SNF Chromatin Remodeling Requires Changes in DNA Topology

Molecular Cell, Vol. 7, 97–104, January, 2001, Copyright 2001 by Cell Press

SWI/SNF Chromatin Remodeling Requires Changes in DNA Topology Igor Gavin,† Peter J. Horn, and Craig L. Peterson* Program in Molecular Medicine Department of Biochemistry and Molecular Biology University of Massachusetts Medical School Worcester, Massachusetts 01605

Summary ySWI/SNF complex belongs to a family of enzymes that use the energy of ATP hydrolysis to remodel chromatin structure. Here we examine the role of DNA topology in the mechanism of ySWI/SNF remodeling. We find that the ability of ySWI/SNF to enhance accessibility of nucleosomal DNA is nearly eliminated when DNA topology is constrained in small circular nucleosomal arrays and that this inhibition can be alleviated by topoisomerases. Furthermore, we demonstrate that remodeling of these substrates does not require dramatic histone octamer movements or displacement. Our results suggest a model in which ySWI/SNF remodels nucleosomes by using the energy of ATP hydrolysis to drive local changes in DNA twist. Introduction The assembly of DNA into compacted arrays of nucleosomes leads to an accessibility problem for DNA-mediated processes such as transcription, DNA repair, recombination, and DNA replication. One group of enzymes that may have evolved to contend with the inhibitory effects of nucleosome assembly is the ATP-dependent family of chromatin remodeling enzymes. These enzymes have been identified and purified from yeast (ySWI/SNF, RSC, ISW1, ISW2), Drosophila (brm, NURF, CHRAC, ACF), Xenopus (x-Mi2, ISWI-D), and humans (hSWI/SNF, NURD, hACF) (reviewed in Kingston and Narlikar, 1999; Muchardt and Yaniv, 1999; Vignali et al., 2000). Each member of the ATP-dependent family of chromatinremodeling enzymes contains an ATPase subunit that is related to the SWI2/SNF2 subfamily of the DEAD/H superfamily of nucleic acid–stimulated ATPases (Eisen et al., 1995). Furthermore, each enzyme can use the energy of ATP hydrolysis to alter chromatin structure and to enhance the binding of proteins to nucleosomal DNA binding sites (Kingston and Narlikar, 1999; Muchardt and Yaniv, 1999; Boyer et al., 2000b). Furthermore, in the case of the ySWI/SNF, Drosophila Brahma, and hSWI/SNF complexes, remodeling is required for transcriptional regulation of target genes in vivo (de la Serna et al., 2000; Deuring et al., 2000; for review see Kingston and Narlikar, 1999). The chromatin-remodeling activity of SWI/SNF-like complexes has been demonstrated by a number of in * To whom correspondence should be addressed (e-mail: craig. [email protected]). † Present address: Biochip Technology Center, Argonne National Laboratory, 9700 S. Cass Avenue, Argonne, IL 60439.

vitro assays. The biochemical outcome of this reaction includes: (1) changes in the rotational path of nucleosomal DNA on the surface of a histone octamer (Cote et al., 1994; Kwon et al., 1994), (2) enhanced accessibility of nucleosomal DNA to transcription factors (Cote et al., 1994; Kwon et al., 1994; Utley et al., 1997) and restriction enzymes (Logie and Peterson, 1997), (3) loss of negative supercoils from circular chromatin (Kwon et al., 1994; Jaskelioff et al., 2000), (4) movements of histone octamers in cis (Whitehouse et al., 1999; Jaskelioff et al., 2000) and in trans (Lorch et al., 1999; Whitehouse et al., 1999), and (5) formation of dinucleosome-like particles (Schnitzler et al., 1998). Recent studies have also indicated that the ability of ySWI/SNF to move histone octamers in cis and to enhance the accessibility of nucleosomal DNA does not involve disruption of the histone octamer but seems to be due solely to changes in DNA– histone interactions (Bazett-Jones et al., 1999; Boyer et al., 2000a). In spite of the abundance of information on the effect of ySWI/SNF remodeling on nucleosome structure, how ATP hydrolysis is coupled to these changes is not known. We have proposed that ySWI/SNF may disrupt nucleosome structure by using the energy of ATP hydrolysis to rotate nucleosomal DNA along its long axis (Boyer et al., 2000a). Other models have proposed that SWI/SNF-like enzymes may use the energy of ATP hydrolysis to induce a novel nucleosome conformation (Schnitzler et al., 1998), or that SWI/SNF-like enzymes may track along the DNA which then destabilizes histone–DNA interactions (discussed in Pazin and Kadonaga, 1997). In the present paper, we exploit a quantitative nucleosome-remodeling assay to test predictions of current models for ySWI/SNF mechanism. We find that ySWI/SNF- remodeling requires changes in DNA topology and that this reaction can lead to enhanced DNA accessibility in the absence of dramatic histone octamer movements or displacement. Results ySWI/SNF-Dependent Remodeling Requires DNA Folding into a Nucleosome-like Structure Although members of the SWI2/SNF2 family of ATPases show similarity to the SF-II superfamily of DNA helicases (Eisen et al., 1995), ySWI/SNF and related complexes do not appear to catalyze the unwinding of the DNA double helix (Cote et al., 1994, 1998). However, several models have proposed that SWI/SNF-like enzymes may possess helix-translocating activity. In these models, the ATP-dependent tracking of an SWI/SNF-like enzyme along DNA might disrupt protein–DNA interactions either due to the generation of positive supercoils ahead of the translocating enzyme or by direct displacement (discussed in Pazin and Kadonaga, 1997; see also Havas et al., 2000). In either case, these models predict that the action of a SWI/SNF-like enzyme is not specific for nucleosomes per se, but that the tracking of the enzyme along DNA has a general destabilizing effect on protein– DNA interactions.

