A Quantitative Characterization of Nucleoplasmin/Histone Complexes Reveals Chaperone Versatility (original) (raw)

Nucleoplasmin (NP) is an abundant histone chaperone in vertebrate oocytes and embryos involved in storing and releasing maternal histones to establish and maintain the zygotic epigenome. NP has been considered a H2A-H2B histone chaperone, and recently it has been shown that it can also interact with H3-H4. However, its interaction with different types of histones has not been quantitatively studied so far. We show here that NP binds H2A-H2B, H3-H4 and linker histones with K d values in the subnanomolar range, forming different complexes. Post-translational modifications of NP regulate exposure of the polyGlu tract at the disordered distal face of the protein and induce an increase in chaperone affinity for all histones. The relative affinity of NP for H2A-H2B and linker histones and the fact that they interact with the distal face of the chaperone could explain their competition for chaperone binding, a relevant process in NP-mediated sperm chromatin remodelling during fertilization. Our data show that NP binds H3-H4 tetramers in a nucleosomal conformation and dimers, transferring them to DNA to form disomes and tetrasomes. This finding might be relevant to elucidate the role of NP in chromatin disassembly and assembly during replication and transcription. Xenopus laevis early embryogenesis is a process characterized by rapid cell division and transcriptional quiescence that depends on parental stored proteins, including histones 1. Histone chaperones bind these basic ligands to store or escort them to their final destinations 2 , and to modulate the post-translational modifications that regulate their chromatin remodelling activity 3-6. Nucleoplasmin (NPM2, here called NP), a member of the nucleophosmin/nucleoplasmin family of histone chaperones 7 , is involved in H2A-H2B storage and chromatin remodelling 8. NP forms stable homopentamers comprised of 22 kDa subunits that fold into a two domain structure (Fig. 1A): an eight-stranded β-barrel N-terminal core domain (residues 16-120) with a jelly roll topology (Fig. 1B) 7,9-12 , and the C-terminus tail that adopts a disordered conformation 13. NP contains three acidic tracts, A1, A2 (or polyGlu) and A3, the last two being part of the C-terminal intrinsically disordered domain (Fig. 1A) that builds the so-called distal face of the protein pentamer (Fig. 1C). This domain also contains the positively charged, bipartite nuclear localization sequence (NLS) (Fig. 1A) 7,14,15. Post-translational modifications (PTMs), in particular phosphorylation, of NP activates its chromatin decondensation activity, enhancing its ability to remove linker histones from DNA 16,17 , and increases its affinity for H2A-H2B and H5 18,19. NP co-immunoprecipitates not only with H2A-H2B but also with H3-H4 in Xenopus oocyte and egg extracts, suggesting that it can interact with both histones 20. Electron microscopy (EM) analysis of full-length, native NP from Xenopus laevis eggs (eNP), isolated and in complexes with H2A-H2B, H3-H4 or histone octamers shows the highly acidic distal face of the chaperone contacting all types of histones 19,20. Interestingly, the interaction of eNP with H2A-H2B, H3-H4 or the histone octamer results in different complexes. As seen by EM and analytical ultracentrifugation, one eNP pentamer can bind 5 H2A-H2B dimers at the distal face, whereas a larger ellipsoidal complex is formed with the H3-H4 tetramer either alone or as part of the histone octamer, in which the basic ligands are wrapped by the distal faces of two NP pentamers 20 .