Npm2 increases nucleosome occupancy and decreases euchromatin. (A) On the left, one-cell X. laevis embryos were microinjected with equivalent volumes of XB or recombinant Npm2 protein (to increase the Npm2 concentration by 3.5 µM). On the right, one-cell X. laevis embryos were microinjected with 1 ng wild-type Npm2 mRNA or an equivalent volume of water. Embryos were allowed to develop to stage 10–10.5 and arrested in G2 with cycloheximide. Isolated nuclei were digested with MNase for the indicated times, and purified DNA was separated on 2% agarose gels and stained with ethidium bromide. On average, 60 embryos were microinjected per condition. Band intensities were quantified and used to calculate the average nucleosome number per fragment (see Materials and methods). Representative experiments are shown. (B) The MNase digestion assays described in A were repeated three times for Npm2 protein microinjection and three times for Npm2 mRNA microinjection. Maximum fold changes in nucleosome occupancy between control and Npm2-microinjected embryos were quantified for each experiment and averaged. (C) One-cell X. laevis embryos were microinjected with equivalent volumes of XB or recombinant Npm2 protein (to increase the Npm2 concentration by 3.5 µM), allowed to develop to stage 10–10.5, and arrested in G2 with cycloheximide. On average, 25 embryos were microinjected per condition. Isolated nuclei were stained with an acetyl-histone H3 antibody, and representative images are shown. Total integrated nuclear acetyl-histone H3 staining intensity was measured for ≥168 nuclei per condition. One representative experiment of two is shown. Two-tailed Student’s t tests assuming equal variances; **, P ≤ 0.01; ***, P ≤ 0.001. Error bars represent SD.