Molecular Cell 98

Figure 1. ySWI/SNF-Dependent Remodeling Requires DNA Folding into a Nucleosome (A) ySWI/SNF-dependent remodeling of nucleosomal arrays and nonspecific DNA–histone complexes. HincII (500 U/ml) and ySWI/SNF (2 nM) were added to remodeling reactions that contained either a 11-mer nucleosomal array (1 nM) or a nonspecific histone–DNA substrate (1 nM). Reactions were incubated at 37⬚C for 20 min followed by addition of ATP to half of the reaction mixture. Aliquots were taken at the indicated times, and the reaction was stopped by addition of 5⫻ stop solution (0.5% SDS, 25 mM EDTA, 1 mg/ml proteinase K). Purified DNA was loaded on a 1% agarose gel to separate digestion products from uncut DNA fragments. The amount of radioactivity in each band was quantified, and the percentage of uncut arrays was calculated. The average from at least three experiments is plotted as a function of time. The standard deviation did not exceed 10%. (B) ySWI/SNF ATPase activity is equally stimulated by DNA, nonspecific histone–DNA complexes, and nucleosomal arrays. ySWI/SNF ATPase reactions were performed at an ATP concentration of 100 ␮M (Logie and Peterson, 1999). Reactions were stopped by spotting onto thin layer chromatography (TLC) paper. Inorganic phosphate was separated from ATP, quantified, and the percentage of hydrolyzed ATP was calculated. The average from at least three experiments was plotted as a function of time. The standard deviation did not exceed 10%. (C) Competition of ySWI/SNF remodeling activity by nonspecific histone–DNA complexes and nucleosomal arrays. Remodeling reactions contained HincII (500 U/ml), 1.5 nM ySWI/SNF, and 1 nM 32P-labeled 11-mer nucleosomal arrays. Reactions either lacked an unlabelled competitor DNA (closed circles) or contained 5 nM nonspecific histone–DNA complexes (squares) or nucleosomal (open circles) competitor DNAs.

To investigate this prediction of the tracking models, we tested the ability of ySWI/SNF to stimulate restriction endonuclease cleavage of DNA that is associated with nonspecifically bound histones. In these experiments, an end-labeled DNA fragment was mixed with histones after a rapid salt dilution to generate nonspecific histone–DNA complexes. The DNA fragment contains a unique HincII restriction site within the central repeat of 11 tandem copies of a 5S rDNA nucleosome positioning sequence (Logie and Peterson, 1997). HincII digestion of the nonspecific histone–DNA complexes is biphasic (Figure 1A and data not shown); the first, rapid phase of the reaction represents digestion of DNA harboring HincII sites not occluded by histones, and the second, slow phase represents digestion of the histone-bound HincII sites. After 20 min of incubation with HincII and ySWI/SNF complex, ATP was added and the restriction digestion was monitored for an additional 10 min. As shown in Figure 1A, addition of ATP (⫹SWI/SNF) did not change the rate of HincII cleavage of the nonspecific histone–DNA template (squares versus diamonds). In contrast, the rate of digestion of bona fide nucleosomal arrays was stimulated ⵑ30-fold by addition of ySWI/ SNF and ATP (Figure 1A, triangles versus “X”). Two results indicate that the defect in remodeling the nonspecific histone–DNA complexes is not due to an inability of ySWI/SNF to gain access to the DNA template: (1) the nonspecific histone–DNA complexes were able to

stimulate the ATPase activity of ySWI/SNF (Figure 1B, triangles versus squares and “X”), and (2) the nonspecific histone–DNA complexes were nearly as effective as nucleosomal arrays in competing for the ySWI/SNFdependent remodeling of a 32P-labeled 11-mer nucleosomal array (Figure 1C). Thus, ySWI/SNF remodeling but not substrate binding or ATPase activity appears to require a cannonical nucleosome structure. Furthermore, these results are inconsistent with simple models in which ySWI/SNF disrupts DNA–histone contacts by translocating along DNA. This requirement for nucleosome assembly is also consistent with our recent observation that arrays of histone (H3-H4)2 tetramers are poor substrates for six different members of the ATP-dependent class of remodeling enzymes (Boyer et al., 2000b). Local Changes in DNA Topology Are Required for SWI/SNF-Dependent Remodeling Recently we proposed that ySWI/SNF may remodel nucleosome structure by using the energy of ATP hydrolysis to rotate nucleosomal DNA along its long axis (Boyer et al., 2000a). If ySWI/SNF remodeling involves such ATP-dependent changes in DNA topology, then remodeling might be inhibited on nucleosomal substrates where DNA topology is constrained. Thus, we decided to investigate the ability of SWI/SNF to remodel small, circular nucleosomal substrates where changes in DNA twist may not easily diffuse throughout the rest

