NDF/GLYR1 Promotes RNA Polymerase II Processivity via Pol II Binding and Nucleosome Destabilization
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ABSTRACT:
PROVIDER: PRJNA1248046 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| Other | ||||
| SRR33035468.fastq.gz | Fastqsanger.gz | |||
| SRR33035470.fastq.gz | Fastqsanger.gz | |||
| SRR33035471.fastq.gz | Fastqsanger.gz | |||
| SRR33035472.fastq.gz | Fastqsanger.gz |
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Project description:The Nucleosome Destabilizing Factor (NDF) facilitates transcription through chromatin, but its precise mechanism remains incompletely understood. Here, we identify a critical region (amino acids 140-160) within NDF that specifically interacts with phosphorylated RPB1, the largest subu-nit of elongating RNA Polymerase II (Pol II). Mutations in this region disrupt Pol II interaction and impair Pol II elongation both in vitro and in cells, yet do not affect NDF's ability to destabilize nu-cleosomes, establishing a functional separation between these two activities. Cellular studies re-veal that NDF knockout cells display faster Pol II elongation rates but produce fewer nascent tran-scripts, demonstrating NDF's primary role in maintaining transcriptional processivity through-out gene bodies. Our findings demonstrate that NDF uses distinct mechanisms to ensure productive transcription elongation rather than simply enhancing elongation speed, offering new insights in-to how transcription efficiency is maintained in chromatin.
2025-05-28 | GSE294088 | GEO
Project description:The Nucleosome Destabilizing Factor (NDF) facilitates transcription through chromatin, but its precise mechanism remains incompletely understood. Here, we identify a critical region (amino acids 140-160) within NDF that specifically interacts with phosphorylated RPB1, the largest subu-nit of elongating RNA Polymerase II (Pol II). Mutations in this region disrupt Pol II interaction and impair Pol II elongation both in vitro and in cells, yet do not affect NDF's ability to destabilize nu-cleosomes, establishing a functional separation between these two activities. Cellular studies re-veal that NDF knockout cells display faster Pol II elongation rates but produce fewer nascent tran-scripts, demonstrating NDF's primary role in maintaining transcriptional processivity through-out gene bodies. Our findings demonstrate that NDF uses distinct mechanisms to ensure productive transcription elongation rather than simply enhancing elongation speed, offering new insights in-to how transcription efficiency is maintained in chromatin.
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Project description:NDF/GLYR1 Promotes RNA Polymerase II Processivity via Pol II Binding and Nucleosome Destabilization
| PRJNA1248047 | ENA
Project description:RNA polymerase II (Pol II) elongation is a critical step in gene expression. Here we find that NDF, which was identified as a bilaterian nucleosome-destabilizing factor, is also a Pol II transcription factor that stimulates elongation with plain DNA templates in the absence of nucleosomes. NDF binds directly to Pol II and enhances elongation by a different mechanism than does transcription factor TFIIS. Moreover, yeast Pdp3, which is related to NDF, binds to Pol II and stimulates elongation. Thus, NDF is a Pol II-binding transcription elongation factor that is localized over gene bodies and is conserved from yeast to humans.
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Project description:Eukaryotic gene expression requires factors that assist RNA polymerase in transcribing through nucleosomes. Human FACT complex (Facilitates chromatin transcription) was originally discovered as an important transcription elongation factor that modifies nucleosome structures and lowers the nucleosome energy barrier for Pol II passage. However, the mechanism by which FACT is recruited to transcribed regions is unknown. Here, we show that the Nucleosome Disassembly Factor (NDF)-mediated FACT phase separation is a key mechanism for FACT recruitment to these regions. In vitro, we found that NDF physically interacts with FACT and works synergistically to enhance Pol II transcription through nucleosomes. This interaction leads to the formation of gel-like NDF-FACT condensates under physiological conditions. Using single-molecule assays, we found that an NDF-FACT condensate forms right before Pol II approaches the nucleosome. The formation of these condensates not only increases local protein concentrations and retention time but also destabilizes nucleosomes. In human stem cells, NDF and FACT condensates are largely colocalized and have characteristics of liquid-liquid phase separation (LLPS). Moreover, cells expressing a single-point mutation in the NDF-FACT interaction domain, which disrupts the formation of NDF-FACT condensates, exhibit slower growth compared to wild-type cells, along with transcription defects and a reduced number of hSpt16 condensates and chromatin occupancy in the nucleus. Our data highlight the importance of phase separation in regulating gene expression and present a novel mechanism by which cellular factors work together to improve transcription efficiency on chromatin.
