Decoupling nucleosome recognition from DNA binding dramatically alters the properties of the Chd1 chromatin remodeler

Abstract

Chromatin remodelers can either organize or disrupt nucleosomal arrays, yet the mechanisms specifying these opposing actions are not clear. Here, we show that the outcome of nucleosome sliding by Chd1 changes dramatically depending on how the chromatin remodeler is targeted to nucleosomes. Using a Chd1-streptavidin fusion remodeler, we found that targeting via biotinylated DNA resulted in directional sliding towards the recruitment site, whereas targeting via biotinylated histones produced a distribution of nucleosome positions. Remarkably, the fusion remodeler shifted nucleosomes with biotinylated histones up to 50 bp off the ends of DNA and was capable of reducing negative supercoiling of plasmids containing biotinylated chromatin, similar to remodelling characteristics observed for SWI/SNF-type remodelers. These data suggest that forming a stable attachment to nucleosomes via histones, and thus lacking sensitivity to extranucleosomal DNA, seems to be sufficient for allowing a chromatin remodeler to possess SWI/SNF-like disruptive properties.

Figure 1.

The Chd1–streptavidin remodeler slides nucleosomes in a biotin- and distance-dependent manner. Nucleosome sliding ability of Chd1[ΔDBD/+mSA] was tested using substrates lacking and possessing DNA-conjugated biotin moieties +11, +16 and +28 bp from the edge of the nucleosome. FAM-labelled centred nucleosomes (100 nM) were incubated with the indicated amounts of Chd1[ΔDBD/+mSA] for 0.5, 5 and 50 min under sliding conditions, and the reactions were then stopped with EDTA and free biotin. Reactions were monitored by native PAGE, where faster band migration is indicative of repositioning the histone octamer towards DNA ends.

Figure 2.

The Chd1–streptavidin remodeler buries biotinylated DNA sites on the nucleosome. (A) Schematic of APB-H2B cross-links generated on FAM-labelled 29-N-30 nucleosomes containing biotinylated DNA. Mapping was performed using a Ser53Cys variant of H2B, which forms a single cross-link to each strand, 18 nucleotides 3′ from the edge of the 145-bp Widom 601 sequence, corresponding to 47 bp from the 5′-FAM label of unshifted 29-N-30 nucleosomes. (B) Histone–DNA contact mapping reactions. All reactions contained 150 nM nucleosomes and 0 or 50 nM remodeler as indicated and they were carried out at room temperature with 2 mM ATP and 5 mM MgCl₂ for 20 min. Cross-linking sites for Chd1[ΔDBD/+mSA] are labelled with filled arrowheads and brackets, which were only observed in the direction of the biotinylated DNA sites (shaded arrows). Cross-linking sites for Chd1_ΔNC are labelled by open arrowheads and were observed on either side of the initial cross-link position (white arrows). (C) A cartoon representation of the cross-links and corresponding positions of the histone octamer on the DNA after sliding by Chd1[ΔDBD/+mSA]. (D) Nucleosomes with biotinylated DNA associated with Chd1[ΔDBD/+mSA] more poorly after sliding. To compare binding before and after sliding, DNA-biotinylated nucleosomes (100 nM) were first incubated with Chd1[ΔDBD/+mSA] (100 nM) at room temperature in the presence of ATPγS (no sliding) or ATP (sliding), as indicated. After 50 min, EDTA was then added to all reactions, and biotin was added as indicated. The reactions were then placed on ice for 2 h after additional Chd1[ΔDBD/+mSA] was added to yield final concentrations of 0, 100, 100, 200, 400, 600 and 800 nM remodeler and then analysed by native PAGE.

Figure 3.

The Chd1–streptavidin remodeler can move nucleosomes off the ends of DNA and independently of extranucleosomal DNA. (A) Schematic of nucleosomes showing biotinylation sites on flexible histone tails. (B) FAM-labelled nucleosomes (150 nM) containing biotinylated H3 (top) or H2A (bottom) were incubated with Chd1[ΔDBD/+mSA] (50 nM) in sliding buffer with or without ATP for the indicated times, and then stopped with EDTA and excess free biotin. (C) Schematic showing the locations of initial cross-links for the FAM-labelled 0-N-63 nucleosome. (D) Histone–DNA contact mapping was used to determine the locations of the histone octamer for 0-N-63 nucleosomes before and after sliding with Chd1_ΔNC and Chd1[ΔDBD/+mSA]. With biotinylated histones, cross-links were observed throughout the DNA fragment, with negative numbers (−18, −29, −40/41), indicating the distances shifted off of the ‘0’ DNA end, and positive numbers (+19 to +63), indicating the distances shifted onto extranucleosomal DNA. Mapping with H2B-Cys53 prevented visualization of octamer movement off the 5′-FAM labelled end, as the cross-linking residue loses contact with DNA. However, movement off of the FAM-labelled DNA end was confirmed by monitoring the Cy3-labelled top strand (not shown). (E) Schematic representations for the mapping reactions shown in (D), highlighting the histone octamer locations and corresponding cross-links of the 0-N-63 nucleosome after sliding with Chd1[ΔDBD/+mSA]. (F) Schematic showing the locations of cross-links for the double-labelled FAM/Cy3 0-N-2 nucleosome. (G) Histone–DNA contact mapping as performed in (D), using 0-N-2 H2A-biotinylated nucleosomes. Mapping is shown for both the bottom strand (FAM-labelled) and top strand (Cy3-labelled). Shaded arrows indicate the distances that the octamer was shifted from the starting position. (H) Schematic representations for the directions and magnitude of sliding for mapping reactions of 0-N-2 nucleosomes shown in (G).