SWI/SWF Action Requires Changes in DNA Topology 99

Figure 2. Changes in DNA Topology Are Required for SWI/SNF Remodeling (A) A schematic representation of linear and circular trinucleosomal array substrates. Predicted nucleosome positions are shown as ellipses relative to restriction sites. The hatch mark between each nucleosome indicates the position of EcoRI sites. (B) ySWI/SNF (2 nM) and MspI (2500 U/ml) were incubated in remodeling reactions that contained either the linear or circular trinucleosomal array (1 nM). The fraction of uncut array was quantified with time as described in the legend to Figure 1. (C) Linear arrays, circular arrays, and DNA equally stimulate ySWI/SNF ATPase activity at similar concentrations. Reactions were carried out at an ATP concentration of 50 ␮M for 10 min. (D) Addition of eukaryotic topoisomerases rescue ySWI/SNF remodeling of small chromatin circles. Topoisomerase I (Amersham; 0.3 U/␮l) or topoisomerase II (USB; 1 U/␮l) were added to remodeling reactions that contained linear or circular trinucleosomal array, ATP, and MspI. Remodeling was initiated by addition of ySWI/SNF at t ⫽ 12 min. The fraction of uncut array was quantified with time as described in the legend to Figure 1.

of the molecule, and remodeling may be inhibited as a consequence. We assembled nucleosomes onto a 627 bp DNA fragment that contains three tandem 5S rDNA nucleosome-positioning sequences that each contain an MspI restriction site about 20 bp from the predicted nucleosomal dyad (Figure 2A). To circularize this DNA fragment, ligations were performed in the presence of ethidium bromide at a concentration that yielded primarily topoisomers with ⌬Lk⫽⫺3 (data not shown; Zivanovic et al., 1986). Since each nucleosome will constrain one negative supercoil, we predicted that a ⌬LK⫽⫺3 might facilitate nucleosome assembly (Goulet et al., 1988) and that the saturated, circular array will have a relaxed DNA topology (see Figure 5). Both linear and circular 627 bp DNAs were reconstituted into nucleosomes at the same

concentrations of DNA and histone octamers. These reconstituted arrays showed similar positioning of nucleosomes as determined by MNase and restriction enzyme digestions (see Figure 4). Also, consistent with similar levels of nucleosome deposition and positioning within the linear and circular arrays, both nucleosomal templates showed the same biphasic kinetics of MspI digestion in the absence of ySWI/SNF (Figure 2B, diamonds versus triangles). We then monitored ySWI/SNF remodeling of these two substrates by the stimulation of MspI digestion during the slow, nucleosomal phase of array cleavage. After 10 min of preincubation with ySWI/SNF and MspI, ATP was added and the reaction was monitored for an additional eight minutes. As shown in Figure 2B, addition of ATP dramatically increased the rate of digestion of linear arrays (squares), most of which

Molecular Cell 100

were digested within the first two minutes. On the other hand, circular arrays were resistant to ySWI/SNF remodeling, as there was almost no stimulation of MspI digestion (Figure 2B, “X”). The inability of ySWI/SNF to remodel the small circular substrates was not due to a defect in DNA binding, as the ATPase activity of ySWI/ SNF was equally stimulated by the linear and circular arrays at the same concentrations (Figure 2C). Furthermore, the linear and circular arrays were equivalent competitors for ySWI/SNF remodeling of linear, 11-mer nucleosomal arrays (data not shown). These data indicate that ySWI/SNF can interact with the circular nucleosomal substrates, but that ATP hydrolysis cannot drive nucleosome remodeling. If the block to ySWI/SNF remodeling is due to the constrained topology of the small nucleosomal circles, then addition of topoisomerases might rescue ySWI/ SNF activity. To investigate this possibility, we tested both eukaryotic topoisomerase I and topoisomerase II for their ability to stimulate MspI digestion of small circular arrays in the presence of ySWI/SNF. As shown in Figure 2D, addition of topoisomerase I (stars) or topoisomerase II (triangles) had no effect on the MspI digestion kinetics of circular chromatin in the absence of SWI/ SNF. However, when either topoisomerase I (circles) or topoisomerase II (“X”) was present during the ySWI/SNF reaction, cleavage of circular arrays was dramatically increased. In contrast, addition of topoisomerases had no effect on the efficiency of ySWI/SNF remodeling of the linear nucleosomal arrays (data not shown). Thus, relief of topological stress by topoisomerases restores the ability of ySWI/SNF to remodel small circular nucleosomal arrays. Previous studies have shown that hSWI/SNF and ySWI/SNF can remodel large, circular minichromosomes (Kwon et al., 1994; Jaskelioff et al., 2000), and, in the case of ySWI/SNF, enhanced restriction enzyme cleavage of these large circles does not require topoisomerases (Jaskelioff et al., 2000). However, these prior studies did not investigate whether the circularization of nucleosomal arrays leads to a decreased rate of SWI/ SNF remodeling compared to linear chromatin. To investigate this possibility, we compared the rates of nucleosome remodeling for circular and linear nucleosomal arrays reconstituted on a 3.1 kb plasmid that contains a single 5S rDNA positioning sequence harboring a unique HincII at the predicted nucleosomal dyad (Jaskelioff et al., 2000). In the absence of ATP (⫹SWI/SNF), the linear and circular arrays had similar kinetics of HincII digestion (Figure 3, diamonds versus triangles). When ATP was added to the reactions, the rate of digestion of the linear and circular arrays was stimulated equally well by ySWI/SNF (Figure 3, squares versus “X”). Thus, there is no inherent defect in the remodeling of large circular chromatin. Furthermore, these results are consistent with ySWI/SNF using the energy of ATP hydrolysis to change DNA topology within small domains. The size of this domain might be equivalent to a single nucleosome, consistent with the ability of SWI/SNF-like enzymes to remodel mononucleosome substrates (Cote et al., 1994; Kwon et al., 1994; Utley et al., 1997; Cote et al., 1998; Jaskelioff et al., 2000).

