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. 2015 Aug 28;43(21):10200–10212. doi: 10.1093/nar/gkv841

Figure 2.

Figure 2.

Magnesium ion induces the selective binding of HP1γ to H3K9me tetra-nucleosomes. (A) Effect of MgCl2 on the binding of HP1γ to H3K9me3 tetra-nucleosomes. Unmethylated (unme) or H3K9me3 tetra-nucleosomes (K9me3) were incubated with GST-HP1γ (HP1γ) or GST-HP1α (HP1α) bound to GSH-Sepharose in the presence of 3 mM MgCl2, 50 mM NaCl. Unbound (U) and bound nucleosomes (B) are visualized (left panel), and the nucleosome core histones indicated by brackets were quantitated as in Figure 1C. Relative amounts of core histones in the bound fraction per input (%) were calculated and shown as mean ± S.E. (n = 3) (right panel). (B) MgCl2 concentration-dependent selective binding of HP1γ to H3K9me3 tetra-nucleosomes. The binding activity of HP1γ to unmethylated H3 (open circles) or H3K9me3 tetra-nucleosomes (closed circles) was determined and shown as mean ± S.E. (n = 3) under the indicated concentration of MgCl2 in the presence of 50 mM NaCl by pull-down assay as described in Figure 1C. (C) MgCl2 concentration-dependent binding of HP1γ to native oligo-nucleosomes with H3K9me3 modification. GST-HP1γ (HP1γ) or GST (GST) bound to GSH-Sepharose was mixed with native oligo-nucleosomes and then pulled down under the condition including 50 mM NaCl, in the absence or presence of 3 mM MgCl2. The H3K9me3 and histone H4 in the unbound (U) and bound fractions (B) were analyzed as in Figure 1A (left panel). The amounts of H3K9me3 in the HP1γ-bound fractions over the input (%) are shown as mean ± S.E. (n = 3) (right panel). (D) Schematic illustration of reconstituted mono-nucleosomes. The position of the nucleosomes on DNA is indicated by ellipses. Length (bp) of linker and nucleosome core region DNA are shown below. (E) Effect of MgCl2 on the binding of HP1γ to H3K9me3 mono-nucleosomes. The binding of ummethylated H3 and H3K9me3 mono-nucleosomes (4 pmol of nucleosome core particle) to GST-HP1γ (80 pmol) bound to GSH-Sepharose in 10 μl of the reaction mixture was examined as in Figure 1C, with 50 mM NaCl, in the absence or presence of 3 mM MgCl2 (left panel). The binding activity of HP1γ to tetra-nucleosomes reconstituted with unmethylated H3 (open circles) or H3K9me3 (closed circles) was determined and shown as mean ± S.E. (n = 3) under the indicated concentration of MgCl2 by pull-down assay as described in Figure 1C.