Figure 1: DNA linkers regulate methylation by DNMT3A2/3B3.

a Sequences of engineered di-nucleosome linkers. Positions of CpG dinucleotides in the sense and antisense strands are numbered based on their location relative to the upstream nucleosome and are colored red and blue, respectively. b-c Bisulfite sequencing assay showing the percentage of CpG methylation at each linker CpG position. Specific cytosines are indicated with colored arrows. Top: nucleosome substrates, bottom: naked DNA substrates. n=2 for 5bp, 8bp, 10bp, 15bp, 20bp, 25bp linkers. n=1 for 35bp linker. d Electromobility shift assays for DNMT3A2/3B3 binding to the di-nucleosome substrates used in a-c. e Electromobility shift assay for DNMT3A2/3B3 binding to 10-8-10 and 10-25-10 di-nucleosomes. f Manually built models of di-nucleosomes with 20bp and 35bp linkers. CpG positions that are accessible and oriented for methylation are indicated with solid blue and red arrows. CpG positions that are inaccessible and oriented between the di-nucleosome are shown with empty blue and red arrows. g Distribution of linker lengths in mouse ES cells ranging from 0–80bp. Distributions for all linkers in the cell (gray line) and at promoter regions +/− 1000bp from the transcription start site (colored lines). Linker length distributions at promotor regions are separated in quartiles based on gene expression level (blue = no expression, green = bottom 25–50%, yellow = top 25–50%, red = top 25%). Linker lengths that inhibit methylation are indicated with a transparent red box, and linker lengths that allow methylation are indicated with a transparent green box. See also Figure S1.