Figure 3. ySWI/SNF Changes DNA Topology in Small Domains Nucleosomal arrays were reconstituted on a 3.1 kb plasmid (pCL115) that contains a single 5S rDNA nucleosome-positioning sequence. Reconstitutions were performed with either the circular plasmid or with the linear plasmid precut with ScaI. Remodeling reactions contained ySWI/SNF (2 nM), nucleosomal substrate (1 nM), and HincII (500 U/ml), for which there is a unique site within the 5S rDNA sequence. The fraction of uncut array was quantified with time as described in the legend to Figure 1.

ySWI/SNF-Dependent Nucleosome Remodeling Does Not Require a Change in Nucleosome Positioning ySWI/SNF can use the energy of ATP hydrolysis to “slide” histone octamers along DNA in cis (Jaskelioff et al., 2000; Whitehouse et al., 1999) or to transfer histone octamers onto acceptor DNAs in trans (Whitehouse et al., 1999). One possibility is that the inability of ySWI/ SNF to enhance the DNA accessibility of small, circular nucleosomal arrays is due to topological constraints that inhibit octamer movements. In this model, topoisomerases may rescue ySWI/SNF remodeling by facilitating the ability of ySWI/SNF to redistribute nucleosome positions. To test these ideas, we compared nucleosome positioning within both linear and circular trinucleosomal arrays before and during a ySWI/SNF remodeling reaction. To map nucleosome positioning, linear or circular nucleosomal substrates were digested with micrococcal nuclease (MNase), and the positions of DNA cleavages were mapped relative to one end of the linear template or to the ligation junction of the circular template (Figure 4). In the case of linear chromatin (Figure 4A), three regions of protection were flanked by two hypersensitive sites in the chromatin samples (lanes 2–4) compared to naked DNA (lanes 11–13). These protected regions are consistent with three nucleosomes that occupy the rDNA positioning sequences within the array (see Figure 2A). A similar pattern, although less well defined, was observed for circular arrays: two hypersensitive sites appeared in circular chromatin at approximately the same location as in linear arrays (compare Figure 4B, lanes 4–6 with Figure 4A, lanes 2–4), and two sites of strong cleavage in naked DNA (marked with asterisks) were protected in circular chromatin (compare Figure 4B, lanes 4–6 with lanes 2 and 3). The similar location of cleavages and protections within linear and circular arrays indicate that nucleosomes occupy similar positions on both types of substrates. When ySWI/SNF was added to the linear arrays, numerous ATP-dependent cleavages were detected in regions previously protected by nucleosomes (Figure 4A, compare lanes 8–10

SWI/SWF Action Requires Changes in DNA Topology 101

with lanes 5–7), and the digestion pattern was similar to but clearly distinct from naked DNA (Figure 4A, lanes 11–13). These results are consistent with an ATP-dependent redistribution of nucleosome positions within the linear arrays, similar to our previous analysis of 11-mer nucleosomal arrays (Jaskelioff et al., 2000). In contrast, addition of ySWI/SNF, ATP, and topoisomerase I to the circular arrays did not significantly change the MNase digestion pattern (Figure 4B, compare lanes 7–9 with lanes 4–6), although ySWI/SNF was able to enhance MspI digestion of these circular substrates to the same degree as linear substrates (data not shown; see also Figure 2). These results suggest that ySWI/SNF can enhance the accessibility of nucleosomal DNA without an obligatory movement of the histone octamer. We also used restriction enzyme digestion of the circular nucleosomal arrays to confirm the correct positioning of nucleosomes prior to ATP-dependent remodeling and to test whether ySWI/SNF action leads to dramatic changes in nucleosome positioning on these substrates. When nucleosomes occupy the major translational positioning frame on a 5S DNA repeat, an MspI site is protected by the nucleosome and an EcoRI site is located in the accessible, linker region between nucleosomes (see schematic, Figure 2A). In the absence of remodeling activity, ⵑ70% of the MspI sites are occluded by nucleosomes on both the circular and linear trinucleosomal arrays (Figure 4C; see also Figure 2B). Conversely, 75% of the EcoRI sites within the circular arrays are freely accessible to digestion (Figure 4C). Similar EcoRI accessibility is also seen for linear 3-mer and 11-mer nucleosomal arrays (data not shown; see Jaskelioff et al., 2000). Thus, these results confirm that the bulk of circular nucleosomal arrays maintain the translational positioning of nucleosomes depicted in Figure 2A. Previously, we used EcoRI digestion of linear 11-mer nucleosomal arrays to follow the ATP-dependent redistribution of nucleosome positions (Jaskelioff et al., 2000). In these studies, we observed a dramatic ATPand ySWI/SNF-dependent occlusion of EcoRI sites that persisted in the absence of continuous ATP hydrolysis. We used this same experimental protocol to test whether ySWI/SNF causes similar nucleosome redistri-

Figure 4. ySWI/SNF Enhances the Accessibility of Small Circular Arrays in the Absence of Dramatic Histone Octamer Movements (A) MNase digestion analysis of nucleosome positions within linear trinucleosomal arrays. End-labeled nucleosomal arrays or free DNA were digested with increasing concentrations of MNase (triangles above lanes). DNA was purified and electrophoresed on a 4% acrylamide gel. Derived nucleosome positions are shown by ellipses. Note that in the presence of ySWI/SNF and ATP, the MNase digestion pattern of the linear arrays is similar to but clearly distinct from that of the free, linear DNA. S, linear array substrate; M, 100 bp DNA ladder. (B) MNase digestion of circular trinucleosomal arrays. Arrays were reconstituted onto DNA circles that contained a 32P label at the point of ligation. Circular arrays were incubated with increasing concentrations of MNase (triangles above lanes), and the purified DNA was cut with AlwI prior to loading on the gel. Cleavage with

