the ATP-utilizing chromatin assembly and remodeling factor (ACF) (Ito et al., 1997). Each of these complexes. The ATPase ISWI is a subunit of several distinct ...
Molecular Cell, Vol. 3, 239–245, February, 1999, Copyright 1999 by Cell Press
ISWI Is an ATP-Dependent Nucleosome Remodeling Factor Davide F. V. Corona,*§ Gernot La¨ngst,*§ Cedric R. Clapier,* Edgar J. Bonte,* Simona Ferrari,* John W. Tamkun,† and Peter B. Becker*‡ * European Molecular Biology Laboratory Gene Expression Programme 69117 Heidelberg Germany † Department of Biology University of California Santa Cruz, California 95064
Summary The ATPase ISWI is a subunit of several distinct nucleosome remodeling complexes that increase the accessibility of DNA in chromatin. We found that the isolated ISWI protein itself was able to carry out nucleosome remodeling, nucleosome rearrangement, and chromatin assembly reactions. The ATPase activity of ISWI was stimulated by nucleosomes but not by free DNA or free histones, indicating that ISWI recognizes a specific structural feature of nucleosomes. Nucleosome remodeling, therefore, does not require a functional interaction between ISWI and the other subunits of ISWI complexes. The role of proteins associated with ISWI may be to regulate the activity of the remodeling engine or to define the physiological context within which a nucleosome remodeling reaction occurs. Introduction Transcription, replication, DNA repair, and other reactions involving chromatin substrates require that repressive chromatin structures are altered to allow regulatory proteins to interact with DNA (for review see Felsenfeld, 1996; Kadonaga, 1998). Nucleosome remodeling by large multisubunit complexes is an important mechanism by which specific chromatin sites are rendered accessible. These complexes increase the accessibility of nucleosomal DNA in a reaction that involves ATP hydrolysis (reviewed by Kingston et al., 1996; Pazin and Kadonaga, 1997; Tsukiyama and Wu, 1997; Cairns, 1998; Varga-Weisz and Becker, 1998). Genetic studies in yeast led to the identification of the first chromatin remodeling factor, the large 11 subunit SWI/SNF complex that counteracts chromatin repression of a subset of inducible genes (Hirschhorn et al., 1992; Peterson and Herskowitz, 1992; Burns and Peterson, 1997; reviewed in Peterson, 1996). Complexes related to SWI/SNF have been identified in yeast (RSC), flies (BRM complex), and man (the BRG1 and hbrm complexes), and all those tested were able to alter nucleosome structure in vitro to facilitate the binding of tran‡ To whom correspondence should be addressed (e-mail: becker@
embl-heidelberg.de). § These authors contributed equally to this work.
scription factors to nucleosomal DNA (Muchardt and Yaniv, 1993; Cote et al., 1994; Imbalzano et al., 1994; Kwon et al., 1994; Cairns et al., 1996; Wang et al., 1996; Papoulas et al., 1998). Chromatin remodeling factors in higher eukaryotes have been implicated in diverse biological processes, including transcriptional activation, cell cycle control, proviral integration, and the control of cell fate (reviewed in Tamkun, 1995). Biochemical studies in Drosophila have identified additional chromatin remodeling factors that also utilize the energy of ATP to increase the accessibility of nucleosomal DNA including the nucleosome remodeling factor (NURF) (Tsukiyama et al., 1995), the chromatin accessibility complex (CHRAC) (Varga-Weisz et al., 1997), and the ATP-utilizing chromatin assembly and remodeling factor (ACF) (Ito et al., 1997). Each of these complexes contains the ATPase ISWI, a