Direct Observation of H3–H4 Octasome by High‐Speed AFM

Abstract

Despite evidence that histone H3 and H4 proteins may act as the precursor for orientating the DNA sequence to form nucleosome structures, there is no direct evidence of the formed compact structure. Here, it is demonstrated that a histone H3-H4 octasome could be constructed without the involvement of histone H2A-H2B under in vitro reconstitution conditions. Atomic force microscopy was used to obtain the first direct observation of the octasome structure, which exhibited a similar core-protein size as that of a nucleosome but with a shorter core histone-binding DNA region. The octasome also displayed a one-step histone-dissociation pattern under heat treatment, distinct micrococcal nuclease and peplomycin accessibility, which suggests a different wrapping pattern to that in nucleosomes.

DNA accessibility of chromatosomes quantified by automated image analysis of AFM data

“…Yet, evaluation of large datasets obtained with single molecule techniques is particularly important for robust statistical analysis. However, the high number of nucleosome studies where structural parameters were determined manually 24,31–34 or image analysis was performed with a commercial (closed source) software 35–40 indicates that open source methods for automated image analysis of nucleosome data are needed.…”

Direct Observation of Dynamic Interactions between Orientation‐Controlled Nucleosomes in a DNA Origami Frame

“…The attachment of the nucleosome to the DNA frame is mediated by nucleic acid hybridization, and so 18‐nt single‐stranded DNA connectors were added to both ends of this template by PCR amplification with two modified primers (Table S1). Reconstituted nucleosomes were prepared with template dsDNA and recombinant histone octamer, using an in vitro salt dialysis method reported previously [25] . The production of reconstituted nucleosomes was confirmed by native PAGE gel electrophoresis (6 % native PAGE) and a clear nucleosome gel shift was detected (Figure S1b).…”

“…Reconstituted nucleosomes were prepared with template dsDNA and recombinant histoneo ctamer,u sing an in vitro salt dialysis method reported previously. [25] The production of reconstituted nucleosomes was confirmedb yn ative PAGE gel electrophoresis (6 %n ative PAGE) and ac lear nucleosome gel shift was detected ( Figure S1b). In addition, we observed nucleosomesu sing AFM by treating the mica with cationic 3-aminopropyl triethoxysilane (APS), which is required to adsorb intact nucleosomeo nto the surfacef or AFM observation.…”

“…Nucleosome reconstitution:N ucleosomes were reconstituted by a protocol previously reported. [25] Briefly,am ixture of different ratio of template DNA (400 nm)a nd histone octamer (1:1.2, 480 nm)i n 20 mm HEPES-KOH buffer (pH 7.5) was dialyzed using ad ialysis cup (MWCO 8000, Bio-tech) in high-salt buffer (20 mm HEPES-KOH, pH 7.5;2 m NaCl) for 2h ours, then in low-salt buffer (20 mm HEPES-KOH, pH 7.5;0m NaCl) overnight at 4 8C. After the dialysis, concentration of nucleosome was determined by measuring the absorbance of DNA at aw avelength of 260 nm.…”

Nucleosome structural variations in interphase and metaphase chromosomes

“…However, many structural variants of nucleosomes can exist in vitro (Lavelle and Prunell, 2007) (Fig. 1A): hexasomes, di-tetrasomes, reversomes (right-handed octameric nucleosome), overlapping dinucleosomes, split nucleosomes, and hemisomes (Arimura et al, 2012;Bancaud et al, 2007;Furuyama et al, 2013;Kato et al, 2017;Zlatanova et al, 2009;Zou et al, 2018). Even within octameric left-handed nucleosomes assembled on the high-affinity nucleosome positioning sequence, Widom 601 (Lowary and Widom, 1998), form multiple structural arrangements of histone octamers, which can be observed by cryo-electron microscopy (cryo-EM) (Bilokapic et al, 2018a(Bilokapic et al, , 2018b.…”