Relative positioning and properties of DNA protection by the H2A.Z and H3K4me3 nucleosomes. (A) Chromatin samples from HeLa cells that express C-terminal Flag-tagged H2A or H2AZ histones were treated with 280 U/mL MNase for 1 h at room temperature. After deproteinization, DNA fragments were run on 7% acrylamide gel. The 1-kb ladder is shown on both sides of the gel. Results are shown for the input samples and for the samples enriched for H2A and H2A.Z nucleosomes (IP). The type of nucleosomes for which the input samples were later enriched is indicated in parentheses. The average difference between the lengths of H2A and H2A.Z fragments in the IP samples (indicated with arrows) at intermediate MNase concentration is estimated as 22 ± 8 bp based on the analysis of seven independent replicates. (B) Distribution of H2A.Z tag density around the positions of H3K4me3 enriched nucleosomes. The black and gray lines represent the tag distributions at positive and negative DNA strands. In addition to the peaks at positions ±73, which correspond to nucleosome termini, there are two inner peaks indicating shortened DNA protection by H2A.Z nucleosomes. The center of the nucleosome fragment is indicated with the dyad symbol. Upper diagram illustrates the possible positioning of 17-bp (gray) fragments relative to the original 147-bp nucleosome sequence (black); the thick gray lines correspond to the predominant digestion scenario; and the thin lines represent positioning of 117-bp fragments that are less likely according to our results. (C) Histogram of the distances between the termini (5′–5′ and 3′–3′ end distances) of the H2A.Z nucleosome fragments predicted based on the 117-bp interpeak distance, and 147-bp H3K4me3 predictions. Dashed lines indicate distances of zero and 30 bp. Two pronounced maxima at distances zero and 30 bp correspond to the predominant digestion scenario depicted in the upper panel of B. (D) Histogram of the distances between 5′–5′ and 3′–3′ ends calculated for the 147-bp H2A.Z and H3K4me3 nucleosome predictions.
