Fig. 4.

Histone glycation changes chromatin architecture in vitro and in cellulo. a Reconstituted nucleosomal 12mer arrays were incubated with 1 mM MGO for 12 h and analyzed by APAGE (agarose polyacrylamide native gel), either with EtBr or western blot utilizing the indicated antibodies to show the stability of the arrays upon MGO treatment. b Nucleosomal arrays were treated with 100 mM MGO (corresponding to 1:30 sites:MGO stoichiometry) for 12 h at 4 °C. Left: Representative single-molecule unfolding trajectories of MGO-treated (red) and untreated (blue) array obtained by an optical tweezer experiment. Right: Histograms of forces required to unravel each nucleosome in the MGO-treated nucleosomal array (red) and untreated (blue) array. Statistical analysis was based on sampling more than 50 single-molecule data and performing a t test. c Left: magnesium compaction assay of nucleosomal arrays treated with different concentrations of MGO. (+) 10 mM (corresponding to 1:3 sites:MGO stoichiometry); (+++) 100 mM MGO (corresponding to 1:30 sites:MGO stoichiometry). Right: array treated with low MGO concentration for different time points. (+) 2 mM MGO (corresponding to 1:0.6 sites:MGO stoichiometry). Error bars represent standard deviation from three different experiments. d MNase digestion of nucleosomal arrays treated with increasing amounts of MGO. Samples were separated on a native gel and stained with EtBr. e ATAC-seq performed on 293T cells either untreated or treated with 0.5 mM MGO for 12 h. Heatmaps showing the density of mapped ATAC-seq reads at consensus nucleosome peaks and 1000 bp up and downstream from the nucleosome dyad. Line graphs above show the same data as the mean read depth over all nucleosome peaks. f Line graph of the sum of ATAC-seq reads over RefSeq annotated transcriptional start sites show a decrease in chromatin accessibility in MGO-treated cells. g Schematic representation of the results presented in (b–d)