Nucleosome remodeling by the human SWI/SNF complex requires transient global disruption of histone-DNA interactions

Abstract

We utilized a site-specific cross-linking technique to investigate the mechanism of nucleosome remodeling by hSWI/SNF. We found that a single cross-link between H2B and DNA virtually eliminates the accumulation of stably remodeled species as measured by restriction enzyme accessibility assays. However, cross-linking the histone octamer to nucleosomal DNA does not inhibit remodeling as monitored by DNase I digestion assays. Importantly, we found that the restriction enzyme-accessible species can be efficiently cross-linked after remodeling and that the accessible state does not require continued ATP hydrolysis. These results imply that the generation of stable remodeled states requires at least transient disruption of histone-DNA interactions throughout the nucleosome, while hSWI/SNF-catalyzed disruption of just local histone-DNA interactions yields less-stable remodeled states that still display an altered DNase I cleavage pattern. The implications of these results for models of the mechanism of SWI/SNF-catalyzed nucleosome remodeling are discussed.

FIG. 1.

DNA fragments used for nucleosome reconstitution. The 215-bp fragment is an extension of the 154-bp fragment. The positions of relevant restriction enzyme sites and the region of DNA assembled into the nucleosome (oval) are shown.

FIG. 2.

Nucleosomes containing APB-modified H2B (H2BG26C-APB) are remodeled efficiently by hSWI/SNF. Nucleosomes were reconstituted with either wild-type (WT) H2B (lanes 4 to 10) or H2B26C-APB (lanes 12 to 18) and then subjected to hSWI/SNF remodeling. Lane 1 shows the G-specific reaction of the radiolabeled 154-bp 5S DNA fragment used for reconstitution. Lane 2 shows the DNase I digestion pattern of the naked 5S DNA fragment (uncross-linked DNA [FD]). Lanes 3 and 11 show 5S DNA prior to DNase I digestion. Lanes 4 and 12 and 5 and 13, respectively, show the DNase I cleavage pattern of nucleosomes incubated in the absence of hSWI/SNF or in the presence of 1.3 μg of SWI/SNF, but without ATP. Lanes 6 to 10 and 14 to 18, respectively, show the DNase I footprint of nucleosomes incubated with 270, 405, 540, and 810 ng and 1.3 μg of hSWI/SNF in the presence of ATP.

FIG.3.

hSWI/SNF activity results in a marginal loss of interactions between H2B and nucleosomal DNA. (A) Effect of hSWI/SNF remodeling on total cross-link formation. Nucleosomes were reconstituted with H2B26C-APB and the 154-bp 5S DNA fragment and then incubated with hSWI/SNF in the presence or absence of ATP. Samples were then irradiated with UV light as indicated, and the amounts of H2B-DNA cross-linked species (X-L) and uncross-linked DNA (FD) were analyzed by SDS-PAGE and autoradiography. Lanes 1 and 2 show cross-linking in nucleosomes incubated in the absence of SWI/SNF, without or with subsequent UV irradiation, respectively. Lanes 3 and 4 show cross-linking in nucleosomes incubated with hSWI/SNF in the absence or presence of ATP, respectively, followed by UV irradiation. The error on determination of percent cross-linking (%X-L) is ±5%. (B) Sites of interaction between H2B and nucleosomal DNA before and after hSWI/SNF remodeling. Nucleosomes were prepared as in panel A, and the sites of H2B26C-APB cross-linking to nucleosome DNA were determined by mapping piperidine-induced DNA strand breaks on sequencing gels (see Materials and Methods). Lanes 1 and 2 show the G-specific and DNase I cleavage reactions of the naked 154-bp 5S DNA fragment (FD+DN′se); lane 3 shows piperidine-treated, uncross-linked 5S DNA (FD) as a background control. Lanes 4 to 6 show cross-links generated in nucleosomes incubated in the absence of hSWI/SNF, in the presence of SWI/SNF, but without ATP, and in the presence of hSWI/SNF and ATP. Numbers indicate the locations of major sites of cross-linking within the 5S sequence (Fig. 1). The locations of the nucleosome (oval) and nucleosome dyad (arrow) are indicated

FIG. 4.