AlwI generates the unique, end-labeled DNA fragments (see Figure 2A for schematic). Reactions contained ySWI/SNF, topoisomerase II, and ATP where indicated. C, circular; L, linear; N, nicked DNA probe. Asterisks indicate hypersensitive sites on the naked, circular DNA probe. Note that the MNase digestion pattern of the circular arrays is similar both in the presence or absence of ATP. S, circular array substrate; M, 100 bp DNA ladder. (C) Restriction enzyme accessibility of circular trinucleosomal arrays. Circular arrays were incubated with topoisomerase II, ySWI/ SNF, and either MspI (500 U/ml) or EcoRI (500 U/ml). ATP was added where indicated. For reactions that contained apyrase, circular arrays were first incubated with topoisomerase II, ySWI/SNF, and ATP for 5 min, then apyrase (10 U/ml) was added to remove ATP, and this treatment was followed by addition of either MspI or EcoRI. The amount of radioactivity in all topoisomers was quantified, and the percentage of uncut array was calculated. Data shown is representative of three independent experiments. Note that the bulk of the minichromosomes contain an accessible EcoRI site and that ySWI/SNF action does not lead to a significant decrease in EcoRI accessibility.

Molecular Cell 102

butions on the small circular arrays. First, we incubated the circular arrays with ySWI/SNF, EcoRI, topoisomerase II, and ATP. Under these conditions, remodeling led to a small, additional increase in EcoRI accessibility (Figure 4C). To further test whether ySWI/SNF might generate stable changes in EcoRI accessibility, ySWI/ SNF was first allowed to remodel the circular arrays, and then the reaction was stopped by addition of apyrase prior to addition of EcoRI. In these reactions, we observed only negligible (⬍5%) ATP-dependent decreases in EcoRI digestion following inactivation of the remodeling reaction (Figure 4C). These results are consistent with the MNase digestion studies shown in Figure 4B, and together these results strongly indicate that ySWI/SNF can enhance the accessibility of the circular nucleosomal arrays in the absence of dramatic nucleosome movements. Furthermore, since the majority of MspI sites revert to an occluded state following inactivation of ySWI/SNF (Figure 4C), these studies also indicate that histone octamers are not evicted from the circular templates. ySWI/SNF-Dependent Change in the Topology of Circular Arrays Does Not Result from Histone Octamer Eviction One of the biochemical hallmarks of ySWI/SNF remodeling is the ATP-dependent decrease in the number of negative supercoils constrained by nucleosomes in a large, circular array (Kwon et al., 1994). These changes require the combined actions of ySWI/SNF and a topoisomerase (Jaskelioff et al., 2000) and they persist after ATP or SWI/SNF removal (Imbalzano et al., 1996). Although our MNase and restriction enzyme mapping studies suggest that nucleosomes are not lost from the circular nucleosomal arrays, we wished to further test the possibility that the loss of negative supercoils from circular chromatin reflects the eviction of histone octamers. Alternatively, ySWI/SNF-dependent changes in DNA topology may cause a partial unwrapping of nucleosomal DNA, and the released supercoils may be removed by a topoisomerase. In the latter case, the subsequent inactivation of ySWI/SNF might yield arrays of nucleosomes with unfolded DNA, similar to the reaction products observed by EM (Bazett-Jones et al., 1999). To test these two possibilities, we analyzed the topology and MspI digestion kinetics of our circular trinucleosome substrates. We reasoned that if the ATPdependent loss of supercoils from circular chromatin is due to histone octamer eviction, then topoisomers with less supercoils (those who may have lost one or two histone octamers) will be digested more rapidly than those that retain three negative supercoils. If the decrease in superhelicity is due to unfolding of nucleosomal DNA and all nucleosomes are retained, then the restriction sites may still be occluded. In this case, the restriction endonuclease will cleave all circles at the same rate regardless of their topology, and it will not change the topoisomer distribution. As shown in Figure 5 (lane 1) most of our reconstituted trinucleosome circles have three negative supercoils (one per nucleosome). Addition of topoisomerase II and ATP to these minichromosomes did not significantly change the distribution of topoisomers (Figure 5, lane

Figure 5. ySWI/SNF-Dependent Loss of Supercoiling of Small Circular Arrays Does Not Result from Eviction of Histone Octamers Analysis of the MspI accessibility of minichromosome topoisomers. Circular trinucleosomal arrays were incubated with topoisomerase II in the absence (lanes 1–3) or presence (lanes 4–7) of MspI and in the presence (lane 1 and lanes 3–7) or absence (lane 2) of ySWI/ SNF. All reactions were incubated in the absence of MspI for 5 min at 37⬚C, and then, where indicated, MspI was added to half of the mixture, and all reactions were incubated for an additional 20 min. For reactions shown in lanes 6 and 7, apyrase (10 U/ml) was added after the initial 5 min incubation prior to MspI addition. DNA was purified and electrophoresed on a 4% acrylamide gel (30:1 acrylamide:bisacrylamide ratio). The migration of topoisomers and linear DNA is shown on the left. Identity of the topoismers was determined by electrophoresis of topoisomer standards isolated by ligating circles in the presence of different concentrations of ethidium bromide. M, 32P-labeled 100 bp DNA molecular weight standard (GIBCO–BRL).

