NASP maintains histone H3–H4 homeostasis through two distinct H3 binding modes

Abstract

Histone chaperones regulate all aspects of histone metabolism. NASP is a major histone chaperone for H3–H4 dimers critical for preventing histone degradation. Here, we identify two distinct histone binding modes of NASP and reveal how they cooperate to ensure histone H3–H4 supply. We determine the structures of a sNASP dimer, a complex of sNASP with an H3 α3 peptide, and the sNASP–H3–H4–ASF1b co-chaperone complex. This captures distinct functionalities of NASP and identifies two distinct binding modes involving the H3 α3 helix and the H3 αN region, respectively. Functional studies demonstrate the H3 αN-interaction represents the major binding mode of NASP in cells and shielding of the H3 αN region by NASP is essential in maintaining the H3–H4 histone soluble pool. In conclusion, our studies uncover the molecular basis of NASP as a major H3–H4 chaperone in guarding histone homeostasis.

A specific role for Importin-5 and NASP in the import and nuclear hand-off of monomeric H3

“…NASP can chaperone a monomer of H3 in vitro (31), bind to an epitope obscured in the H3-H4 interface (28, 29), and pulldown supra-stoichiometric amounts of H3 over H4 when isolated from HeLa cell extracts (21). To probe the NASP-associated histone pool at endogenous levels, we tagged NASP at its endogenous locus with TEV-cleavable (TEVcs) eGFP using CRISPR (Fig.…”

“…3E). This was intriguing as only the shorter sNASP isoform was expressed as the eGFP-fusion and may relate to a low level of dimerisation with the endogeous protein, which has been suggested from the crystal structure (29, 30) and biochemical analysis (41). Interestingly, only the s, and not the t, isoform co-migrated with the H3 monomer band.…”

“…The role of NASP as a holdase is easily reconciled with the idea of NASP overseeing a reservoir of soluble histones (27, 63), which would be compatible with its acting in the nucleus. In addition, the presence of NASP in downstream complexes (16, 19), mediated by interactions outside of the TPR-H3 peptide interface (29–31), supports NASP guiding the transition from monomeric import to a heterodimer capable of DNA deposition (31).…”

“…Interestingly, in NASP pulldowns we find histone H4K5Ac peaking in bands with sparse HAT1, suggesting a catalytic turnover of HAT1, but with retention of RBBP7, which may be related to its interactions with H3 N-terminal tail (66). The low levels of ASF1 detected in complex with HAT1 is notable and suggests a transitory state, potentially involving competition between the TPR domain of NASP and ASF1 for the H3 alpha 3 helix (29, 34, 35).…”

“…Subsequently, NASP has been shown to interact predominantly with H3 and H4 (15, 16, 19, 27). NASP displays multivalent interactions with its histone cargo, binding to a peptide epitope at the C-terminus of H3 via a canonical TPR-peptide interaction (28, 29), and to an N-terminal region of H3 via a site on the surface of the TPR domain (27, 29, 30). The latter interaction is structurally compatible with H3-H4 dimer binding in conjunction with ASF1.…”