FACT caught in the act of manipulating the nucleosome

The organization of genomic DNA into nucleosomes profoundly affects all DNA-related processes in eukaryotes. The histone chaperone ‘FAcilitates Chromatin Transcription’ (FACT; consisting of subunits SPT16 and SSRP11) promotes both disassembly and reassembly of nucleosomes during gene transcription, DNA replication, and repair2. The mechanism by which FACT causes these opposing outcomes is unknown. Here we report two cryo-EM structures of human FACT in complex with partially assembled sub-nucleosomes, with supporting biochemical and hydrogen–deuterium exchange (HDX) data. FACT is engaged in extensive interactions with nucleosomal DNA and all histones. The large DNA-binding surface on FACT appears to be protected by the C-terminal domains of both subunits, and this inhibition is released by interaction with H2A–H2B, allowing FACT–H2A–H2B to dock onto a (H3–H4)2–DNA complex3. SPT16 binds nucleosomal DNA and tethers H2A–H2B through its C-terminal domain by acting as a placeholder for DNA. SSRP1 also contributes to DNA binding, and can assume two conformations, depending on whether a second H2A–H2B dimer is present. Our data suggest a compelling mechanism for how FACT maintains chromatin integrity during polymerase passage, by facilitating H2A–H2B dimer removal, stabilizing intermediate ‘sub-nucleosomal’ states, and promoting nucleosome reassembly. Our findings reconcile discrepancies regarding the many roles of FACT and underscore the dynamic interactions between histone chaperones and nucleosomes.