Journal: The Journal of Cell Biology
Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Figure Lengend Snippet: Structure and stability of H3.X- and H3.Y-containing nucleosomes. (A) In silico homology model of H3.X (purple, left) and H3.Y (light blue, right) protein structures in overlay with the crystal structure of H3.2 (dark blue). (B) Crystal structure of nucleosome with H3.2 exchanged by in silico homology models of H3.X (purple, left) and H3.Y (light blue, right), respectively. (C) IP of mononucleosomes generated from HeLa cells transfected with empty vector, HA-H3.1, -H3.X, and -H3.Y shows incorporation of novel H3 variants into nucleosomes. Bioanalyzer evaluation of purified DNA after IP of MNase-treated chromatin (unbound and bound material) shows digestion of chromatin to mononucleosomes and their successful precipitation (left; see also Fig. S2 A for DNA size and quality). Silver stain of 15% SDS-PAGE with α-HA IPs of mononucleosomes revealed successful binding of HA-tagged H3 variants (asterisks) and pull-down of core histones (top, right). Immunoblot of immunoprecipitates with α-HA (red) and α-H3 C-terminal (green) antibodies visualized by the Odyssey infrared imaging system (bottom, right). Notice that endogenous H3 is coimmunoprecipitated with all H3 variants analyzed. (D) FRAP experiments to evaluate nucleosomal stability of novel H3 variants using spinning disk confocal microscopy. HeLa Kyoto cells were transiently transfected with GFP, GFP-H3.1, -H3.3, -H3.X, and -H3.Y constructs. A small nuclear area was photobleached (box) and the recovery of the fluorescent signal was monitored over 1 min and up to 8 h (see Fig. S2, B–D, for long-term FRAP). Depicted is a short-term FRAP series (selected time points are shown) of GFP-tagged H3 variants compared with GFP alone. Bar, 5 µm. (E) Quantification of short-term FRAP experiment. Mean curves of 10–20 individual cells are shown. Standard deviations were very small (in the range of ± 0.02) and were omitted for clarity (for details see Fig. S2 D). All GFP-H3 variants show almost no recovery within the first 60 s after bleaching, which indicates that all expressed fusion protein was stably incorporated into nucleosomes. In contrast, GFP alone recovers to almost 100% within 5 s.
Article Snippet: Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM).
Techniques: In Silico, Generated, Transfection, Plasmid Preparation, Purification, Silver Staining, SDS Page, Binding Assay, Imaging, Confocal Microscopy, Construct, Stable Transfection