Pieces of the Puzzle: Individual hFACT Subdomains Coordinate to Remodel Nucleosomes

Abstract

Chromatin is destabilized through a variety of mechanisms that enhance transcription by targeting DNA-protein interactions involved in nucleosome formation. We focus on specific disruptions due to the histone chaperone protein FACT (facilitates chromatin transcription), a heterodimer in humans (hFACT) comprised of Spt16 and SSRP1 subunits. Combined atomic force microscopy (AFM) and optical tweezers (OT) experiments yield nucleosomal stability and dynamics related to the affinity and function of hFACT and its component domains. We first find that hFACT binds strongly to nucleosomes (Kd = 21 +/- 5 nM), displacing the DNA from the H2A/H2B dimers and disrupting remaining interactions with a measured loss of nearly half of the tetramer-DNA binding energy. Within the SSRP1 subunit is an HMGB domain, comprised of a conserved DNA binding motif that we show binds directly to the bent DNA near the entry/exit from the nucleosome. Binding disrupts the H2A/H2B dimer - DNA interaction and leads to the release of the outer turn of DNA from the nucleosome, consistent with our previous work. In contrast, the MD and CTD domains of the Spt16 subunit serve to weakly stabilize the tetramer-DNA interaction, even after displacement of the dimer-DNA contacts. AFM experiments quantify dimer loss due to Spt16 MD and CTD. Finally, we find that nucleosomes can rewind DNA even after several cycles of force disruption, but only in the presence full hFACT. Thus, these domains function in concert, with the HMGB domain binding to DNA and the Spt16 MD and CTD binding to the histones. The result both increases DNA accessibility by displacing histones, and chaperones the residual structure, promoting reassembly.