Biochemical characterization of the placeholder nucleosome for DNA hypomethylation maintenance

Abstract

DNA methylation functions as a prominent epigenetic mark, and its patterns are transmitted to the genomes of offspring. The nucleosome containing the histone H2A.Z variant and histone H3K4 mono-methylation acts as a "placeholder" nucleosome for DNA hypomethylation maintenance in zebrafish embryonic cells. However, the mechanism by which DNA methylation is deterred by the placeholder nucleosome is poorly understood. In the present study, we reconstituted the placeholder nucleosome containing histones H2A.Z and H3 with the Lys4 mono-methylation. The thermal stability assay revealed that the placeholder nucleosome is less stable than the canonical nucleosome. Nuclease susceptibility assays suggested that the nucleosomal DNA ends of the placeholder nucleosome are more accessible than those of the canonical nucleosome. These characteristics of the placeholder nucleosome are quite similar to those of the H2A.Z nucleosome without H3K4 methylation. Importantly, the linker histone H1, which is reportedly involved in the recruitment of DNA methyltransferases, efficiently binds to all of the placeholder, H2A.Z, and canonical nucleosomes. Therefore, the characteristics of the H2A.Z nucleosome are conserved in the placeholder nucleosome without synergistic effects on the H3K4 mono-methylation.

Keywords: Chromatin; DNA methylation; H2A.Z; H3K4 methylation; Histone; Nucleosome.

Fig. 1

Preparation of the placeholder nucleosome. A. The purified nucleosomes were analyzed by native-PAGE with ethidium bromide staining. B. The purified nucleosomes were analyzed by SDS-PAGE with Coomassie Brilliant Blue staining.

Fig. 2

Thermal stability of the placeholder nucleosome. A. Schematic representation of the thermal stability assay. In this assay, thermal dissociations of the histones from the nucleosomes are detected using SYPRO Orange fluorescent dye, which emits substantial fluorescence when it binds to the exposed hydrophobic surfaces of the denatured histones. B. Normalized fluorescent intensity curves of thermal dissociations of the histones from the nucleosomes. The error bars indicate standard deviations. The experiments were performed four times. C. Plots of the derivatives of the curves in panel B.

Fig. 3

Accessibility of the nucleosomal DNA in the placeholder nucleosome. A. Schematic representation of the MNase susceptibility assay. In this assay, DNA ends detached from histones in the nucleosomes were preferentially digested by MNase. After the digestion reaction, the resulting DNA fragments were analyzed by native-PAGE. B. A representative gel image of the MNase susceptibility assay. The canonical (lanes 2–6), H2A.Z (lanes 7–11), and placeholder (12–16) nucleosomes containing the 147 base-pair Widom 601 DNA were treated with MNase for 0, 3, 6, 12, and 15 min at 37 °C. After the incubation, the reaction was stopped by adding stop solution containing proteinase K, SDS, and EDTA. The resulting DNA fragments were analyzed by native-PAGE with ethidium bromide staining. C. Graphic representation of the MNase susceptibility assay. The experiments were repeated three times, and the average values were plotted with the standard deviation values. D. Schematic representation of the DNaseI treatment assay. In this assay, the DNA strands exposed to the solvent are preferentially digested by DNaseI. After the digestion reaction, the resulting DNA fragments are analyzed by denaturing urea-PAGE. E. A representative gel image of the DNaseI treatment assay. The canonical (lanes 2–7), H2A.Z (lanes 8–13), and placeholder (lanes 14–19) nucleosomes containing the 147 base-pair Widom 601 DNA were treated with DNaseI for 0, 2, 4, 6, 8, and 10 min at 26 °C. The digestion reaction was stopped by adding stop solution containing proteinase K, SDS, and EDTA. The resulting DNA fragments were denatured and analyzed by urea-PAGE with SYBR Gold staining.

Fig. 4

Linker histone H1 binding to the placeholder nucleosome. A. Schematic representation of the H1 binding assay. In this assay, increasing amounts of H1 are mixed with the nucleosomes in the presence of Nap1. After an incubation, the complex formation is analyzed by native-PAGE. B. The predicted nucleosome positioning on the 193 base-pair DNA is presented by an ellipse. A gray box indicates the 145 base-pair Widom 601 sequence region. C. A representative gel image of the H1 binding assay. Increasing amounts of H1 (0, 0.23, 0.32, 0.41, 0.50, and 0.60 μM) were mixed with the canonical (lanes 1–6), H2A.Z (lanes 7–12), and placeholder (lanes 13–18) nucleosomes (0.1 μM) containing the 193 base-pair Widom 601 DNA in the presence of Nap1 (0.3 μM). After an incubation at 25 °C for 30 min, the reaction mixtures were analyzed by native-PAGE with ethidium bromide staining.