The histone chaperone sNASP binds a conserved peptide motif within the globular core of histone H3 through its TPR repeats

Abstract

Eukaryotic chromatin is a complex yet dynamic structure, which is regulated in part by the assembly and disassembly of nucleosomes. Key to this process is a group of proteins termed histone chaperones that guide the thermodynamic assembly of nucleosomes by interacting with soluble histones. Here we investigate the interaction between the histone chaperone sNASP and its histone H3 substrate. We find that sNASP binds with nanomolar affinity to a conserved heptapeptide motif in the globular domain of H3, close to the C-terminus. Through functional analysis of sNASP homologues we identified point mutations in surface residues within the TPR domain of sNASP that disrupt H3 peptide interaction, but do not completely disrupt binding to full length H3 in cells, suggesting that sNASP interacts with H3 through additional contacts. Furthermore, chemical shift perturbations from 1H-15N HSQC experiments show that H3 peptide binding maps to the helical groove formed by the stacked TPR motifs of sNASP. Our findings reveal a new mode of interaction between a TPR repeat domain and an evolutionarily conserved peptide motif found in canonical H3 and in all histone H3 variants, including CenpA and have implications for the mechanism of histone chaperoning within the cell.

Evidence for the nuclear import of histones H3.1 and H4 as monomers

“…A previous study demonstrated that sNASP can bind both monomeric H3 and an H3‐H4 dimer in complex with ASF1 in vitro (Bowman et al , ). Interaction with the monomeric H3 is mediated in part through NASP's TPR domain binding to the C‐terminus of H3 (Bowman et al , ). As importins interact with the N‐terminal region of histones, the C‐terminal region of H3 would be free to hand‐off to NASP.…”

“…The rationale behind the experiment was as follows: as RbAp46 and HAT1 bind to H4 epitopes within the H3.1-H4 heterodimer (Murzina et al, 2008;Song et al, 2008) (Fig 4G), if tethered histones were folded with their endogenous counterpart, we would expect to see the recruitment of RbAp46 and HAT1 to both tethered H3.1 and tethered H4, whereas if histones were monomeric, we would expect to see recruitment to tethered H4, but not H3.1. Conversely, as sNASP interacts directly with H3 as a monomer and as an H3.1-H4 heterodimer (Bowman et al, 2016(Bowman et al, , 2017, we would expect to see recruitment to both tethered H3.1 and H4 if a heterodimer was present, but only to tethered H3.1 if the histones were monomeric. As ASF1 contacts both H3 and H4 through independent binding sites (English et al, 2005;Natsume et al, 2007), we may expect to see recruitment to both independent of the histone's oligomeric status.…”

Histone chaperone networks shaping chromatin function

“…3Ab) has been characterized as a reader module for the H3 tail 25,26 , whereas the unrelated TPR domain of sNASP (Hif1 in yeast) 27 (FIG. 3B) also binds the H3 α3 helix 28 . HJURP 29 , Scm3 (REF.…”

“…Curiously, sNASP, RBAP46 and ASF1 seem to be capable of engaging histones H3–H4 in a single co-chaperone complex 87,89–91 . However, ASF1 and sNASP may have overlapping binding sites at the H3–H4 tetramerization interface 22,23,28 . Therefore, for sNASP and ASF1 to engage histones at the same time (the interaction between ASF1 and sNASP is histone dependent 87 ), additional H3–H4-binding sites may be present in sNASP to allow the partial handover of the H3–H4 tetramerization interface to ASF1.…”

A Poly-ADP-Ribose Trigger Releases the Auto-Inhibition of a Chromatin Remodeling Oncogene

“…Fluorescence two-hybrid (F2H) assay F2H assays were performed as previously described (Bowman et al, 2016;Czarna et al, 2013). Briefly, assays were performed in borosilicate 8-well Lab-Teks (Thermo Scientific).…”