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. 2017 Sep 12;6:e28836. doi: 10.7554/eLife.28836

Figure 3. Human histone H3 mutants ‘X’ and ‘Y’ form obligate heterodimers in vitro.

(A) Size exclusion (Superdex 200 Increase 3.2/300) gel filtration profiles showing the purification of octamer refolding reactions containing wild-type and heterodimer ‘X’ and ‘Y’ histone H3. Octamer refolding in the presence of both ‘X’ and ‘Y’ H3 yields histone octamers with a characteristic elution profile, similar to refolding reactions containing wild-type H3, while reconstitutions with only H3X or only H3Y form aggregates. (B) Native PAGE analysis of nucleosome reconstitution experiments with purified histones from (A). H3X/Y octamer reconstitutions readily form nucleosomes when assembled onto DNA, while the aggregates formed in octamer refolding reactions containing either ‘X’ or ‘Y’ alone do not form histone octamers and therefore cannot form nucleosomes in vitro.

Figure 3.

Figure 3—figure supplement 1. Generation of asymmetric X/Y nucleosomes for in vitro studies.

Figure 3—figure supplement 1.

(A) Purified recombinant histones. (B) Replicate size exclusion (Superdex 200 Increase 3.2/300) gel filtration profiles showing the purification of octamer refolding reactions containing wild-type and heterodimer ‘X’ and ‘Y’ histone H3. As in Figure 3A, with additional replicate shown. (C) Coomassie Blue G staining of affinity-purified chromatin composed of asymmetric nucleosomes. Chromatin was washed with buffer containing 0.1% Triton X100 and 0.5 M NaCl and purified by binding to streptavidin-agarose in the same buffer. Strains expressed the indicated combination of X-Y asymmetric H3s. 10 μl (lanes 1, 4, 6), 5 μl (lanes 2, 5, 7) or 2.5 μl (lane 3) of the bound material was analyzed on a 15% SDS-PAGE gel.