Visualizing both nucleosome disassembly and reassembly during FACT chaperone activity.

Abstract

The histone chaperone FACT (FAcilitates Chromatin Transcription) enhances transcription in eukaryotic cells, targeting DNA-protein interactions. FACT, a heterodimer in humans, comprises SPT16 and SSRP1 subunits. We measure nucleosome stability and dynamics in the presence of FACT and critical component domains. Optical tweezers quantify FACT/subdomain binding to nucleosomes, displacing the outer wrap of DNA, disrupting direct DNA-histone (strong site) interactions, altering the energy landscape of unwrapping and increasing the kinetics of DNA-histone disruption. Atomic force microscopy reveals nucleosome remodeling and DNA unwinding. Single molecule multi-channel fluorescence observes component histones as they disassemble under tension and are lost. Yet if tension is released, reassembly is also observed and surprisingly both loss and reassembly are accelerated by key domains of FACT. We resolve this ambiguity, noting that all histone-DNA kinetics are faster with FACT. Of the individual domains we studied, two were shown to influence nucleosome structure and dynamics with distinctly contradictory functions; while the SSRP1 HMGB domain displaces DNA, SPT16 MD/CTD stabilizes DNA-H2A/H2B dimer interactions. However, only intact FACT tethers disrupted DNA to the histones, and supports rapid nucleosome reformation over several cycles of force disruption/release. These results demonstrate how key FACT domains combine to catalyze nucleosome disassembly and reassembly.