat domain (CTD) of Pol II. Interestingly, an intrinsically disordered region (IDR) derived from the Plant FCA can substitute for the alpha-helical region of Pcf11 in its silencing capacity and support animal development, demonstrating a causal relationship between phase separation and Pcf11’s function. Further exploration into how Pcf11 causes Pol II stalling revealed a direct interaction between Pcf11’s CTD-interaction domain (CID) and a CTR region of Spt5. The dephosphorylation of Spt5-CTR by PP1/PNUTS is essential for Pcf11’s recruitment during Pol II termination. It appears that the phosphorylated state of CTR inhibits Pcf11 condensate formation, while the phosphorylation of Pol II CTD at Threonine 4 enhances Pcf11’s ability to undergo phase separation. In conclusion, we propose a model in which Pcf11 regulates transcriptional termination by slowing down Pol II elongation, a process that is aided by phase separation with the unphosphorylated Spt5. The ability of Pcf11 to stall Pol II, utilized by the piRNA pathway to initiate heterochromatin formation, might represent a universal strategy for nascent RNA-mediated epigenetic control.

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Pcf11/Spt5 condensates stall RNA polymerase II to facilitate termination and piRNA-guided heterochromatin formation [RNA-Seq]

2025-01-27 | GSE261847 | GEO