Architecture of the SWI/SNF-nucleosome complex

Abstract

The SWI/SNF complex disrupts and mobilizes chromatin in an ATP-dependent manner. SWI/SNF interactions with nucleosomes were mapped by DNA footprinting and site-directed DNA and protein cross-linking when SWI/SNF was recruited by a transcription activator. SWI/SNF was found by DNA footprinting to contact tightly around one gyre of DNA spanning approximately 50 bp from the nucleosomal entry site to near the dyad axis. The DNA footprint is consistent with nucleosomes binding to an asymmetric trough of SWI/SNF that was revealed by the improved imaging of free SWI/SNF. The DNA site-directed cross-linking revealed that the catalytic subunit Swi2/Snf2 is associated with nucleosomes two helical turns from the dyad axis and that the Snf6 subunit is proximal to the transcription factor recruiting SWI/SNF. The highly conserved Snf5 subunit associates with the histone octamer and not with nucleosomal DNA. The model of the binding trough of SWI/SNF illustrates how nucleosomal DNA can be mobilized while SWI/SNF remains bound.

FIG. 1.

DNA footprinting of SWI/SNF bound to nucleosomes after recruitment by Gal4-VP16. (A) Purified SWI/SNF was separated by 4 to 20% SDS-PAGE and stained with Coomassie blue. E1 and E2 are the first two fractions from the M2-agarose column. (B) Nucleosomes (Nucl.) were assembled with a radiolabeled 242-bp DNA probe (601 nucleosome positioning sequence flanked by 34 and 61 bp of extranucleosomal DNA). The positions of the nucleosome (oval), flanking DNA, and one or two Gal4 binding sites (2G) are shown. Nucleosomes (55 nM with 0.14 nM of the nucleosomes contains Gal4 binding sites) were incubated with 2.5 nM SWI/SNF in the presence or absence of Gal4-VP16 (75 nM) for 30 min at 30°C. Nucleosomes alone (lane 1) or with SWI/SNF (lanes 3 and 4) and/or Gal4-VP16 (lanes 2 and 4) added were separated on a native 4% polyacrylamide gel and visualized by phosphorimaging. (C) DNA footprinting with the hydroxyl radical was performed on complexes of the nucleosome plus Gal4-VP16 with (gray) or without (black) SWI/SNF. Quantification of the different lanes from a 6% denaturing polyacrylamide gel after normalization is overlaid for comparison. The region protected by SWI/SNF is indicated below by either a solid black (strongest) or gray (moderate) bar. The open bar indicates the region in which SWI/SNF binding enhances cutting, and the numbering refers to the number of base pairs from the right (+) or left (−) of the dyad axis.

FIG. 2.

cryoEM reconstruction of SWI/SNF and model of the SWI/SNF-nucleosome complex. Panels A, C, and E show three different views of the SWI/SNF structure obtained from cryoEM. Panels B, D, and F are the models of the SWI/SNF-nucleosome complex obtained by fitting the crystal structure of the nucleosome low pass filtered to 25 Å into the putative nucleosome binding surface of SWI/SNF. Features of the nucleosome binding face of SWI/SNF are a trough whose base (TB) is met by a high wall (HW), a low wall (LW), and a back wall (BW). The base slopes very gently up into the high wall and down to the nearly rimless front; junctions with the low and back walls are more pronounced. Along the rim of the trough are two prominent features, labeled lip (L) and clamp (CL). The dyad axis of the nucleosome is indicated by an arrow (B, D, and F).

FIG. 3.

Swi2/Snf2 is cross-linked most extensively to the nucleosomal region, and Snf6 is cross-linked most extensively to the extranucleosomal region closest to the Gal4 binding site. (A) The 601 DNA sequence is shown, along with the 20 regions modified by the incorporation of a photoreactive nucleotide and radiolabel. The numbering is the same as that in Fig. 1, and the highlighted region is the core nucleosome region. Those stretches of sequence shown below have photoreactive nucleotides, indicated by “^,” and the incorporated α-³²P-deoxynucleotide is indicated by “*.” Numbering refers to the location of the photoreactive nucleotide(s) relative to the dyad axis. (B to D) Binding of SWI/SNF to nucleosomes was done as indicated in the legend of Fig. 1. SWI/SNF-nucleosome complexes were cross-linked, DNA digested, and analyzed by 4 to 20% SDS-PAGE and phosphorimaging. Nucleosomes were reconstituted with different DNA photoaffinity probes and SWI/SNF with (even lanes) and without (odd lanes) Gal4-VP16 added. The * in panel C indicates a band that does not correlate to a SWI/SNF subunit, and the * in panel D indicates labeled bands due to trace amounts of undigested DNA that were not consistently observed. (E) The relative efficiency of cross-linking Swi2/Snf2 or Snf6 to DNA was obtained by dividing the labeling intensity of these two subunits at different positions with that of Swi2/Snf2 at bp −18/−17.

FIG. 4.

Snf6 is the subunit of SWI/SNF most proximal to the Gal4-VP16 binding site. The same DNA probes as those in Fig. 3 were used in binding reactions with SWI/SNF without (A) and with (B) Gal4-VP16 added but without prior assembly into nucleosomes. Samples were analyzed as described in the legend to Fig. 3, and the relative electrophoretic mobilities of the SWI/SNF subunits are shown.

