Fig. 5 | Nature Communications

Fig. 5

From: Reversible histone glycation is associated with disease-related changes in chromatin architecture

Fig. 5

Histone glycation is repaired by DJ-1 in vitro and in cellulo. a Either non-glycated or glycated biotin-H3 tail (residues 1-18) was incubated with either recombinant DJ-1 or a 293T lysate at 4 °C for 2 h. Subsequently, peptides were enriched by streptavidin bead pull-down and analyzed by anti-DJ-1. b Full-length H3 was either treated or not treated with 1 mM of MGO (corresponding to 1:2 sites:MGO stoichiometry) for 2 h at 37 °C after which, either wild-type DJ-1 or catalytically inactive mutant C106A DJ-1 was added for an additional hour. Samples were analyzed on SDS-PAGE with the indicated antibodies. c NCPs were treated with MGO in the presence or absence of either wild-type DJ-1 or catalytically inactive mutant C106A DJ-1. Samples were analyzed by SDS-PAGE followed by a western blot with the indicated antibodies. d 293T cells were either not transfected, or transfected with wild-type or catalytically inactive mutant C106A DJ-1, and then grown in the absence or presence of 0.5 mM MGO for 12 h after which histones were extracted in a high salt buffer and analyzed by western blot with the indicated antibodies. e Mg2+ compaction experiments of nucleosomal arrays treated with MGO in the presence or absence of wild-type DJ-1. Error bars represent the standard deviation from three different experiments. f shRNA of DJ-1. 293T cells were transfected with shRNA plasmid and 24 h later were harvested. Total cell lysate was analyzed by western blot with anti-DJ-1 and anti-Actin as a loading control. g 293T cells transfected with shRNA DJ-1 were incubated with 0, 0.25, 0.5 and 1 mM of MGO for 12 h at 37 °C, after which histones were extracted with high salt and analyzed by western blot with the indicated antibodies

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