protein related to SWI2/ SNF2, the ATPase subunit of the SWI/SNF complex (Elfring et al., 1994). Potential homologs of ISWI have been identified in all eukaryotes examined to date, suggesting that it plays an important, conserved role in counteracting chromatin repression. Since most biochemical studies of chromatin remodeling factors have utilized purified complexes, little is known about the specific functions of their individual subunits. One current view is that ATPase subunits of these complexes serve as “engines” of the complexes, while other subunits mediate interactions with DNA, nucleosomes, DNA-binding proteins, or other molecules and structures within nuclei. The activity profiles of the ISWI complexes differ in detail, presumably reflecting their distinct subunit composition. In order to study the roles of ISWI in diverse nucleosome remodeling complexes, we characterized the activities of the isolated ATPase after expression in bacteria. Remarkably, we find that ISWI per se, out of the context of a multisubunit complex, is able to remodel nucleosomes, rearrange nucleosomal positions, and to function as a chromatin assembly factor. Since these activities so far have only been found associated with large multisubunit remodeling complexes, our finding that a single ATPase is capable of nucleosome remodeling necessitates substantial modification of current concepts. Results The ATPase Activity of ISWI Is Stimulated by Nucleosomes but Not by Free DNA We expressed the full-length D. melanogaster ISWI with an N-terminal FLAG tag (rISWI) in E. coli and purified the protein to homogeneity. As a negative control for ATP-dependent effects, we expressed in parallel an ISWI derivative in which lysine 159 was replaced by arginine. This residue is part of a conserved adenine nucleotide binding fold, GXGKT, that is shared by a diverse group of proteins requiring ATP for function (Sung et al., 1988; Walker et al., 1988; Henikoff, 1993; Laurent et al., 1993; Elfring et al., 1994). The corresponding mutation in SWI2/SNF2 abolishes its DNA-stimulated ATPase activity as well as its ability to support
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Figure 1. ISWI Is a Nucleosome-Stimulated ATPase (A) ATPase assays contained either of the following: 1.3 pmol of rISWI (160 ng), an equivalent volume of the control protein preparation, or 0.6 pmol of CHRAC. The substrates of the reaction were either 3.75 pmol (360 ng) of a 145 bp fragment (D) or the equivalent amount of nucleosomal DNA (N). The asterisk points to the signal derived from free phosphate generated during the 30 min assay. (B) A comparison of the ATPase activity of similar amounts of rISWI-wt and the K159R mutant derivative of rISWI. In order to monitor any residual ATPase activity of the K159 mutant, the reaction was run for 60 min. (C) The ISWI ATPase is not stimulated by free DNA. The following DNA substrates (100 ng) were included into standard ATPase assays (30 min reactions) containing recombinant rISWI or purified CHRAC: nucleosomal DNA (N), a 3 kb supercoiled plasmid (SC), a 146 bp PCR fragment (PCR), a synthetic four-way junction (4WJ), phage lambda DNA (l, approx. 50 kb), polyA1 RNA (RNA). The ATPase activity is displayed as the percentage of ATP hydrolyzed during the assay. (D) Competition of histone N-terminal “tails” with nucleosomes. ATPase assays as in (B) contained either only buffer (B), 1 pmol of nucleosomes (N), or nucleosomes plus 50 pmol of individual Drosophila histone N termini (as indicated) fused to GST, or as a control, the GST moiety alone (GST).