Prior cross-linking of H2B to nucleosomal DNA does not inhibit hSWI/SNF remodeling as detected by DNase I digestion assay. Nucleosomes reconstituted with H2BG26C-APB and the radiolabled 215-bp 5S DNA fragment were irradiated with UV and then incubated with hSWI/SNF in the presence or absence of ATP, and remodeling of cross-linked and uncross-linked nucleosomes was analyzed by DNase I footprinting. Lane 1, G-specific reaction of naked 5S DNA. Lane 2, DNase I digestion of naked 5S DNA. Lanes 3 and 9 show DNase I digestion of uncross-linked and cross-linked 5S nucleosomal DNA, respectively. Lanes 4 and 10 show DNase I digestion of uncross-linked nucleosomes incubated with 245 ng of hSWI/SNF in the absence of ATP, respectively. Lanes 5 to 8 and 11 to 14 show DNase I digestion of cross-linked and uncross-linked 5S nucleosomal DNA as indicated, incubated with 245 ng of hSWI/SNF and ATP. DNase I was added either at the same time as hSWI/SNF (lanes 5 and 11) or 2 (lanes 6 and 12), 7 (lanes 7 and 13), or 27 (lanes 8 and 14) min after the addition of hSWI/SNF, and the digestions were allowed to proceed for 3 min.

FIG. 5.

A single cross-link severely inhibits SWI/SNF-catalyzed nucleosome remodeling as detected by restriction enzyme digestion analysis. Nucleosomes reconstituted with H2B26C-APB and the 215-bp 5S DNA fragment were irradiated with UV light and then digested with either SacI or EcoRV restriction enzymes as described in Materials and Methods. (A) Time course of SacI digestion for cross-linked and uncross-linked nucleosomal DNA. Digested nucleosome samples were loaded onto an SDS-PAGE gel to separate cross-linked (XL) and uncross-linked (UX) DNAs, and the extent of enzyme digestion was determined by autoradiography. Lane 1 shows 5S nucleosomes without UV irradiation or SacI digestion. Lanes 2 to 7 show products of SacI digestion of UV-irradiated nucleosomes incubated without hSWI/SNF. Lanes 8 to 13 and 14 to 19, respectively, show SacI digests ofUV-irradiated 5S nucleosomes incubated with either 270 or 570 ng of hSWI/SNF. (B) Plot of SacI digestion of 5S nucleosomal DNA. The amount of undigested nucleosomal DNA remaining at each time point was calculated from the gel shown in panel A, and the natural log of the percent remaining uncut was plotted for cross-linked and uncross-linked fractions versus time of digestion. The data were fit to linear trend lines calculated in Excel. Solid symbols with solid trend lines are SacI digestion profiles of uncross-linked nucleosomes, and open symbols with dotted trend lines are SacI digestion profiles of cross-linked nucleosomes. Diamonds represent SacI digestions in the absence of hSWI/SNF; squares and circles, respectively, represent SacI digestions in the presence of 270 or 570 ng of hSWI/SNF. (C) Plot of EcoRV digestion of 5S nucleosomal DNA. A plot of the loss of nucleosomal DNA substrate (cross-linked and uncross-linked) by EcoRV cleavage over time is shown as in panel B. Symbols and trend lines are as in panel B.

FIG. 6.

Accumulation of stably remodeled nucleosomes. Nucleosomes reconstituted with the 215-bp 5S DNA fragment were treated with hSWI/SNF and ATP for various times and then subjected to EcoRV digestion, and the extent of cleavage was determined as in Fig. 5. The percentage of DNA uncut versus the time of digestion is plotted for reactions without hSWI/SNF (diamonds) and with 254 ng of hSWI/SNF for 0.5, 2, 5, and 10 min (squares, triangles, solid circles, and open circles, respectively). The solid line represents the single exponential fit to 0.5-min data.

FIG. 7.

Extent of accumulation of remodeled species is related to SWI/SNF concentration. Nucleosomes reconstituted with the 215-bp 5S DNA fragment were incubated with various amounts of hSWI/SNF for 15 min, and then the extent of remodeling was examined by digestion with EcoRV. (A) Plot of digests. Digests of reaction mixtures incubated with 0, 0.2, 0.5, 0.8, and 1.0 μl of 245 ng of hSWI/SNF per μl, plotted as diamonds, squares, triangles, solid circles, and open circles, respectively, are shown. (B) Plot of extent of EcoRV digestion at the 10-min time point versus the amount of hSWI/SNF present in the incubation.

FIG. 8.

Cross-linking occurs within remodeled nucleosomes. Nucleosomes containing cross-linkable H2B and the 215-bp 5S DNA fragment were treated with hSWI/SNF and ATP and then irradiated, and the extent of EcoRV accessibility in cross-linked and uncross-linked fractions was determined. In some cases, remodeling reaction mixtures were treated with apyrase before the irradiation step. The digestion profiles for unirradiated samples incubated with SWI/SNF without ATP and with ATP (solid and open diamonds, respectively) are shown for reference. The profiles for cross-linked and uncross-linked fractions from the SWI/SNF remodeled, irradiated sample (solid and open triangles, respectively) and cross-linked and uncross-linked fractions from remodeled samples treated with apyrase before irradiation (solid and open circles, respectively) are shown.