FACT orchestrates the interplay between chromatin structure and transcription

FACT has been identified as a histone chaperone that enables transcription through chromatin in vitro, but its role in regulating chromatin structure and transcription in vivo remains unclear. In this study, we have investigated the function and molecular mechanism of FACT in unprecedented detail, by using a rapid depletion system in combination with high-resolution genomic analyses. We show that acute depletion of FACT leads to changes in 3D chromatin structure and a concomitant multilayered transcriptional defect, including loss of promoter-proximal pausing, deregulated elongation and increased drop-off of RNA Pol II. Integration of in vitro transcription assays shows that FACT stimulates RNA Pol II pausing by stabilizing nucleosomal architecture, while simultaneously promoting transcription through the +1 nucleosome. In addition to providing detailed mechanistic insight into the seemingly contradictory functions of FACT and the regulation of promoter-proximal pausing, our study indicates a direct coupling of chromatin structure and transcription. Overall design: Tiled-Micro-Capture-C (Tiled-MCC). Details are described in the Methods section of the paper.