transcriptional activation in vivo (Laurent et al., 1993; Richmond and Peterson, 1996). The wild-type (rISWI-wt) and the mutated (rISWI-K159R) enzymes were prepared with similar concentrations and purity (data not shown). To evaluate the functional status of the recombinant enzymes, ATPase assays were conducted in the presence of a range of potential substrates. rISWI-wt displayed a low constitutive level of ATPase activity in a standardized reaction when compared to control protein preparations (Figure 1A, lanes 1 and 7); this activity was stimulated only poorly in the presence of a 146 bp DNA fragment (Figure 1A, lane 2; Figure 1B, lanes 1 and 2). However, if the same amount of DNA was wrapped around a histone octamer, an approximately 10-fold increase in ATPase activity was reproducibly observed (Figure 1A, lane 3; Figure 1B, lane 3). A time course of ATP hydrolysis by ISWI established that the reaction kinetics were linear through the first hour (data not shown), hence all ATPase assays were done within this time. The rate of nucleosome-stimulated ATP hydrolysis under these conditions was estimated to be in the order of 75 molecules of ATP per minute (average hydrolysis during the first 5 min in the presence of 1 mM ATP). Mutating the adenine nucleotide binding pocket of ISWI (K159R) abolished its ATPase activity (Figure 1B, lanes 4–6), which demonstrates that the observed activity was indeed due to rISWI rather than a copurifying contaminant. The ATPase of purified CHRAC, in contrast, was stimulated by nucleosomes as well as free DNA (Figure 1A, lanes 4–6). As previously reported, DNA stimulates the ATPase activity of topoisomerase II that is present in an enzymatically active form in CHRAC (Varga-Weisz et al., 1997). The fact that roughly equivalent amounts of rISWI or ISWI in CHRAC (see legend to Figure 1A) yielded equivalent levels of nucleosomestimulated ATPase demonstrated that rISWI was highly active. The substrate requirements for eliciting the ATPase
activity were further examined. Since DNA is distorted when wound around a nucleosome, we assayed whether the ISWI ATPase was affected by various DNA substrates or RNA. Neither a supercoiled plasmid, a small PCR fragment, phage lambda DNA, synthetic cruciform DNA, nor messenger RNA was able to stimulate the ATPase of rISWI significantly, while all DNA substrates, but not the RNA, stimulated an ATPase of CHRAC (Figure 1C). Next, we explored whether histones alone were able to induce ATP hydrolysis. Histone octamers, purified to homogeneity from Drosophila embryo chromatin, dissociate under the low salt reaction conditions of the ATPase assay into H2A/H2B dimers and H3-H4 tetramers (Eickbush and Moudrianakis, 1978). The rISWI ATPase was not stimulated by this mixture of histones even when four times more free histones were added than the amount of nucleosomal histones that elicited a clear ATPase reaction (data not shown). Similarly, fusion proteins displaying the N-terminal “tail” domains of individual histones attached to a GST moiety did not trigger any ATPase activity (data not shown). However, these histone tail–GST fusion proteins, but not the GST moiety alone, inhibited the ATPase activity when added in a 50-fold excess to a reaction containing nucleosome substrates (Figure 1D). The significance of the observation that the N terminus of H4 inhibited the ATPase less than the other N termini is unclear at present, but this result excludes nonspecific effects of a net positive charge of histone N termini. These results are consistent with an earlier report on the inhibitory effect of histone tail–GST fusion proteins on the ATPase activity of NURF (Georgel et al., 1997) and might indicate that ISWI recognizes a particular feature on the histone N termini that would not be sufficient by itself to elicit the ATPase reaction. However, since we failed to detect an interaction between ISWI and histone tail–GST fusion proteins in classical “pull-down” assays, an indirect effect (e.g., a masking of a nucleosomal feature by interaction of a
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histone tail) cannot be excluded. We conclude that rISWI responds with ATP hydrolysis to a specific feature that is unique to a nucleosome core particle and cannot be found in either free DNA or free histones.
