Electron microscopy analysis of ATP-independent nucleosome unfolding by FACT

Sivkina et al. present a biochemical and biophysical characterization of the interaction of S. cerevisiae histone chaperone FACT with the nucleosome core particle. They show that FACT adopts a more open geometry in the presence of Nhp6, and together they unfold nucleosomes to an almost extended conformation, suggesting a mechanism for FACT-facilitated disassembly of nucleosomes. FACT is a histone chaperone that participates in nucleosome removal and reassembly during transcription and replication. We used electron microscopy to study FACT, FACT:Nhp6 and FACT:Nhp6:nucleosome complexes, and found that all complexes adopt broad ranges of configurations, indicating high flexibility. We found unexpectedly that the DNA binding protein Nhp6 also binds to the C-terminal tails of FACT subunits, inducing more open geometries of FACT even in the absence of nucleosomes. Nhp6 therefore supports nucleosome unfolding by altering both the structure of FACT and the properties of nucleosomes. Complexes formed with FACT, Nhp6, and nucleosomes also produced a broad range of structures, revealing a large number of potential intermediates along a proposed unfolding pathway. The data suggest that Nhp6 has multiple roles before and during nucleosome unfolding by FACT, and that the process proceeds through a series of energetically similar intermediate structures, ultimately leading to an extensively unfolded form.