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	Figure 1. Structural properties of Nucleoplasmin.(A) Schematic representation of the primary structure of native egg and oocyte NP, full length recombinant NP (rNP) and the two deletion mutants rNPΔ150–200 and rNPΔ120–200 used in this study. The core (residues 1–120; light gray) and tail (residues 121–200; white) domains are also shown. Phosphorylation sites (circles) and the location of the three acidic tracts (A1, A2, A3; black boxes) and the NLS (dark grey box) are highlighted. (B) Crystal structure of the N-terminal core domain of one NP monomer (amino acids 16–120). The location of the A1 acidic tract (dotted line), and of the distal, and lateral protein faces is indicated (PDB 1K5J). (C) Electron microscopy reconstruction of eNP. Side view of the final volume of the three-dimensional reconstruction of eNP19.
	Figure 2. Phosphorylation and exposure of the polyGlu tract modulates NP affinity for H2A–H2B and linker histones.(A) Normalized fluorescence intensity change (Eq. 1) of H2A–H2BT112C-Alexa 488 at increasing concentration of eNP (filled circles), oNP (empty circles), rNPΔ150–200 (gray squares), rNP (filled diamonds), and rNPΔ120–200 (empty diamonds). In this particular experiment histone concentration was 1 nM and NP concentration is given for the protein pentamer. Values of the mean ± SEM from three independent experiments are shown. The Kd values obtained from fitting the experimental data to eqs. 2 and 3 are included in Table 1. (B) Determination of the saturation stoichiometry of the NP/H2A–H2B complexes. Normalized change in the fluorescence intensity (Eq. 1) as a function of the NP pentamer/H2A–H2BT112C-Alexa molar ratio. The intersection of the linear phase with the plateau gives the molar ratio at which pentameric eNP (filled circles), oNP (empty circles), rNPΔ150–200 (gray squares), and rNP (filled diamonds) are saturated with H2A–H2BT112C. (C) Fluorescence competition assays in which complexes of eNP (0.1 nM)/H2A–H2B-Alexa 488 (1 nM) are competed with increasing concentrations of unlabelled H1 (filled circles) or H5 (empty circles). Controls of the fluorescence intensity of H2A–H2BT112C-Alexa 488 alone (gray circles), and in the presence of 0.3 μM H1 (filled squares), 0.3 μM H5 (empty squares) or 0.1 nM eNP (gray diamonds). D) Normalized fluorescence change (Eq. 1) of eNP/ (circles) or rNP/H2A–H2BT112C-Alexa (squares) complexes titrated with increasing concentrations of unlabelled H1 (filled symbols) or H5 (empty symbols). NP and H2A–H2BT112C-Alexa concentrations were 0.1 and 1 nM, respectively for the eNP/H2A–H2B complex, and 0.5 and 5 nM for the rNP/H2A–H2B complex. Experimental data were fitted to eqs. 6 and 7. Values of the mean ± SEM from three independent experiments are shown. The apparent Kd values are shown in Table 1.
	Figure 3. Nucleoplasmin binds H3C110A-H4 dimers and tetramers, forming different types of complexes.Fluorescence intensity change as a function of the eNP/H3-H4 molar ratio for the different H3-H4 variants: H3C110A-H4T71C-Alexa 488 (empty circles), H3C110E-H4T71C-Alexa 488 (gray circles), and cross-linked H3C110AK115C-H4T71C-Alexa 488 (filled circles). Histone concentration was kept constant at 10 nM. Experimental data are means ± SEM from three independent experiments.
	Figure 4. NP affinity for H3-H4 dimers and cross-linked tetramers.(A) Titration of H3C110E-H4T71C-Alexa 488 with different NP variants. Normalized fluorescence intensity change (Eq. 1) of H3C110E-H4T71C-Alexa 488 as a function of eNP (filled circles), oNP (empty circles), rNPΔ150–200 (gray squares), rNP (filled diamonds), or rNPΔ120–200 (empty diamonds) concentration. In this particular experiment histone concentration was 1 nM and NP concentration is given for the pentamer. (B) Normalized fluorescence intensity change (Eq. 1) of H3C110E-H4T71C-Alexa 488 as a function of the NP pentamer/histone molar ratio. The saturation stoichiometry of samples containing eNP (filled circles) or rNP (empty circles) is estimated from the intersection of the linear phase with the plateau. Histone concentration was kept constant and 10-fold higher than the apparent Kd estimated for each complex. Data corresponding to the eNP/H2A–H2BT71C-Alexa 488 complexes (gray squares) are also shown for the sake of comparison. (C) Normalized fluorescence intensity change (Eq. 1) of cross-linked H3C110AK115C-H4T71C-Alexa 488 (2 nM) as a function of eNP (filled circles) or rNP (empty circles) concentration. (D) Saturation stoichiometry of complexes formed by cross-linked H3C110AK115C-H4T71C and eNP (filled circles) or rNP (empty circles). Other details as in B. Binding data (A,C) were fitted to the ligand-depleted model described in Materials and Methods using eqs. 4 and 5. The estimated Kd values are shown in Table 1. Data in A-D are means ± SEM from at least three independent experiments.
	Figure 5. NP stabilizes a tetrameric H3-H4 conformation.FRET analysis of eNP/H3-H4T71C complexes formed upon incubation of H3-H4T71C labelled with Alexa 350 (0.5 μM) or 488 (0.5 μM) with eNP. (A) Emission spectra of H3C110A-H4T71C-Alexa in 0.15 M NaCl (gray solid line), 2 M NaCl (gray dashed line), and of eNP/H3C110A-H4T71C complexes obtained in 0.15 M NaCl at the following molar ratios: 1/1 (black dotted line), 1/2 (black dashed line), and 1/3 (black solid line). Excitation wavelength was 359 nm. (B) Emission spectra of H3C110E-H4T71C-Alexa. Other details as in A. (C) Comparison of fluorescence energy transfer of the samples shown in A and B, expressed as the ratio of the emission at 519 and 442 nm. The values for the same histone mixtures in 0.15 M NaCl, 2 M NaCl, and for the eNP/H3-H4 complexes in 0.15 M NaCl and 3 M guanidine HCl are also shown.
	Figure 6. Directed sulfhydryl reactive cross-linking suggests that NP stabilizes a nucleosomal H3-H4 conformation.(A) Cross-linking of eNP/H3C110AK115C-H4T71C complexes revealed by electrophoretic separation of the reaction components and western blotting with anti-H3 (upper panel) and anti-NP (lower panel). (B) Same as in A for eNP/H3C110EK115C-H4T71C complexes. Cross-linked bands of eNP or free histones in 0.15 M NaCl (A,B) and histones in the nucleosome (B) are also shown as controls. (C) Cross-linking efficiency of the samples analysed in A and B estimated as described in the Materials and Methods section. (D) Identification of the different H3 and H4 cross-linked adducts by SDS-PAGE.
	Figure 7. NP facilitates transfer of H3-H4 tetramers to DNA.(A,C) EMSA studies of disome and tetrasome formation by native H3-H4 or cross-linked H3C110AK115C-H4T71C. 0.8 μM histones were incubated with increasing eNP concentrations for 1 h before addition of 0.4 μM DNA. After 2 h at room temperature and 30 min at 42 °C, samples were analysed by Native-PAGE. The position of the disome, tetrasome and free DNA bands is indicated. (B,D) Quantification of the disome and tetrasome bands shown in A and C by densitometry. The NP-mediated disome and tetrasome assembly was estimated as the ratio of the intensity of the disome or tetrasome bands in the presence and absence of eNP. Means ± SEM from at least three independent experiments.

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