2), consistent with the relaxed conformation of circles assembled into three nucleosomes. In contrast, addition of ySWI/SNF to these reactions reduced the superhelicity: the number of plasmids with ⌬Lk⫽⫺3 was significantly decreased, while topoisomers with ⌬Lk⫽⫺2, ⌬Lk⫽⫺1 and ⌬Lk⫽0 were dramatically increased (Figure 5, compare lane 3 with lane 2). Furthermore, in the continued presence of ATP, the loss of negative supercoils is accompanied by an increased accessibility of the nucleosomal MspI sites (Figure 5, compare lanes 5 and 4; see also Figure 4C). When the ySWI/SNF and topoisomerase II reaction was stopped by removal of ATP with apyrase and then MspI was added (Figure 5, lane 6), all topoisomers were cleaved to an extent similar to that observed in the absence of ATP (lane 4). Importantly, circles with 2 or fewer negative supercoils were not preferentially cleaved, which would be expected if the loss of supercoils was due to eviction of histone octamers (Figure 5, compare lanes 6 and 3). Furthermore, after inactivation of ySWI/SNF, the total amount of uncut array (including all topoisomers) was nearly equivalent to that of reactions in which ATP had not been added (Figure 5, compare lanes 6 and 4; see also Figure 4C). This suggests that the ySWI/SNF reaction does not generate accessible MspI sites that persist in the absence of ATP hydrolysis (see also Jaskelioff et al., 2000); and furthermore, these results indicate that the ATP-dependent loss of supercoils is not sufficient to generate increased nucleosomal accessibility to restriction enzymes. Thus, these data indicate that the ATP-dependent loss of supercoiling is not due to the loss of histone octamers, but rather the results support

SWI/SWF Action Requires Changes in DNA Topology 103

models in which ATP-dependent changes in local DNA topology can lead to a nucleosome with unfolded DNA, consistent with previous EM studies (Bazett-Jones et al., 1999). Discussion Here we have investigated the ability of ySWI/SNF to enhance restriction enzyme cleavage of small, circular substrates that contain only three nucleosomes. On these topologically constrained minicircles, ySWI/SNF remodeling is dependent on addition of a topoisomerase, suggesting that the remodeling reaction creates topological stress that must be relieved in order to generate accessible nucleosomal DNA. Based on previous results, one might have predicted that the role of the topoisomerase is to facilitate ySWI/SNF-dependent movement of histone octamers in cis or in trans. However, our results indicate that ySWI/SNF can enhance restriction enzyme accessibility of circular chromatin in the absence of an obligatory eviction or dramatic movement of histone octamers. We favor a model in which ySWI/SNF uses the energy of ATP hydrolysis to drive local changes in DNA twist which generates “accessible” nucleosomes. This accessible state can then lead indirectly to movements of nucleosomes in cis or in trans on linear or large circular chromatin. Thus, in this type of model, ATP-dependent remodeling is not equivalent to the movement of histone octamers. This conclusion is not entirely novel but has been suggested by previous studies demonstrating that SWI/SNF can successfully remodel nucleosomes in which the histone octamer is immobilized on DNA by chemical cross-linking (Cote et al., 1998; Lee et al., 1999), or where nucleosomes are assembled on very short DNA fragments where movements are not observed (Cote et al., 1994; Imbalzano et al., 1996; Jaskelioff et al., 2000). ySWI/SNF Action Involves Changes in DNA Topology Two distinct types of remodeling assays have demonstrated that chromatin remodeling by SWI/SNF-like enzymes involves changes in DNA topology. First, the incubation of circular chromatin templates with SWI/SNF and a topoisomerase decreases the number of negative supercoils constrained by nucleosomes. This reaction is not the result of nucleosome loss (Figure 5) but must reflect either an unwrapping of nucleosomal DNA or a major change in the path of DNA around the histone octamer (a change in writhe). An ATP-dependent unwrapping of nucleosomal DNA is consistent with recent EM studies that indicate a loss of ⵑ40 bp of DNA from remodeled nucleosomes (Bazett-Jones et al., 1999). SWI/SNF remodeling may disrupt the 20 base pairs of DNA at both the exit and entry termini of nucleosomal DNA, since these DNA regions are less tightly bound to the histone octamer (Luger and Richmond, 1998) and are released first during low-energy transitions (Simpson, 1979; Polach and Widom, 1995). Importantly, these putative “unwrapped” nucleosomes are not equivalent to a fully remodeled nucleosome species, since they still harbor DNA that is inaccessible to restriction enzyme digestion (Figure 5). Thus, such species may represent

