dinucleosome variant  (New England Biolabs)

Journal: The Journal of Biological Chemistry

Article Title: Nucleosome linker DNA methylation by DNMT3A/DNMT3B3 is controlled by nucleosome binding and multimerization of DNMT3 complexes on DNA

doi: 10.1016/j.jbc.2026.111154

Figure Lengend Snippet: Schematic drawing of the multimerization modes of DNMT3 complexes and of the dinucleosome substrates used in this study. A , protein/protein interaction interfaces present on DNMT3A, DNMT3B, DNMT3L, and DNMT3B3 subunits. The RD interface also provides the DNA binding site. B , protein multimerization of different DNMT3 complexes. DNMT3A and DNMT3B can form large homomultimers; homotetramers with one central RD interface are the smallest catalytically active species. DNMT3A/3L and DNMT3A/3B3 form defined heterotetramers. C , multimerization of DNMT3 complexes binding next to each other on DNA. D , dinucleosomes used as methylation substrates in this study. The CpG sites in the linker DNA region are highlighted and annotated. The regions used for bisulfite sequencing of the top and bottom DNA strand are indicated. The parts of the 70 bp linker removed in the 58(1) and 58(2) linkers are indicated. The NNN part between CpG 5 and CpG 6 denotes an internal bar code, used to discriminate the NGS data from pooled substrates. NNN = TGT in Linker-70, TCA in Linker-58(1), CTA in Linker-58(2). NGS, next generation sequencing.

Article Snippet: For the methylation experiments with different dinucleosome substrates, each dinucleosome variant was digested with MluI-HF (NEB) for 15 min at 37 °C in 7 μl NEB Cutsmart buffer (50 mM KOAc/20 mM Tris-acetate pH 7.9, 10 mM magnesium acetate, 100 μg/ml bovine serum albumin).

Techniques: Binding Assay, Methylation, Methylation Sequencing, Next-Generation Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Nucleosome linker DNA methylation by DNMT3A/DNMT3B3 is controlled by nucleosome binding and multimerization of DNMT3 complexes on DNA

doi: 10.1016/j.jbc.2026.111154

Figure Lengend Snippet: Linker methylation patterns of all CpG sites in the three dinucleosomes by DNMT3AC/3B3C. A , example of top strand and bottom strand methylation data. Values were normalized to the average methylation of linker CpG sites. B , relative linker methylation levels observed in five independent experiments. In panel A and B , no methylation data were indicated for the missing CpG sites 1 to 3 in Linker-58(1), and 6 to 8 in Linker-58(2). C , difference in the relative methylation levels of equivalent CpG sites between Linker-70 and Linker-58(1) or 58(2). The regions missing in the Linker-58 nucleosomes are shaded in gray . Error bars represent the propagated standard deviations. D , p values for the significance of the methylation differences shown in panel C determined by two-sided t test with equal variance using the individual data points. Significant p values ( p < 0.05/26, considering multiple testing) are shaded in gray .

Article Snippet: For the methylation experiments with different dinucleosome substrates, each dinucleosome variant was digested with MluI-HF (NEB) for 15 min at 37 °C in 7 μl NEB Cutsmart buffer (50 mM KOAc/20 mM Tris-acetate pH 7.9, 10 mM magnesium acetate, 100 μg/ml bovine serum albumin).

Techniques: Methylation

Journal: The Journal of Biological Chemistry

Article Title: Nucleosome linker DNA methylation by DNMT3A/DNMT3B3 is controlled by nucleosome binding and multimerization of DNMT3 complexes on DNA

doi: 10.1016/j.jbc.2026.111154

Figure Lengend Snippet: DNMT3AC/3B3C methylation of linker CpG sites determined by competitive methylation of all three dinucleosome substrates. A , average linker methylation levels of all three dinucleosomes, methylated in competition in one reaction tube. Exemplary methylation profiles are shown in . The table provides p values for the pairwise comparison of the methylation levels determined by two-sided t test with equal variance. Significant p values ( p < 0.05/3, considering multiple testing) are shaded in gray . B , enrichment of sequence reads with many methylation events in the entire data set. The distribution of the total number of methylation events per sequence read was determined. Based on the average methylation level of the pool, an expected number of sequence reads with each number of methyl groups was calculated by binomial statistics and the observed/expected ratio (obs/exp) determined.

Article Snippet: For the methylation experiments with different dinucleosome substrates, each dinucleosome variant was digested with MluI-HF (NEB) for 15 min at 37 °C in 7 μl NEB Cutsmart buffer (50 mM KOAc/20 mM Tris-acetate pH 7.9, 10 mM magnesium acetate, 100 μg/ml bovine serum albumin).

