NDF-FACT phase separation condensates are required for RNA Polymerase II transcription elongation through chromatin [CUT&Run]

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE273679 | GEO | 2025/08/05

REPOSITORIES: GEO