either an intermediate or a stable byproduct of nucleosome remodeling. Whereas previous studies have shown that SWI/SNF action can lead to changes in DNA topology, our current studies indicate that the ability of SWI/SNF to enhance nucleosomal DNA accessibility requires changes in DNA topology. Since SWI/SNF is not a topoisomerase and, thus, cannot directly change the linking number of DNA, SWI/SNF action must involve changes in the writhe or twist of nucleosomal DNA. One possibility is that SWI/ SNF changes the path of DNA on the surface of the histone octamer or alters the orientation of the entry and exit angles of nucleosomal DNA. Alternatively, we favor a model in which SWI/SNF uses the energy of ATP hydrolysis to drive local changes in DNA twist that diffuse throughout the nucleosome and weaken both wraps of histone–DNA interactions. This reaction would lead to a nucleosome in which the histone octamer is inherently more mobile and DNA more accessible to nucleases and transcription factors. Since this accessible nucleosomal state requires continuous ATP hydrolysis (Figure 4C; see also Jaskelioff et al., 2000), our data suggests that ySWI/SNF must continuously alter DNA twist in order to generate a remodeled state. Such continuous changes in DNA topology might have a high energy cost on topologically constrained minicircles, and, thus, remodeling would not be proficient on these chromatin templates. This type of model is supported by recent studies which demonstrate that SWI/SNF-like enzymes can use the energy of ATP hydrolysis to generate unconstrained superhelical torsion in DNA and nucleosomal substrates (Havas et al., 2000). Our proposed model for ATP-dependent remodeling may be mechanistically related to how the bacterial DNA helicase, PcrA, couples ATP binding and hydrolysis to localized DNA helix destabilization (Velankar et al., 1999; Soultanas et al., 2000). PcrA is a member of helicase superfamily I, and it contains the seven DNA helicase motifs that are also found in SWI2/SNF2 family members. Recent functional and structural studies of PcrA have suggested that this helicase has two complementary but distinct ATP-dependent functions: ATP-dependent ssDNA tracking activity and ATP-dependent DNA duplex destabilization. In the latter case, the binding of ATP induces PcrA to bind double-stranded DNA in such a way as to destabilize the DNA helix. Subsequent ATP hydrolysis leads to the translocation of the enzyme one nucleotide along the adjacent ssDNA tail and the release of the distorted DNA duplex. We propose that SWI/ SNF may harbor only one of the two ATP-dependent activities of PcrA: ATP-dependent DNA duplex destabilization. Consistent with this view, the ATPase subunit of SWI/SNF, SWI2/SNF2, shares significant structural homology to the 2B domain of PcrA (crucial for ATPdependent DNA duplex destabilization) but lacks homology to the domain of PcrA involved in ssDNA translocation (C. Smith and C. L. P., unpublished observations). Experimental Procedures DNA Probes A DNA fragment containing three tandem 5S rDNA repeats from Lytechinus variegatus was isolated from pLH6 by digestion with BglII and BamHI, gel purified, and labeled with ␥ATP[32P] in an exchange

Molecular Cell 104

reaction. To make DNA circles, the fragment was ligated at a concentration of 100 ng/ml with 400 U/ml T4 DNA ligase in the presence of 1 ␮g/ml ethidium bromide at 16⬚C overnight (Zivanovic et al., 1986). Circles were concentrated, extracted with phenol/chloroform and purified on G-25 spin column. For MNase experiments, the pLH6 plasmid was isolated from a dam⫺ bacterial strain. The plasmid was digested with BamHI and PstI to make a linear DNA probe. The resulting DNA fragment had an extra 13 bp at the BglII terminus and was labeled at the BamHI site with Klenow fragment using dATP[32P]. Nucleosome Reconstitutions and Enzymatic Assays Small circular nucleosomal arrays were reconstituted from purified chicken histone octamers at a DNA concentration of 20 ␮g/ml using equal mole to mole histone to DNA ratios by stepwise salt dialysis (Logie and Peterson, 1997). 208-11 nucleosomal arrays were prepared as described (Logie and Peterson, 1997), and the extent of nucleosome reconstitution was monitored by EcoRI analysis (Logie and Peterson, 1997). Nonspecific DNA–histone complexes were made at a concentration of 40 ␮g/ml by adding 208-11 DNA to an equal mass of histone octamers in 10 mM Tris, 1 mM EDTA. Remodeling assays were performed, and ySWI/SNF complex was purified as described previously (Logie and Peterson, 1999). MNase digestion was performed as described (Jaskelioff et al., 2000). DNA from circular templates was cleaved with AlwI before loading on a 4% polyacrylamide gel. Acknowledgments We would like to thank Anthony Imbalzano for comments on the manuscript, Colin Logie for providing DNA and chromatin templates, and members of the Peterson lab for helpful discussions throughout the course of this work. These studies were supported by grants from the NIH to C. L. P (GM49054) and P. J. H. (5F32GM20229). Received November 9, 2000; revised December 27, 2000. References Bazett-Jones, D.P., Cote, J., Landel, C.C., Peterson, C.L., and Workman, J.L. (1999). The SWI/SNF complex creates loop domains in DNA and polynucleosome arrays and can disrupt DNA-histone contacts within these domains. Mol. Cell. Biol. 19, 1470–1478. Boyer, L.A., Shao, X., Ebright, R.H., and Peterson, C.L. (2000a). Roles of the histone H2A–H2B dimers and the (H3–H4)(2) tetramer in nucleosome remodeling by the SWI-SNF complex. J. Biol. Chem. 275, 11545–11552. Boyer, L.A., Logie, C., Bonte, E., Becker, P.B., Wade, P.A., Wolffe, A.P., Wu, C., Imbalzano, A.N., and Peterson, C.L. (2000b). Functional delineation of three groups of the ATP-dependent family of chromatin remodeling enzymes. J. Biol. Chem. 275, 18864–18870. Cote, J., Quinn, J., Workman, J.L., and Peterson, C.L. (1994). Stimulation of GAL4 derivative binding to nucleosomal DNA by the yeast SWI/SNF complex. Science 265, 53–60. Cote, J., Peterson, C.L., and Workman, J.L. (1998). Perturbation of nucleosome core structure by the SWI/SNF complex persists after its detachment, enhancing subsequent transcription factor binding. Proc. Natl. Acad. Sci. USA 95, 4947–4952. de La Serna, I.L., Carlson, K.A., Hill, D.A., Guidi, C.J., Stephenson, R.O., Sif, S., Kingston, R.E., and Imbalzano, A.N. (2000). Mammalian SWI-SNF complexes contribute to activation of the hsp70 gene. Mol. Cell. Biol. 20, 2839–2851. Deuring, R., Fanti, L., Armstrong, J.A., Sarte, M., Papoulas, O., Prestel, M., Daubresse, G., Verardo, M., Moseley, S.L., Berloco, M., et al. (2000). The ISWI chromatin-remodeling protein is required for gene expression and the maintenance of higher order chromatin structure in vivo. Mol. Cell 5, 355–365. Eisen, J.A., Sweder, K.S., and Hanawalt, P.C. (1995). Evolution of the SNF2 family of proteins: subfamilies with distinct sequences and functions. Nucleic Acids Res. 23, 2715–2723.

