Fig. 7

Functional regulation of Npm by intramolecular interactions. a Npm domain map and truncations used in these experiments. Truncation names and residue numbers shown on right. b Equilibrium tryptophan fluorescence binding curves derived from the Npm tail and truncations binding to either H2A/H2B or H3/H4. Points represent average values of three replicates for each binding curve. Standard error of the points is included during the calculation of the binding curves and dissociation constants. c Dissociation constants (K _D) and standard errors (±) of Npm tail and truncations derived from b. d Competitive pull-down assays using various truncations of the Npm tail and StrepII-tagged H2A/H2B dimers. Lanes 1–4, 10% inputs. Lanes 5–8, resin only controls. Lanes 9–12, standard pull-down assays. Lanes 13–18, competitive pull-down assays. Presence of Npm truncation bands in competitive pull-down lanes indicate higher affinity for H2A/H2B. e Chromatin assembly assay comparing the full-length Npm (residues 1–195) and tail domain (119–195). Concentrations of chaperone used in each indicated as molar ratio to histone octamer. f Chromatin assembly assay comparing full-length Npm (residues 1–195), ∆Cterm10 (1–185), ∆Cterm16 (1–179), and core+A2 (1–145) truncations at 11:1 molar ratio of Npm to histone octamer. g Quantification of chromatin assembly assays in f. 4–7 replicates of each shown with standard errors. One-way ANOVA and multiple group comparison of chromatin assembly activity of truncations to full-length Npm. ****p < 0.0001. h In vitro TTLL4 glutamylation assay using core+A2 truncation ± H2A/H2B. Glutamylation of Npm readout by anti-glutamylation western blot (top). Membrane stained in Direct Blue 71 (bottom). Histone binding inhibits Npm glutamylation on glutamates 127, 128, 129, and 131. i In vitro TTLL4 glutamylation assay using C-terminal truncations of Npm. Glutamylation of Npm readout by anti-glutamylation western blot (top). Membrane stained in Direct Blue 71 (bottom). The C-terminal 10 amino acids inhibit Npm glutamylation on glutamates 127, 128, 129, and 131. j Histone aggregate removal assay of linear DNA + H2A/H2B run on native TBE-PAGE with various truncations of Npm added at 2:1 molar ratio of Npm:H2A/H2B. Lower band indicates free DNA, upper bands indicate H2A/H2B:DNA aggregates. Core+A2 (1–145) is the only truncation capable of removing aggregates