Human Fact Proteins Facilitate Both Disassembly And Reassembly Of Nucleosomes

Chromatin structure disruption enhances 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), comprising Spt16 and SSRP1 subunits. Combined atomic force microscopy (AFM), optical tweezers (OT) and single molecule fluorescence (SMF) experiments yield nucleosomal stability and dynamics information related to the affinity and function of hFACT and its component domains. We first find that hFACT binds strongly to nucleosomes, 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, containing a conserved DNA binding motif that we show binds directly to the bent DNA near the entry/exit from the nucleosome. Binding disrupts DNA interactions with the histone tails and leads to the release of the outer turn of DNA from the nucleosome, consistent with prior studies of HMGB domains. 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. Furthermore, we find that nucleosomes can reform even after several cycles of force disruption, but only in the presence full hFACT. AFM experiments reveal the extent of nucleosomal remodeling while SMF quantifies the kinetics of dimer and octamer loss under the influence of hFACT. Thus, hFACT domains must function in concert, with the HMGB domain binding to DNA and the Spt16 MD and CTD binding to histones. The result both increases DNA accessibility by displacing histones, and chaperones the residual structure, facilitating reassembly.