Goulet, I., Zivanovic, Y., Prunell, A., and Revet, B. (1988). Chromatin reconstitution on small DNA rings. I. J. Mol. Biol. 200, 253–266. Havas, K., Flaus, A., Phelan, M., Kingston, R., Wade, P.A., Liley, D.M., and Owen-Hughes, T. (2000). Generation of superhelical torsion by ATP-dependent chromatin remodeling activities. Cell 103, 1133– 1142. Imbalzano, A.N., Schnitzler, G.R., and Kingston, R.E. (1996). Nucleosome disruption by human SWI/SNF is maintained in the absence of continued ATP hydrolysis. J. Biol. Chem. 271, 20726–20733. Jaskelioff, M., Gavin, I.M., Peterson, C.L., and Logie, C. (2000). SWISNF-mediated nucleosome remodeling: role of histone octamer mobility in the persistence of the remodeled state. Mol. Cell. Biol. 20, 3058–3068. Kingston, R.E., and Narlikar, G.J. (1999). ATP-dependent remodeling and acetylation as regulators of chromatin fluidity. Genes Dev. 13, 2339–2352. Kwon, H., Imbalzano, A.N., Khavari, P.A., Kingston, R.E., and Green, M.R. (1994). Nucleosome disruption and enhancement of activator binding by a human SWI/SNF complex. Nature 370, 477–480. Lee, K.M., Sif, S., Kingston, R.E., and Hayes, J.J. (1999). hSWI/SNF disrupts interactions between the H2A N-terminal tail and nucleosomal DNA. Biochemistry 38, 8423–8429. Logie, C., and Peterson, C.L. (1997). Catalytic activity of the yeast SWI/SNF complex on reconstituted nucleosome arrays. EMBO J. 16, 6772–6782. Logie, C., and Peterson, C.L. (1999). Purification and biochemical properties of yeast SWI/SNF complex. Methods Enzymol. 304, 726–741. Lorch, Y., Zhang, M., and Kornberg, R.D. (1999). Histone octamer transfer by a chromatin-remodeling complex. Cell 96, 389–392. Luger, K., and Richmond, T.J. (1998). DNA binding within the nucleosome core. Curr. Opin. Struct. Biol. 8, 33–40. Muchardt, C., and Yaniv, M. (1999). ATP-dependent chromatin remodelling: SWI/SNF and Co. are on the job. J. Mol. Biol. 293, 187–198. Pazin, M.J., and Kadonaga, J.T. (1997). SWI2/SNF2 and related proteins: ATP-driven motors that disrupt protein-DNA interactions? Cell 88, 737–740. Polach, K.J., and Widom, J. (1995). Mechanism of protein access to specific DNA sequences in chromatin: a dynamic equilibrium model for gene regulation. J. Mol. Biol. 254, 130–149. Schnitzler, G., Sif, S., and Kingston, R.E. (1998). Human SWI/SNF interconverts a nucleosome between its base state and a stable remodeled state. Cell 94, 17–27. Simpson, R.T. (1979). Mechanism of a reversible, thermally induced conformational change in chromatin core particles. J. Biol. Chem. 254, 10123–10127. Soultanas, P., Dillingham, M.S., Wiley, P., Webb, M.R., and Wigley, D.B. (2000). Uncoupling of DNA translocation and helicase activity in PcrA: direct evidence for an active mechanism. EMBO J. 19, 3799–3810. Utley, R.T., Cote, J., Owen-Hughes, T., and Workman, J.L. (1997). SWI/SNF stimulates the formation of disparate activator-nucleosome complexes but is partially redundant with cooperative binding. J. Biol. Chem. 272, 12642–12649. Velankar, S.S., Soultanas, P., Dillingham, M.S., Subramanya, H.S., and Wigley, D.B. (1999). Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism. Cell 97, 75–84. Vignali, M., Hassan, A.H., Neely, K.E., and Workman, J.L. (2000). ATP-dependent chromatin-remodeling complexes. Mol. Cell. Biol. 20, 1899–1910. Whitehouse, I., Flaus, A., Cairns, B.R., White, M.F., Workman, J.L., and Owen-Hughes, T. (1999). Nucleosome mobilization catalysed by the yeast SWI/SNF complex. Nature 400, 784–787. Zivanovic, Y., Goulet, I., and Prunell, A. (1986). Properties of supercoiled DNA in gel electrophoresis. The V-like dependence of mobility on topological constraint. DNA-matrix interactions. J. Mol. Biol. 192, 645–660.