Phase separation of NDF-FACT promotes transcription elongation through chromatin

The FACT complex (facilitates chromatin transcription) is an essential factor that plays pivotal roles in various aspects of chromatin biology, including transcription, DNA replication, and DNA repair1-5. During transcription, FACT modifies nucleosome structures, reducing the energy barrier for the passage of RNA polymerases, and maintains chromatin structure after transcription6-16. Despite its critical role, the mechanism of FACT recruitment to transcribed regions in human cells has remained elusive2. Here, we show that Nucleosome Destabilizing Factor (NDF)-mediated phase separation is essential for FACT recruitment. In vitro studies reveal that NDF physically interacts with FACT, synergistically enhancing Pol II transcription through nucleosomes. This interaction leads to the formation of gel-like NDF-FACT liquid-liquid phase separation (LLPS) condensates, which exhibit significantly enhanced biochemical activities in modifying nucleosome energy landscapes, promoting FACT engagement, and ensuring histone retention post-disassembly. In human stem cells, NDF and FACT condensates predominantly colocalize at actively transcribed regions, exhibiting LLPS characteristics. Rapid depletion of NDF using an auxin-inducible degron system significantly reduces FACT puncta and chromatin enrichment. Furthermore, cells harboring a single-point mutation in the NDF-FACT interaction domain, which disrupts FACT condensate formation, show impaired growth, transcriptional defects, and decreased FACT chromatin occupancy. Our findings reveal a novel mechanism by which NDF-mediated phase separation facilitates FACT recruitment and enhances transcription efficiency through chromatin.