Extended Data Fig. 6: Nucleosome octamer dynamics are important for Swi6 nucleosome binding.
From: HP1 reshapes nucleosome core to promote phase separation of heterochromatin
a, Representative non-reducing SDS–PAGE showing nucleosome with H3–H4 histone disulfide-linked (H3•H4 S-S) or reduced (H3•H4 S-H). Around 90% of H3 and H4 are disulfide-linked in both the methylated and unmethylated nucleosomes. These are the cross-linking sites used in Fig. 4. For gel source data, see Supplementary Fig. 1. b, Nucleosome binding assays by fluorescence anisotropy showing reduced binding of Swi6 to H3K9me3 nucleosomes containing H3–H4 disulfide-linked octamer (H3•H4 S-S versus H3•H4 S-H). c, Fluorescence anisotropy measuring Swi6 binding to unmethylated nucleosome, H3•H4 S-H and S-S, showing that disulfide linkages have no effect on Swi6 binding to unmethylated nucleosome. d, Additional residues in the H2B and H4 mutated to Cys for generating dynamically restrained octamers are represented with spheres. e, Representative non-reducing SDS–PAGE showing nucleosome with H2B-H4 histone disulfide-linked (H2B•H4 S-S) or reduced (H2B•H4 S-H). Around 50% of H2B and H4 are disulfide-linked in both H3Kc9me3 and unmethylated nucleosomes. For gel source data, see Supplementary Fig. 1. f, Nucleosome binding assays by fluorescence anisotropy showing reduced binding of Swi6 to H2B-H4 disulfide-linked octamer (H2B•H4 S-S). g, Fluorescence anisotropy experiments showing comparable Swi6 binding to H3cK9me3 non-oxidized, H3cK9me3 oxidized and H2B•H4 S-H mononucleosomes. These results show that the oxidation process does not alter Swi6 binding to Cys-devoid nucleosomes and that the presence of reduced Cys does not affect Swi6 binding either. h, Fluorescence anisotropy measuring Swi6 binding to unmethylated nucleosome, H2B•H4 S-H and S-S, showing that disulfide linkages have no effect on Swi6 binding to unmethylated nucleosomes. i, Nucleosome binding assays by fluorescence anisotropy showing that Zmet2 binding to H2B–H4 disulfide-linked octamer (H2B•H4 S-S) is not significantly affected. FP, fluorescence polarization units. Measurements entailed at least three independent experiments and error bars reflect s.d.