Techniques: Methylation, Comparison, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Nucleosome linker DNA methylation by DNMT3A/DNMT3B3 is controlled by nucleosome binding and multimerization of DNMT3 complexes on DNA

doi: 10.1016/j.jbc.2026.111154

Figure Lengend Snippet: Results of the competitive methylation of dinucleosomes linker regions and free DNA by DNMT3AC, DNMT3AC/3B3C, and DNMT3AC/3B3C-RE mutant. A , relative CpG site methylation levels of free DNA determined in four independent competitive methylation experiments. The sequence and CpG site annotation of the free DNA is provided in A . The 6 most preferred sites based on DNMT3A flanking sequence preferences literature data are shaded in gray . B , relative methylation levels of linker DNA in the Linker-70 dinucleosomes substrate determined in the four independent competitive methylation experiments. C , heatmap comparing the relative DNMT3AC/3B3C activity (rel. Act.) on free DNA and Linker-70 linker DNA with the flanking sequence preferences of the corresponding CpG sites (Pref) sorted by the average of both columns after scaling. Pearson r-values are indicated below. The scatter plots showing the correlations of the heatmaps are provided in , B and C . D , ratio of the average methylation activities of DNMT3AC, DNMT3AC/3B3C, and DNMT3AC/3B3C-RE on dinucleosomes and free DNA. The table provides the p values of pairwise comparisons based on two-sided t test with equal variance. Significant p values ( p < 0.05/3, considering multiple testing) are shaded in gray .

Article Snippet: For the methylation experiments with different dinucleosome substrates, each dinucleosome variant was digested with MluI-HF (NEB) for 15 min at 37 °C in 7 μl NEB Cutsmart buffer (50 mM KOAc/20 mM Tris-acetate pH 7.9, 10 mM magnesium acetate, 100 μg/ml bovine serum albumin).

Techniques: Methylation, Mutagenesis, Sequencing, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Nucleosome linker DNA methylation by DNMT3A/DNMT3B3 is controlled by nucleosome binding and multimerization of DNMT3 complexes on DNA

doi: 10.1016/j.jbc.2026.111154

Figure Lengend Snippet: Effects of the recruitment of DNMT3AC/3B3C to the nucleosome. A , comparison of the flanking sequence preferences of DNMT3A and the relative DNMT3AC/3B3C activity at all linker CpG sites. The error bars show the standard deviation. Individual data points are provided in . Thirteen out of the 26 sites with significant difference and deviations > 10% are shaded gray . p -values were determined by Z-statistics and p < 0.05/26 (considering multiple testing) was indicated as significant. B , Visualization of the sites showing the strongest nucleosomal effects on methylation rates on a dinucleosome model generated by rigid body superposition as described in the Methods section. Top strand CpG (in the notation of this study) are colored red , bottom strand CpG orange . The position of sites with most prominent nucleosome dependent stimulation or repression of activity are highlighted by green (increased activity) or red arrows (reduced activity). The two images show the same model rotated by approximately 90° about the linker DNA axis.

Article Snippet: For the methylation experiments with different dinucleosome substrates, each dinucleosome variant was digested with MluI-HF (NEB) for 15 min at 37 °C in 7 μl NEB Cutsmart buffer (50 mM KOAc/20 mM Tris-acetate pH 7.9, 10 mM magnesium acetate, 100 μg/ml bovine serum albumin).

Techniques: Comparison, Sequencing, Activity Assay, Standard Deviation, Methylation, Generated

Journal: The Journal of Biological Chemistry

Article Title: Nucleosome linker DNA methylation by DNMT3A/DNMT3B3 is controlled by nucleosome binding and multimerization of DNMT3 complexes on DNA

doi: 10.1016/j.jbc.2026.111154

Figure Lengend Snippet: Schematic picture of the multimerization of DNMT3AC/3B3C complex heterotetramers on dinucleosomes. CpG cytosine residues that can easily reach a DNMT3A active site after base flipping are methylated more efficiently than expected by their flanking sequence (symbolized by gray shading), CpG cytosine residues placed such that they cannot bind to an active site are methylated only weakly (symbolized by the white color). The DNMT3 complexes and nucleosome are not drawn to scale.

Article Snippet: For the methylation experiments with different dinucleosome substrates, each dinucleosome variant was digested with MluI-HF (NEB) for 15 min at 37 °C in 7 μl NEB Cutsmart buffer (50 mM KOAc/20 mM Tris-acetate pH 7.9, 10 mM magnesium acetate, 100 μg/ml bovine serum albumin).

Techniques: Methylation, Sequencing