FIG. 6.

Acetylation of nucleophosmin and core histones potentiates their interaction and nucleosome disruption. (A) Effect of acetylation on NPM1 and histone interaction. Indicated amounts of normal (untreated) core histones purified from the HeLa nuclear pellet were tested for their interaction with 1 μg of either NPM1 (lanes 3, 4, and 5) or acetylated NPM1 (lanes 6, 7, and 8) using the Ni-NTA pulldown assay. Ni-NTA beads alone, without NPM1 or acetylated NPM1, were used as a negative control (lane 2). Four micrograms of the sample was loaded as input (lane 1). The top panel is the Coomassie blue-stained gel showing the amounts of the pulled-down proteins, while the bottom panel shows the Western immunoblot (IB) with antihistone H3. (B) Effect of acetylation on NPM1 and acetylated histone interaction. Indicated amounts of hyperacetylated core histones purified from the HeLa nuclear pellet were tested for their interaction with 500 ng of either NPM1 (lanes 3, 4, and 5) or acetylated NPM1 (lanes 6, 7, and 8) using the Ni-NTA pulldown assay. Ni-NTA beads alone, without NPM1 or acetylated NPM1, were used as a negative control (lane 2). Two micrograms of the sample was loaded as input (lane 1). The top panel is the Coomassie blue-stained gel showing the amounts of the pulled-down proteins, while the bottom panel shows the Western blot with antibodies against acetylated histone H3. (C) Acetylated NPM1 transfers histones more efficiently than NPM1. Histone transfer was tested with 500 ng of core histones and increasing concentrations of either NPM1 (lanes 6, 8, and 10) or the acetylated NPM1 (lanes 7, 9, and 11). Lane 1, supercoiled DNA; lane 2, DNA relaxed by TopoI; lanes 3 through 11, relaxed DNA along with core histones (lane 3), NPM1 (lane 4), or acetylated NPM1 alone (lane 5) and 0.5, 1, or 5 pmol (lanes 6 and 7, 8 and 9, and 10 and 11, respectively) of either NPM1 and acetylated NPM1, respectively.