Histone chaperones physically interact with histones to direct proper assembly and disassembly of nucleosomes regulating diverse nuclear processes such as DNA replication, promoter remodeling, transcription elongation, DNA damage, and histone variant exchange. Currently, the best-characterized chaperone-histone interaction is that between the ubiquitous chaperone Asf1 and a dimer of H3 and H4. Nucleosome assembly proteins (Nap proteins) represent a distinct class of histone chaperone. Using pulsed electron double resonance (PELDOR) measurements and protein crosslinking, we show that two members of this class, Nap1 and Vps75, bind histones in the tetrameric conformation also observed when they are sequestered within the nucleosome. Furthermore, H3 and H4 trapped in their tetrameric state can be used as substrates in nucleosome assembly and chaperone-mediated lysine acetylation. This alternate mode of histone interaction provides a potential means of maintaining the integrity of the histone tetramer during cycles of nucleosome reassembly.

Histone chaperones are proteins that interact with histones to regulate the thermodynamic process of nucleosome assembly. sNASP and ASF1 are conserved histone chaperones that interact with histones H3 and H4 and are found in a multi-chaperoning complex in vivo Previously we identified a short peptide motif within H3 that binds to the TPR domain of sNASP with nanomolar affinity. Interestingly, this peptide motif is sequestered within the known ASF1-H3-H4 interface, raising the question of how these two proteins are found in complex together with histones when they share the same binding site. Here, we show that sNASP contains at least two additional histone interaction sites that, unlike the TPR-H3 peptide interaction, are compatible with ASF1A binding. These surfaces allow ASF1A to form a quaternary complex with both sNASP and H3-H4. Furthermore, we demonstrate that sNASP makes a specific complex with H3 on its own in vitro, but not with H4, suggesting that it could work upstream of ASF1A. Further, we show that sNASP and ASF1A are capable of folding an H3-H4 dimer in vitro under native conditions. These findings reveal a network of binding events that may promote the entry of histones H3 and H4 into the nucleosome assembly pathway.

NAP-1 fold histone chaperones play an important role in escorting histones to and from sites of nucleosome assembly and disassembly. The two NAP-1 fold histone chaperones in budding yeast, Vps75 and Nap1, have previously been crystalized in a characteristic homodimeric conformation. In this study, a combination of small angle X-ray scattering, multi angle light scattering and pulsed electron-electron double resonance approaches were used to show that both Vps75 and Nap1 adopt ring-shaped tetrameric conformations in solution. This suggests that the formation of homotetramers is a common feature of NAP-1 fold histone chaperones. The tetramerisation of NAP-1 fold histone chaperones may act to shield acidic surfaces in the absence of histone cargo thus providing a 'self-chaperoning' type mechanism.