Journal: eLife

Article Title: Acetylation of H3K115 is associated with fragile nucleosomes at CpG island promoters and active regulatory sites

doi: 10.7554/eLife.108802

Figure Lengend Snippet: ( A ) Heatmap showing the coverage of H3K115ac, H3K122ac, H3K27ac, and ATAC-seq centred on promoter distal accessible peaks (putative enhancers) in mouse embryonic stem cells (mESCs). Data is grouped in enhancers marked by all three H3 acetylation marks (Group 1; H3K27ac+ H3K122ac+ H3K115ac+), just H3K27ac together with H3K122ac (Group 2) or H3K27ac alone (Group 3). ( B ) Heatmaps showing H3K115ac and ATAC-seq signal for common and dynamic enhancers between ESC and neural progenitor cell (NPC). Loss/gain of H3K115ac correlates with loss/gain in chromatin accessibility. ( C ) mESC enhancers selected based on the presence of two Oct 4 motifs (n=650) within the Tn5-accessible region, with the region between the two motifs scaled to the same length (shaded grey region). Top: MNase-seq signal (black, left y-axis) and ATAC-seq (purple, right y-axis). H3K115ac ChIP-seq (middle panel) and H3K27ac ChIP-seq (bottom panel) on mononucleosomes (monoNuc; green) or subnucleosome-sized fragments (subMuc; blue). ( D ) Immunoblotting for H3, H3.3, and H3K115ac on whole-cell extracts from E14 mESCs, H3.3 knock-out ESCs, and the parental ESC line. Ponceau S staining of histones is shown below as loading control ( and ). A biological replicate blot is shown in . Figure 4—source data 1. Original immunoblots for , indicating the mouse embryonic stem cell (mESC) samples (E14, parental, H3.3KO) and the antibodies used (H4, red; H3, H3.3, and H3K115ac, green). LI-COR collection data shown below. Figure 4—source data 2. Original files for immunoblots for .

Article Snippet: We used a 15 min incubation on ice in the nuclei preparation step, and the Tn5 reaction was performed in 50 μl of custom transposition buffer (10 mM Tris pH 8, 5 mM MgCl 2 and 10% dimethylformamide) with 2.5 μl Tn5 transposase (Illumina, 20034197) at 37°C while mixing at 1000 rpm for 30 min.

Techniques: ChIP-sequencing, Western Blot, Knock-Out, Staining, Control