Figure 5. 5-H-Y binds to chromatin DNA tightly and folds chromatin in vitro and in vivo.

(A) Experimental scheme of the two chromatin folding assays: ultracentrifuge assay (left) and nuclear volume assay (right). (B) 5-H-Y induces chromatin folding. Samples of reconstituted nucleosome fibers were exposed to the indicated concentrations of 5-H-Y (left) or cisplatin (center) and analyzed by sedimentation velocity analytical ultracentrifugation. The integral distribution of diffusion-corrected sedimentation coefficients obtained after analysis of the data by the method of Demeler and van Holde are shown⁶⁰,⁶¹. (Right) Summary of analytical ultracentrifuge-SV results. Values at the 50% boundary are displayed as a function of drug concentration added 5-H-Y (red) or cisplatin (blue). (C) Nuclear volume was decreased by 5-H-Y in a dose-dependent manner. Nuclei treated with 50 μM 5-H-Y showed a 12-fold decrease in the volume. This indicates that 5-H-Y induces chromatin folding. The nuclei treated with 5 mM Mg²⁺ were prepared as a control for the nuclei with highly folded chromatin. The error bars represent the standard deviation. For each point, n = ~100. (D) Volumes between Mg²⁺-pretreated and 5-H-Y-pretreated nuclei after buffer washing. When the volume was normalized by Mg²⁺-pretreated nuclei, although Mg²⁺-pretreated nuclei became large after the washing (relative nuclear volume = 1), 5-H-Y-pretreated nuclei did not change (~0.1). 5-H-Y seems to bind tightly to chromatin DNA, in contrast to Mg²⁺. The error bars represent the standard deviation. (E) 5-H-Y induces chromatin condensation in vivo. HeLa cells were treated with TSA to decondense chromatin and then with 5-H-Y. 5-H-Y induced enrichment of chromatin at nuclear periphery and nucleoli (left) although we cannot exclude the possibility that condensation by 5-H-Y only occur around nucleoli and nuclear periphery. Right plot shows the intensity quantification of nuclear periphery chromatin. **p < 0.01, Chi-square test.