Structural Role of Pob3 CTD in FACT-Mediated Nucleosome Uncoiling Revealed by Electron Microscopy

Histone chaperone FACT plays a key role in chromatin reorganization by mediating ATP-independent nucleosome unwinding. The yeast yFACT complex consists of the Spt16 and Pob3 subunits, which form a heterodimer functionally associated with the nonhistone protein Nhp6. In this study, negative-stain transmission electron microscopy was used to investigate the interaction of the yFACT complex, containing the Pob3 subunit with the C-terminal domain (CTD) removed, with the nucleosome in the presence of Nhp6. As a result of CTD removal, the efficiency of FACT binding to the nucleosome decreased by a factor of two, and the ability to fully unfold the nucleosome was impaired: instead of the almost symmetrical, fully unfolded structures characteristic for wild type yFACT, asymmetrical, partially unfolded structures were observed. The data obtained indicate the key role of Pob3 CTD in ensuring FACT binding to the nucleosome, which is important for understanding the mechanisms of chromatin remodeling and transcription regulation.