Fig. 5
From: Function of HP1BP3 as a linker histone is regulated by linker histone chaperones, NPM1 and TAF-I
Effects of NPM1 and TAF-I on the chromatin binding activity of HP1BP3. (A) Purified GST-NPM1, GST-TAF-Iβ, and GST. The GST proteins (200 ng) were separated by 10% SDS-PAGE and visualized by CBB staining. The positions of molecular weight markers are shown at the right side of the panel. (B) NPM1 and TAF-Iβ inhibit the nonspecific binding of HP1BP3 to NCPs. 5S rDNA NCP (0.1 pmole) was incubated without (lane 1, 3, and 4) or with (lanes 2 and 5–14) Flag-HP1BP3 (0.8 pmole). In lanes 5–9 and 10–14, GST-NPM1 and GST-TAF-Iβ (0.4, 0.8, 1.6, 3.2, and 4.8 pmole) were also added. In lane 15, GST (4.8 pmole) was added as a control. The mixture was incubated at 30 °C for 30 min and loaded on 6% PAGE. Positions of free DNA, NCP, and the HP1BP3-NCP complex are shown at the right side of the panel. (C) Localization of HP1BP3 in U2OS cells treated with siRNAs. U2OS cells were transfected with control siRNA, NPM1 siRNA, and TAF-Iβ siRNA as indicated and the plasmid for the expression of EGFP-HP1BP3 was transfected 48 h after siRNA transfection. The localization of EGFP-HP1BP3 were observed 72–80 h post plasmid transfection under a confocal microscope. A bar shown at the right bottom indicates 10 μm. (D) Expression of NPM1 and TAF-Iβ in siRNA treated cells. U2OS cells treated with siRNAs as in C were subjected to western blotting with antibodies against HP1BP3, NPM1, TAF-Iβ, and actin. Proteins from 1 × 104, 3 × 104, and 1 × 105 cells were loaded for each sample. The band intensity of HP1BP3 and β-actin in lanes 3, 6, and 9 was analyzed by Image J and the ratio HP1BP3/β-actin for lanes 3, 6, and 9 are shown at the bottom of the panel. (E) FRAP analyses of EGFP-HP1BP3. U2OS cells transfected with siRNA and EGFP-HP1BP3 plasmid as in C were grown in glass-based dishes and subjected to FRAP analyses. The EGFP signals were bleached with a 488-nm lase line and the recovery of the EGFP signals was measured with one-second interval for two-minutes and plotted as a function of time (s). For each sample, 10 cells were examined and the data shown were averaged. The error bars indicate ± SD. The recovery rates of HP1BP3 in NPM1- and TAF-I-knockdown cells were statistically analyzed by student t-test and the data for TAF-I knockdown cells (from 14 to 120 s after photo bleach) relative to those of control cells were p < 0.02. (F) Expression of NPM1 in NPM1 KO MEF cells. Proteins from control MEF cells and NPM1 knockout MEF cells (3 × 104 and 1 × 105 cells for lanes 1 and 2, and 3 and 4) were separated by SDS-PAGE and subjected to western blotting with anti-UBF and anti-NPM1 antibodies. (G) FRAP assay with NPM1 KO cells. EGFP-HP1BP3 was transiently expressed in control (black) and NPM1 KO (red) MEF cells and FRAP assay as in E were conducted