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Project description:Eukaryotic gene expression requires factors that assist RNA polymerase in transcribing through nucleosomes. Human FACT complex (Facilitates chromatin transcription) was originally discovered as an important transcription elongation factor that modifies nucleosome structures and lowers the nucleosome energy barrier for Pol II passage. However, the mechanism by which FACT is recruited to transcribed regions is unknown. Here, we show that the Nucleosome Disassembly Factor (NDF)-mediated FACT phase separation is a key mechanism for FACT recruitment to these regions. In vitro, we found that NDF physically interacts with FACT and works synergistically to enhance Pol II transcription through nucleosomes. This interaction leads to the formation of gel-like NDF-FACT condensates under physiological conditions. Using single-molecule assays, we found that an NDF-FACT condensate forms right before Pol II approaches the nucleosome. The formation of these condensates not only increases local protein concentrations and retention time but also destabilizes nucleosomes. In human stem cells, NDF and FACT condensates are largely colocalized and have characteristics of liquid-liquid phase separation (LLPS). Moreover, cells expressing a single-point mutation in the NDF-FACT interaction domain, which disrupts the formation of NDF-FACT condensates, exhibit slower growth compared to wild-type cells, along with transcription defects and a reduced number of hSpt16 condensates and chromatin occupancy in the nucleus. Our data highlight the importance of phase separation in regulating gene expression and present a novel mechanism by which cellular factors work together to improve transcription efficiency on chromatin.
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Project description:Eukaryotic gene expression requires factors that assist RNA polymerase in transcribing through nucleosomes. Human FACT complex (Facilitates chromatin transcription) was originally discovered as an important transcription elongation factor that modifies nucleosome structures and lowers the nucleosome energy barrier for Pol II passage. However, the mechanism by which FACT is recruited to transcribed regions is unknown. Here, we show that the Nucleosome Disassembly Factor (NDF)-mediated FACT phase separation is a key mechanism for FACT recruitment to these regions. In vitro, we found that NDF physically interacts with FACT and works synergistically to enhance Pol II transcription through nucleosomes. This interaction leads to the formation of gel-like NDF-FACT condensates under physiological conditions. Using single-molecule assays, we found that an NDF-FACT condensate forms right before Pol II approaches the nucleosome. The formation of these condensates not only increases local protein concentrations and retention time but also destabilizes nucleosomes. In human stem cells, NDF and FACT condensates are largely colocalized and have characteristics of liquid-liquid phase separation (LLPS). Moreover, cells expressing a single-point mutation in the NDF-FACT interaction domain, which disrupts the formation of NDF-FACT condensates, exhibit slower growth compared to wild-type cells, along with transcription defects and a reduced number of hSpt16 condensates and chromatin occupancy in the nucleus. Our data highlight the importance of phase separation in regulating gene expression and present a novel mechanism by which cellular factors work together to improve transcription efficiency on chromatin.
2025-08-05 | GSE273680 | GEO
Project description:Eukaryotic gene expression requires factors that assist RNA polymerase in transcribing through nucleosomes. Human FACT complex (Facilitates chromatin transcription) was originally discovered as an important transcription elongation factor that modifies nucleosome structures and lowers the nucleosome energy barrier for Pol II passage. However, the mechanism by which FACT is recruited to transcribed regions is unknown. Here, we show that the Nucleosome Disassembly Factor (NDF)-mediated FACT phase separation is a key mechanism for FACT recruitment to these regions. In vitro, we found that NDF physically interacts with FACT and works synergistically to enhance Pol II transcription through nucleosomes. This interaction leads to the formation of gel-like NDF-FACT condensates under physiological conditions. Using single-molecule assays, we found that an NDF-FACT condensate forms right before Pol II approaches the nucleosome. The formation of these condensates not only increases local protein concentrations and retention time but also destabilizes nucleosomes. In human stem cells, NDF and FACT condensates are largely colocalized and have characteristics of liquid-liquid phase separation (LLPS). Moreover, cells expressing a single-point mutation in the NDF-FACT interaction domain, which disrupts the formation of NDF-FACT condensates, exhibit slower growth compared to wild-type cells, along with transcription defects and a reduced number of hSpt16 condensates and chromatin occupancy in the nucleus. Our data highlight the importance of phase separation in regulating gene expression and present a novel mechanism by which cellular factors work together to improve transcription efficiency on chromatin.
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Project description:Eukaryotic gene expression requires factors that assist RNA polymerase in transcribing through nucleosomes. Human FACT complex (Facilitates chromatin transcription) was originally discovered as an important transcription elongation factor that modifies nucleosome structures and lowers the nucleosome energy barrier for Pol II passage. However, the mechanism by which FACT is recruited to transcribed regions is unknown. Here, we show that the Nucleosome Disassembly Factor (NDF)-mediated FACT phase separation is a key mechanism for FACT recruitment to these regions. In vitro, we found that NDF physically interacts with FACT and works synergistically to enhance Pol II transcription through nucleosomes. This interaction leads to the formation of gel-like NDF-FACT condensates under physiological conditions. Using single-molecule assays, we found that an NDF-FACT condensate forms right before Pol II approaches the nucleosome. The formation of these condensates not only increases local protein concentrations and retention time but also destabilizes nucleosomes. In human stem cells, NDF and FACT condensates are largely colocalized and have characteristics of liquid-liquid phase separation (LLPS). Moreover, cells expressing a single-point mutation in the NDF-FACT interaction domain, which disrupts the formation of NDF-FACT condensates, exhibit slower growth compared to wild-type cells, along with transcription defects and a reduced number of hSpt16 condensates and chromatin occupancy in the nucleus. Our data highlight the importance of phase separation in regulating gene expression and present a novel mechanism by which cellular factors work together to improve transcription efficiency on chromatin.
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