Figure 2. ISWI Is a Nucleosome Remodeling Factor (A) Nucleosome remodeling in the NURF assay. The nucleosome remodeling reaction is described in the text. The reactions contained ATP (throughout), GAGA factor as indicated, and the following remodeling factors: 0.15 pmol CHRAC, 0.13 pmol rISWI, rISWI-K159R, or 4 ml crude embryo extract, and a chromatin substrate equivalent of roughly 1 pmol nucleosomes. DNA fragments of diagnostic lengths: Arrow, mononucleosome; filled circle, dinucleosome; asterisk, atypical particle indicative of remodeling. The upper panel indicates the status around the GAGA boxes of the hsp26 promoter, while the lower panel shows the control hybridization of the same membrane with a vector-derived DNA probe. (B) TTF-1-dependent nucleosome remodeling at the rDNA promoter. TTF-1 binding to the rDNA promoter was assayed in the context of chromatin-bound remodeling machines (panel 1) or after endogenous remodeling factors had been removed by Sarkosyl treatment
Nucleosome Remodeling by rISWI Facilitates the Binding of Transcription Factors to Chromatin The degree of nucleosome stimulation of the ISWI ATPase was comparable to the effect of nucleosomes on the ATPase activity of CHRAC and NURF complexes (see above; Tsukiyama and Wu, 1995). We therefore explored whether rISWI was also able to carry out established nucleosome remodeling reactions. We first tested rISWI in the assay that was initially used to define NURF (Tsukiyama et al., 1994). This assay monitors the change in nucleosomal occupancy of a particular DNA element upon interaction of a transcription factor. Chromatin was reconstituted in a Drosophila embryo extract on a plasmid containing the hsp26 promoter featuring binding sites for the GAGA factor. Chromatin-bound remodeling machines were inactivated with Sarkosyl, and the chromatin was then purified via gel filtration. Access of GAGA factor to its binding sites in chromatin relies on concomitant ATP-dependent nucleosome remodeling (Tsukiyama et al., 1994). The nucleosome remodeling reaction is monitored by digestion of chromatin with Micrococcal Nuclease (MNase), which cleaves the internucleosomal linker DNA leading to the accumulation of nucleaseresistant nucleosomal DNA fragments of about 145 bp in length (arrow in Figure 2A). Southern blotting of the MNase fragments and hybridization with specific probes reveals the association of a particular DNA sequence with a nucleosome and whether the nucleosomal occupancy changes upon interaction of a transcription factor. The nucleosomal occupancy (arrow in Figure 2A) at the hsp26 promoter did not change significantly if the GAGA factor was added in the absence of remodeling factors (Figure 2A, upper panel 2). However, if a small amount of embryo extract as a source of mixed remodeling factors was included (Figure 2A, upper panel 10), no nucleosomal DNA was recovered: the corresponding DNA was either degraded or was found in atypical fragments (asterisks) characteristic of the remodeled configuration (Wall et al., 1995). Nucleosome remodeling as defined by this assay requires the presence of GAGA factor, a remodeling factor, and ATP and is limited to the target site since rehybridization of the membrane with a vector probe establishes that bulk chromatin is unaffected (Figure 2A, vector control). Purified CHRAC did not alter the nucleosomal occupancy at the GAGA boxes significantly (Figure 2A, upper panel 4), consistent with earlier findings (Varga-Weisz et al., 1997). Remarkably, an equivalent amount of rISWI was able to elicit
(panel 2), in the presence of either CHRAC or rISWI or rISWI-K159R as indicated. TTF-1 binding (box to the left) and aligned nucleosomes (ellipsoids) were visualized by indirect end labeling. (C) TTF-I-dependent nucleosome rearrangements on chromatin reconstituted from pure DNA and histones using polyglutamic acid as a histone carrier. Nucleosome remodeling was assayed analogous to (B) in the absence or presence of TTF-I, CHRAC, rISWI, and rISWI-K159R as indicated.
