Nishikawa J and Ohyama T (2013), Selective association between nucleosomes with ...

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Nucleic Acids Res 2013 Feb 01;413:1544-54. doi: 10.1093/nar/gks1269.

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Selective association between nucleosomes with identical DNA sequences.

Nishikawa J , Ohyama T .

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Self-assembly is the autonomous organization of constituents into higher order structures or assemblages and is a fundamental mechanism in biological systems. There has been an unfounded idea that self-assembly may be used in the sensing and pairing of homologous chromosomes or chromatin, including meiotic chromosome pairing, polytene chromosome formation in Diptera and transvection. Recent studies proved that double-stranded DNA molecules have a sequence-sensing property and can self-assemble, which may play a role in the above phenomena. However, to explain these processes in terms of self-assembly, it first must be proved that nucleosomes retain a DNA sequence-sensing property and can self-assemble. Here, using atomic force microscopy (AFM)-based analyses and a quantitative interaction assay, we show that nucleosomes with identical DNA sequences preferentially associate with each other in the presence of Mg(2+) ions. Using Xenopus borealis 5S rDNA nucleosome-positioning sequence and 601 and 603 sequences, homomeric or heteromeric octa- or tetranucleosomes were reconstituted in vitro and induced to form weak intracondensates by MgCl(2). AFM clearly showed that DNA sequence-based selective association occurs between nucleosomes with identical DNA sequences. Selective association was also detected between mononucleosomes. We propose that nucleosome self-assembly and DNA self-assembly constitute the mechanism underlying sensing and pairing of homologous chromosomes or chromatin.

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	Figure 1. Magnesium ion-induced condensation of octanucleosomes reconstituted in vitro. (A) DNA templates used in the reconstitution and the resulting nucleosomal arrays (also see Supplementary Figure S1). (B) Types of intracondensates and representative AFM images. The types are indicated by the number of globules. A nucleosome and a condensate of nucleosomes are each counted as a single globule. Bars indicate 100 nm. (C) Population ratio of each type of condensate relative to the entire population of octanucleosomes at 0.25, 0.50 or 1.0 mM Mg2+. The population ratio of the 2g type is indicated with a yellow line. ‘NC’ indicates octanucleosomes with no condensation; n ≥ 205 for each array. (D) Four forms of condensate type 2g. Bars indicate 100 nm. (E) Population ratio of each form of 2g relative to the entire population of octanucleosomes at 0.25, 0.50 or 1.0 mM Mg2+. For the 4-4 form of the chimeric array, two data sets are shown. The lower set (dotted line) indicates the data points for the 4-4 form with purely separated nucleosome species, as judged from the number of bridge lines (linker DNA) between the globules: a single bridge line means that each globule is comprised of a single nucleosome species. The upper set (solid line) indicates the data points for all the 4-4 forms. 4-4 forms with unclear linker image are included in calculating the upper data set. Representative AFM images of form 4-4 of the chimeric array are also shown.
	Figure 2. Discrimination of association between nucleosomes in chimeric tetranucleosomal arrays. (A) DNA templates and nucleosomal arrays (see also Supplementary Figure S1). (B) Types of intracondensates and representative AFM images. The types are indicated by the number of globules, as in Figure 1. Bars indicate 100 nm. (C) Population ratio of each type of condensate relative to the entire population of tetranucleosomes at the indicated concentrations of Mg2+. The population ratio of the 3g type is indicated with a yellow line. ‘NC’ indicates tetranucleosomes with no condensation. The population ratios are mean values of three independent determinations. Across the range of Mg2+ concentrations, the total count of tetranucleosomes ranged from 88 to 508 (usually ∼200). (D) Four forms of type 3g. Bars indicate 100 nm. (E and F) Population ratio of each form of condensate relative to the entire population of the 3g-type condensates at the indicated concentrations of Mg2+. The linker DNA lengths were 30 bp (E) and 62 bp (F). The light blue and brown dotted lines indicate 16.7 and 33.3%, respectively (see text). The condensates were generated three times as described above, using at least two independently reconstituted samples. Data are shown as means ± SD (n = 3). For form I of the AABB array and form IV of the ABAB array, the P-values for comparison of the corresponding data for the tetra-601 array (upper) and tetra-603 array (lower) are indicated. P < 0.05; *P < 0.01. Representative AFM images of form I of the AABB array and form IV of the ABAB array are also shown.
	Figure 3. DNA-sequence-based preferential association between nucleosomes. (A) An assay for quantifying association between nucleosomes. Magnetic beads carrying nucleosomes were mixed with Alexa 555-labeled nucleosomes in the presence or absence of Mg2+. The associated nucleosomes were fixed and quantified using fluorescence microscopy. Bars indicate 5 µm. (B) Microscopic images (left) and quantification (right) of the association of Alexa 555-labeled nucleosomes with magnetic beads carrying nucleosomes. The fluorescence intensity generated by the association of Alexa 555-labeled nucleosomes (test) was calibrated by using the intensity of the internal control (IC). For beads used for the IC, see ‘Materials and Methods’ section. They were distinguished from test samples by detecting fluorescence of Alexa 647. The inset in each panel shows the IC bead. Mg2+-induced increase in fluorescence intensity on the beads is shown as a function of Mg2+ concentration. The focus was adjusted on the plane involving the ‘equator line’ of each bead. Bars indicate 5 µm. Data are shown as means ± SEM (n = 7). P < 0.05; *P < 0.01.
	Figure 4. Topology of nucleosome self-assembly and its hypothetical figure in the pairing of homologous chromatin. (A) Possible topologies of associated nucleosomes in the form I and form IV condensates of tetranucleosomal arrays. For 3g, form I and form IV, see Figure 2. Only three nucleosomes in the form I and form IV condensates are extracted. DNAs with the same sequence have the same color. (B) A hypothetical schema depicting nucleosome–nucleosome and DNA–DNA associations in the pairing of homologous chromatin.

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