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.
Highlights
Genomic DNA is highly organized as chromatin in eukaryotes
The indicated amount of purified histone H1.2 was mixed with the placeholder, H2A.Z, or canonical nucleosome (0.1 μM), which was reconstituted with the 193 base-pair Widom 601 DNA in the presence of Nap1 (0.3 μM), in 10 μL of reaction buffer (35 mM Tris-HCl, 70 mM NaCl, 0.01 mM PMSF, 0.05 mM EDTA, 6.5% glycerol, 1.2 mM dithiothreitol, and 1.1 mM 2-mercaptoethanol)
The human histone H3.2 was employed as the canonical H3, because it contains only one Cys residue at position 110
Summary
Genomic DNA is highly organized as chromatin in eukaryotes. The nucleosome is the basic architecture of chromatin. Various histone variants are encoded in genomes, and are usually incorporated into chromatin in a cell-cycle independent manner [11,12]. Numerous histone PTMs, such as lysine/arginine methylation, lysine alkylation, lysine ubiquitination, and serine/threonine phosphorylation, have been identified in the solvent accessible histone tails and inaccessible histone-fold domains in the nucleosome [13,14]. These histone variants and PTMs in the nucleosome function as epigenetic marks, which confer the chromatin states to daughter cells and offspring [15,16]. We prepared the placeholder nucleosome with human recombinant histones, and tested its biochemical properties by thermal stability, micrococcal nuclease sensitivity, DNaseI sensitivity, and linker histone H1 binding assays
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