Figure 4.

Thermal stability assay of the LmaH3 nucleosome. (A) Schematic representation of the nucleosome disruption during the thermal stability assay. In this assay, the fluorescence of SYPRO Orange, a fluorescent dye that hydrophobically binds to denatured histones, is monitored as the histones dissociate from the nucleosomes or the nucleosomes lacking H2A and H2B molecules. (B) The upper panel shows the normalized fluorescence intensity curves of the thermal dissociation of the nucleosomes containing H3.1 (•) or LmaH3 (○). The first and second melting temperatures correspond to the dissociations of the H2A–H2B dimers and the H3–H4 tetramer from the nucleosome, respectively. The bottom panel shows the derivative values of the thermal stability curves presented in the upper panel. The bars indicate standard deviations of triplicate experiments. Three independent experiments were performed and similar results were obtained. The H3.1 and LmaH3 nucleosomes were reconstituted with the 145 bp Widom601L DNA. Gel images showing the purified nucleosomes and the nucleosomal histone contents are presented in Supplementary Figure S3A and B. (C) The upper panel shows the normalized fluorescence intensity curves of the thermal dissociation of the H3–H4–DNA complexes without the H2A–H2B dimer. The bottom panel shows the derivative values of the thermal stability curves presented in the upper panel. The H3.1–H4–DNA complex (•) and LmaH3–H4–DNA complex (○) are shown. The bars indicate standard deviations of triplicate experiments. The H3.1–H4–DNA and LmaH3–H4–DNA complexes without the H2A–H2B dimer were reconstituted with the 145 bp Widom601L DNA. Gel images showing the purified histone–DNA complexes and the histone contents are presented in Supplementary Figure S3C and D.