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profound nucleosome remodeling with almost no “canonical” nucleosome left on the target site (upper panel 6). Changes in nucleosomal occupancy at the GAGA box relied on ATP hydrolysis since the K159R derivative was inactive (upper panel 8). Due to the harsh Sarkosyl treatment, some irregularity is introduced into the nucleosomal arrays. These irregular arrays serve as substrates for CHRAC in a spacing reaction that improves their regularity in the absence of GAGA factor (Figure 2, upper panel 3). rISWI, but not the inactive K159R derivative, also improved the regularity of the MNase ladder around the GAGA box to some extent, but clearly not as well as CHRAC (Figure 2, compare upper panels 1, 3, 5, and 7). To establish whether nucleosome remodeling by rISWI works under various circumstances, we tested whether rISWI was able to synergize with another transcription factor in the context of a ribosomal DNA promoter. Activation of this promoter on chromatin templates relies on a remodeling reaction triggered by the transcription factor TTF-1 (La¨ngst et al., 1997). In the absence of a remodeling factor, TTF-1 is able to interact with its target site, T0, upstream of the rDNA promoter (Figure 2B, panel 2, DNase I hyperreactivity flanking the binding site marked by the shaded rectangle; see also La¨ngst et al., 1998). In the presence of an extract containing remodeling factors, TTF-1 triggered an ATP-dependent nucleosome repositioning of nucleosomes to either side of the bound factor, which was visualized by mapping of MNase cleavage sites by indirect end labeling (Figure 2B, ovals in panel 1). The repositioning of nucleosomes depended on ATP-consuming remodeling activities, since it was abolished by Sarkosyl treatment of chromatin (Figure 2B, panel 2). However, purified CHRAC (panel 3) or rISWI (panel 4) promoted nucleosome rearrangements while rISWI-K159R was inactive (panel 5). In order to rule out that rISWI associated with factors in Sarkosyl-stripped chromatin, effectively reconstituting a larger remodeling complex, we repeated the experiment with a more defined nucleosomal substrate. Chromatin substrates consisting only of DNA and histones were reconstituted using polyglutamic acid as a nonphysiological carrier for histones (Laybourn and Kadonaga, 1991) and tested for TTF-1 and ISWI-dependent nucleosome rearrangements. In the presence of either CHRAC (Figure 2C, panel 2) or rISWI (Figure 2C, panel 3), the diagnostic nucleosome rearrangements occurred while the K159R mutant was inactive as before. These results demonstrate that ATP-dependent nucleosome rearrangements are indeed caused by rISWI. Since rISWI remodeled chromatin in two very distinct settings, the ability to remodel nucleosomes is a general property of the enzyme, independent of the transcription factor that profits from the reaction.
rISWI Can Function as a Chromatin Assembly Factor Whereas all three Drosophila ISWI-containing complexes known to date (NURF, CHRAC, and ACF) are able to increase the accessibility of chromatin by nucleosome remodeling, only CHRAC and ACF are nucleosome spacing factors during chromatin assembly (Ito et al.,
Figure 3. rISWI Functions as a Chromatin Assembly Factor Nucleosomes were assembled from 200 ng purified DNA and histones using dNAP1 as a histone carrier (panel 1) and in the presence of 0.7 pmol of rISWI, 0.7 pmol of rISWI-K159R, or 0.2 pmol of CHRAC as indicated. The resulting nucleoprotein complex was digested with Micrococcal nuclease, the DNA fragments were purified and analyzed by agarose gel electrophoresis and ethidium bromide staining.
1997; Varga-Weisz et al., 1997). Ito et al. (1997) established, using a defined chromatin assembly system, that ACF played a role for both nucleosome assembly and nucleosome spacing. In order to establish whether rISWI alone had properties similar to ACF, we tested it in the context of a NAP-1-dependent nucleosome assembly system analogous to the one described by Ito et al. (1997). MNase digestion of chromatin formed by incubation of NAP-1 and histones with DNA yielded essentially a smear of irregular fragments with few DNA protected by mononucleosomes indicating the absence of nucleosomal arrays (Figure 3, panel 1). Inclusion of rISWI (panel 2) resulted in the accumulation of significantly more mononucleosomal DNA and the appearance of an extended ladder of defined fragments, indicating the presence of nucleosomal arrays. The inclusion of rISWIK159R also resulted in the recovery of more mononucleosomal DNA, but no extensive nucleosomal arrays were seen (Figure 3, panel 3) that established that rISWI utilized energy and therefore played an active role in the chromatin assembly process. CHRAC showed a comparable activity under these conditions (Figure 3, panel 4; data not shown). Discussion A Change of Paradigm: ISWI Is a Single Subunit Nucleosome Remodeling Factor Without exception, ATP-dependent nucleosome remodeling factors have been isolated from cells as large multisubunit entities, an observation that led to the common perception that energy-dependent nucleosome remodeling required the functional synergism between physically associated subunits. Our demonstration that a
