• Figure 1.
    Download figureOpen in new tabFigure 1. Separation of nucleosomes and hexasomes made with the Widom 601 sequence.

    (A) Hexasomes but not nucleosomes migrate differently by native PAGE when flanking DNA is on the left or right of the 601 sequence. These two gels, poured from the same solution, are representative of 0-601-80 and 80-601-0 reconstitutions made using histone octamer. (B) Separation of hexasomes from nucleosomes. Shown is a representative purification over a 7% native acrylamide column using a Prep Cell apparatus. The elution fractions were analyzed by native PAGE. (C) Purified nucleosome and hexasome pools, analyzed by native PAGE. (D) As shown by SDS-PAGE, the hexasome species lack one H2A/H2B dimer. The bar graph is a quantification of gel band intensities from three different nucleosome/hexasome purifications. All histone bands were normalized to histone H4. The H2A and H2B bands often migrate close together, and therefore the relative intensities of H2A/H2B bands are shown summed together. Within each nucleosome/hexasome pair, the intensity of H2A/H2B in hexasomes was 47 ± 6% of that of nucleosomes.

    DOI: http://dx.doi.org/10.7554/eLife.21356.002

    Figure 5.
    Download figureOpen in new tabFigure 5. Oriented hexasomes allow targeted placement of modified H2A/H2B dimers on the nucleosome.

    (A) Analysis of dual labeled 3-601-80 nucleosomes (H2A T120C-Cy3 and DNA-Cy5) by single-molecule FRET (smFRET) reveals multiple species prior to nucleosome sliding by Chd1. Nucleosomes were surface-immobilized by biotin on the 80 bp flanking DNA. Infusion of 300 nM Chd1 and ATP initiated remodeling. (B) Oriented 3-601-80 hexasomes (H2A-Cy3 and DNA-Cy5) uniformly show one dye pair that yields high FRET. Right panel shows relatively poor mobilization of hexasomes by Chd1. (C) Incubation of a two-fold molar excess of unlabeled H2A/H2B dimer with the labeled 3-601-80 hexasomes yielded asymmetric nucleosomes, only possessing the high FRET dye pair. After remodeling with Chd1 and ATP, the FRET population was similar to nucleosome.

    DOI: http://dx.doi.org/10.7554/eLife.21356.010

    Figure 8.
    Download figureOpen in new tabFigure 8. Entry-side H2B-Ubiquitin stimulates nucleosome sliding by Chd1.

    (A) Generation of symmetric and asymmetric nucleosomes with site-specific placement of H2B-Ubiquitin. Nucleosomes were formed from subsaturating H2A/H2B dimer (12 nM) addition to 0-601-80 hexasomes (10 nM). Hexasomes and H2A/H2B dimer contained either unmodified (Wt) or ubiquitinated (Ub) H2B as indicated, and resulting nucleosome and hexasome species were visualized by native PAGE. Shown is a representative from six independent dimer addition experiments. (B) Comparison of remodeling reactions with subsaturating (25 nM) Chd1, using hexasomes (10 nM) and H2A/H2B dimers (12 nM) containing unmodified or Ub-conjugated H2B. Shown are progress curves for remodeling reactions monitored using a Cy3-Cy3 pair at 25 μM ATP. Black traces represent fits to the data. Progress curves are representative of two independent experiments. (C) Representative progress curves of nucleosome sliding reactions monitored by stopped flow using Cy3B-Dabcyl at 25 μM ATP and saturating (400 nM) Chd1. Each progress curve is an average of 3–6 technical replicates. Black traces represent fits to the data. (D) Comparison of observed sliding rates monitored with Cy3B-Dabcyl at 25 μM ATP and saturating Chd1 (400 nM). Error bars show standard deviations from three independent experiments. **** p-value <0.0001.

    DOI: http://dx.doi.org/10.7554/eLife.21356.016

    Figure 9.
    Download figureOpen in new tabFigure 9. Model for nucleosome packing by oriented hexasomes.

    As others have shown, transcription by Pol II through nucleosomes is facilitated by removal of the promoter-distal H2A/H2B dimer (Kulaeva et al., 2009). Our results indicate that Chd1 would slide a hexasome of this orientation upstream. We propose that one or more hexasomes would corral intervening nucleosomes toward the promoter. Alternately, if every transcribed nucleosome were briefly converted to a hexasome, unidirectionally sliding of each hexasome would maintain tight nucleosome packing.

    DOI: http://dx.doi.org/10.7554/eLife.21356.017