Phase-separated NDF-FACT condensates facilitate transcription elongation on chromatin [PRO-Seq]

How the facilitates chromatin transcription (FACT) complex enables RNA polymerase II to overcome chromatin barriers in cells remains poorly understood—especially given the limited direct interactions of FACT with polymerases, DNA or nucleosomes. Here we demonstrate that phase separation, mediated by nucleosome destabilizing factor (NDF), is a key mechanism enabling the function of FACT during transcription elongation. Through biochemical and single-molecule assays, we found that NDF-FACT condensates create specialized biochemical environments that enhance transcription efficiency approximately 20-fold compared with FACT alone. These dynamic condensates form on transcribing RNA polymerase II and travel along chromatin, where they promote efficient nucleosome disassembly at barriers while retaining histones on DNA to preserve chromatin integrity. In human stem cells, disruption of these condensates leads to genome-wide transcriptional defects and chromatin instability, mirroring the effects of FACT depletion. By showing that phase separation enhances FACT function during transcription elongation, our study reveals a key mechanism that preserves chromatin integrity and transcriptional homeostasis in human stem cells. Overall design: Pro-seq analysis of NDF_WT and NDF_K161A mutant cells.