NDF Is a Transcription Factor that Stimulates Elongation by RNA Polymerase II
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE185464 | GEO | 2022/03/10
REPOSITORIES: GEO
Similar Datasets
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 | GSE294087 | 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.
2025-05-28 | GSE294088 | 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.
2025-08-05 | GSE300859 | 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.
2025-08-05 | GSE273681 | 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.
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.
2025-08-05 | GSE273679 | 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.
2025-08-05 | GSE273678 | GEO
Project description:The super elongation complex (SEC) contains the positive transcription elongation factor b (P-TEFb) and a subcomplex, ELL2-EAF1, which stimulates transcription elongation by RNA polymerase II (Pol II). Here we report the cryo-EM structure of ELL2-EAF1 bound to a Pol II elongation complex at 2.8 Å resolution. The ELL2-EAF1 dimerization module directly binds the Pol II lobe, explaining how SEC delivers P-TEFb to Pol II. The same site on the lobe also binds the initiation factor TFIIF, consistent with SEC binding only after the transition from transcription initiation to elongation. Structure-guided functional analysis shows that elongation stimulation requires the dimerization module and an ELL2 protein linker that tethers this module to the Pol II protrusion. Our results show that SEC stimulates elongation allosterically and indicate that this stimulation involves stabilization of a further closed conformation of the Pol II active center cleft.
2021-07-15 | PXD024457 | Pride
Project description:NDF Is a Transcription Factor that Stimulates Elongation by RNA Polymerase II
| PRJNA769120 | ENA
Project description:Elongin is a hetero-trimeric elongation factor for RNA polymerase (Pol) II transcription that is conserved among metazoa. We solved three structures of human Elongin bound to transcribing Pol II using cryo-EM assisted by crosslinking mass spectrometry. The structures show that Elongin subunit ELOA binds the RPB2 side of Pol II and anchors the ELOB-8 ELOC subunit heterodimer. ELOA contains an N-terminal ‘latch’ that binds between the end of the RPB1 bridge helix and the funnel helices, thereby inducing a conformational change near the Pol II active center. The latch is strictly required for the elongation-stimulatory activity of Elongin, but not for its binding to Pol II, indicating that Elongin functions by allosterically influencing the conformational mobility of the active center. Structural comparisons show that Elongin binding to Pol II is incompatible with association of super elongation complex, the PAF1 complex, and RTF1, which also contains a latch element that stimulates Pol II.
2023-10-11 | PXD045446 | Pride