FIG. 5.

DNA cross-linking of SWI/SNF with photoreactive diazirine probes. DNA cross-linking was done as described in the legend to Fig. 3 with photoreactive nucleosomes using the more photoreactive diazirine-containing DNA probes. (A) Samples contained SWI/SNF with (even lanes) and without (odd lanes) Gal4-VP16. (B) All samples contained SWI/SNF and Gal4-VP16.

FIG. 6.

Display of the locations in DNA and histone octamer that were cross-linked to SWI/SNF subunits. (A and B) The DNA protected by SWI/SNF binding is highlighted in red in the nucleosome model, and the position of the Gal4 binding site is highlighted in pink. The black (strong) and dark blue (weak) spots on the DNA indicated the location to which Swi2/Snf2 was cross-linked. The light blue spots indicate the location of Snf6 cross-linked in the extranucleosomal DNA region. The location of Swp29 cross-linking is indicated by green spots (B). (C) The locations of the eight different cysteine sites are shown on the surface of the nucleosome core particle along with that of the DNA position to which Swi2/Snf2 is most efficiently cross-linked (bp −18/−17). Those sites that were >0.3 times as efficiently cross-linked to Swi2/Snf2 as residue 80 of histone H3 are shown in the shaded region (left). The locations of the sites that are most efficiently cross-linked to Snf5, Swp82, and Snf11 or Rtt102 are in the shaded regions depicted on the right.

FIG. 7.

Identification of SWI/SNF subunits bound to the histone octamer surface by site-directed histone cross-linking. (A to C). Nucleosomes reconstituted with different cysteine mutant octamers were modified with PEAS (see Materials and Methods). Modified nucleosomes (4 nM) were bound to SWI/SNF (6 nM) in the presence or absence of Gal4-VP16 (6.4 nM) and cross-linked by UV irradiation. After transfer of the radiolabel by disulfide reduction, the samples were analyzed by 4 to 12% (A and B) or 10% (C) Bis-Tris SDS-PAGE and phosphorimaging. The location of the modification is indicated above the lanes by referring to the histone protein and residue that was changed to cysteine. Gal4-VP16 and/or competitor DNA (2 ng/μl in panels A and B and 1 ng/μl in panel C) were added as indicated, and all reaction mixtures contained SWI/SNF. (D) The relative efficiency of histone cross-linking to SWI/SNF subunits was shown by normalizing the labeling intensity relative to Swi2/Snf2 cross-linking to residue 80 of histone H3. For each experiment, the relative labeling signal of Swi2/Snf2 and other subunits was obtained by dividing the signal to that of Swi2/Snf2 at residue 80 of histone H3. The data presented are the average data for two to three independent experiments for all sites except for H422 and H2B109. This comparison is shown for four primary SWI/SNF subunits (Swi2/Snf2, Snf5, Swp82, and Snf11 or Rtt102) shown to be cross-linked to the histone octamer face.

FIG. 8.

Subunit topology in the nucleosome binding pocket of SWI/SNF. The views in panels A, C, and E are in the same orientation as that shown in Fig. 2A, C, and E. Nucleosomal DNA is displayed with the same color scheme as that in Fig. 6A and B and refers to sites that cross-linked Swi2, Snf6, and Swp29. The histone octamer is not shown for better visualization of the proximity of DNA cross-linking sites to the surface of SWI/SNF. The DNA protected by SWI/SNF binding is shown in red, and that not protected is shown in gray (A to B and E and F). In this model, the linker trajectory is arbitrarily assigned. (C) The locations of particular DNA and histone sites are shown with the nucleosome removed and are based on the SWI/SNF-nucleosome model described above. The legend for the symbols used to mark these locations is shown (D), along with a summary of the SWI/SNF subunits cross-linked at these sites and their relative efficiencies, i.e., strong (++) and weak (+). The proposed path of nucleosomal DNA along the surface of the SWI/SNF trough is shown as a dashed line. Arrows in panels A and E indicate the dyad axis of the nucleosome.

FIG. 9.

Structure-based model for SWI/SNF remodeling. (A) A cutaway view of the SWI/SNF trough surface with a schematic view of the bound nucleosome is shown. The dark gray line represents the DNA in close contact with the high wall surface of SWI/SNF, and the light gray line is the DNA that is more accessible and is located next to the low wall. The position of the translocation and anchor domains of SWI/SNF is indicated by two gray spheres, and the approximate location of the Gal4-VP16 is indicated. (B and C) Remodeling starts with the translocation domain pulling DNA toward it and the concerted displacement of ∼50 bp of DNA from the surface of the nucleosome. The translocation domain continues to pull DNA toward itself and creates a large DNA loop by ratcheting DNA on one side, while the other part of DNA remains fixed to an adjacent site in SWI/SNF. (D and E) The DNA loop is released to move around the nucleosome in a wave-like process. (F) The movement of the DNA loop toward the other entry/exit site causes the movement of the histone octamer to a new position on DNA.