Fig. 3

Histone glycation disrupts nucleosome assembly and stability. a Histones were treated as described in Fig. 2a before used in nucleosome assembly with salt dialysis in the presence of a strong nucleosome positioning DNA sequence (‘601’). Samples were separated on a native gel that was either stained with intercalating agent (EtBr) (top) or transferred and western blotted with anti-biotin (bottom). b Nucleosomes were treated with 0, 2, 30 or 100 mM of MGO (corresponding to 1:8, 1:120 or 1:400 sites:MGO stoichiometry, respectively) for 12 h at 37 °C and then used as templates in a PCR reaction. Products were separated on a 1 % agarose gel and stained with EtBr. c Top: histone-DNA glycation mediated cross-linking was examined using the sensitivity of unmodified nucleosomes to high salt treatment. Nucleosomes were treated with 0, 0.5, 5 or 100 mM of MGO (corresponding to 1:2, 1:20 or 1:400 sites:MGO stoichiometry, respectively) for 72 h at 37 °C after which they were either untreated or treated with 2 M NaCl. Samples were then separated on a native gel and either imaged by EtBr or transferred and analyzed by western blot with the indicated antibodies. Bottom: Schematic representation of untreated versus 100 mM MGO-treated nucleosomes before and after high salt treatment