Figure 4.

The Chd1–streptavidin remodeler can disrupt histone octamers from dinucleosome substrates. (A) Chd1[ΔDBD/+mSA] (200 nM) was incubated with 0-[601]-31-[603]-11 dinucleosomes containing biotinylated H2A (100 nM) at 30°C in sliding buffer, and reactions were stopped after 0.5, 1, 2, 4, 8, 16, 32, 64 and 128 min. (B) A sliding reaction carried out under the same conditions as in (A), but using 200 nM yeast RSC. The sliding gels shown in (A) and (B) are representative of three or more experiments carried out under similar conditions. (C) Analysis of mono- and dinucleosome products by native PAGE. Lanes 21–24 show various octamer:DNA ratios using the dinucleosome DNA template and histone octamers containing biotinylated H2A. Purified dinucleosomes used for sliding reactions are shown in lane 25. The result of remodelling these purified dinucleosomes (100 nM) with Chd1[ΔDBD/+mSA] (100 nM) for 30 min is shown in lane 26. Purification of the remodelling reaction to enrich for faster migrating species is shown in lane 27, the top band of which corresponds well to migration of end-positioned 0-N-190 mononucleosomes (lane 28).

Figure 5.

The Chd1–streptavidin remodeler disrupts nucleosomal wrapping of DNA on chromatinized plasmids. (A) Both Chd1_ΔNC and Chd1[ΔDBD/+mSA] exposed HhaI sites on pre-assembled nucleosomal arrays. A plasmid containing 34 repeats of the 601 array, with or without pre-assembled nucleosomes (5 nM), was incubated without or with remodelers (100 nM) for 10 min, and then either an additional 25 min without Hha I enzyme (−), or for 5, 10, 15, 20 or 25 min with 1 U of Hha I enzyme. (B) Chd1[ΔDBD/+mSA] caused a loss in supercoiling, suggestive of histone disruption. The 10-kb 34-repeat plasmid, with or without pre-assembled nucleosomes, was incubated with or without remodelers for 10 min, and then either another 50 min without topoisomerase (−), or 5 or 50 min with topoisomerase (‘topo incubation’). (C) Analysis of plasmid supercoiling for a 3-kb template in the presence and absence of nucleosomes and various remodelers. A 3-kb plasmid was incubated with or without topoisomerase as naked DNA (lanes 43, 44, 51 and 52) or after pre-assembly into chromatin in various histone : DNA ratios (0.3, 0.5, 0.7, 0.9, 1.1 and 1.3:1.0 for lanes 45–50, and 1.2:1.0 for lanes 53–67). Pre-assembled chromatin samples incubated with Chd1_ΔNC (100 nM) or Chd1[ΔDBD] (10 µM) were quenched with EDTA, sodium dodecyl sulphate and proteinase K after the indicated times. Reactions with Chd1[ΔDBD/+mSA] (100 nM) were stopped with 10 mM biotin and either immediately quenched or allowed additional incubation time for supercoiling reversion. (D) Duplicate samples for reactions shown in (C) analysed in the presence of 2 µg/ml chloroquine. (E) Analysis of topoisomers by 2D agarose gel electrophoresis. Samples were electrophoresed first in the absence (first dimension) and then the presence (second dimension) of 2 µg/ml chloroquine. Left panels show the locations of supercoiled, relaxed and nicked species obtained with naked DNA in the presence and absence of topoisomerase. Middle panels show supercoiled and relaxed species of saturated (top) and unsaturated (bottom) chromatinized plasmids, respectively. Right panels show distributions of supercoiled species after 1 h incubation with Chd1[ΔDBD/+mSA] followed by either 0 (top) or 6 h (bottom) additional time for supercoiling reversion.