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single recombinant ATPase, ISWI, is able to alter nucleosome structure in various ways radically changes the current paradigm. According to the new view, ISWI can be considered the catalytic core, a nucleosome remodeling module, of ISWI-containing remodeling complexes, while other associated proteins may have a role in regulating ISWI activity or integrating the remodeling reaction into a defined physiological context within the nucleus. Association of the ISWI ATPase will integrate a nucleosome remodeling function into a multifunctional entity. A prominent example of a modular remodeling complex is the chromatin accessibility complex (CHRAC), which contains topoisomerase II and three uncharacterized subunits in addition to ISWI. Topoisomerase II can be considered a DNA topology module since it is present as an active dimer in CHRAC but appears not to be involved in the mechanism of nucleosome remodeling (Varga-Weisz et al., 1997; P. Varga-Weisz, E. J. B., and P. B. B., unpublished data). In NURF, ISWI is associated with an enzymatically active pyrophosphatase that apparently also does not contribute to the nucleosome remodeling function (Gdula et al., 1998) and with p55, a module that has also been found associated with CAF-1 and enzymes regulating the acetylation status of histones (Martinez-Balbas et al., 1998). A comparison of the activity profiles of ISWI-containing nucleosome remodeling machines suggests that the activity of ISWI itself could be influenced by the different molecular environment in each complex. While ACF and CHRAC can function as nucleosome spacing factors, NURF is unable to do so (Ito et al., 1997; VargaWeisz et al., 1997; for reviews see Cairns, 1998; VargaWeisz and Becker, 1998). In contrast to ACF and NURF, CHRAC has so far not been shown to facilitate transcription of chromatin templates (Ito et al., 1997; Mizuguchi et al., 1997). rISWI reproduced major activities of the three ISWI-containing complexes to a remarkable degree: it was active in the established NURF assay, and its activity compared favorably with CHRAC when TTF-1induced nucleosome rearrangements were monitored. It also showed activity in the chromatin assembly reaction that originally identified ACF. While our study does not address the detailed mechanism of nucleosome remodeling by ISWI, our findings suggest that the phenomenologically distinct site-specific nucleosome remodeling, nucleosome alignment, and nucleosome assembly reactions may be mechanistically related.
ISWI Recognizes and Responds to a Defined Nucleosomal Structure The ATPase activity of rISWI can be used as a measure for productive substrate recognition. rISWI was stimulated to a much greater extent by nucleosomes than by free DNA or free histones, in interesting contrast to the STH ATPase whose activity responds to free DNA and nucleosomes equally well (Cairns et al., 1996). rISWI therefore recognizes a feature of nucleosomes that is not found on free histones nor free DNA. Since histones can only be assayed as dimers and tetramers as opposed to octamers under physiological ionic conditions, it is unclear whether rISWI responds to a feature of the histone octamer or of the nucleosome (i.e., a structure
involving both the protein and DNA moiety). The interference of histone N termini with ATPase induction by nucleosomes points to an involvement of these N-terminal domains in substrate recognition, as was already suggested earlier in the context of NURF (Georgel et al., 1997). The ability to express ISWI in a functionally active form will allow the definition of the interaction surface on ISWI as well as the recognition site on the nucleosome. ATPase Modules in Other Complexes Involved in Chromatin Metabolism ISWI is not the only ATPase module that endows a larger complex with nucleosome remodeling activity. Kingston and coworkers (Phelan et al., 1999 [this issue of Molecular Cell]) have shown that both BRG1 and hBRM have some intrinsic nucleosome remodeling activity that can be further stimulated by the BAF155, BAF170, and INI1 subunits in the human SWI/SNF-like complexes. ISWI (SNF2L) and SWI2/SNF2-like ATPases define subfamilies of the larger SWI2/SNF2 family of DNA-stimulated ATPases (Eisen et al., 1995). Several other ATPases of this family have recently been suggested to function via modulating chromatin structure. Among those is CHD1 and related proteins that contain, in addition to the SWI2/SNF2 ATPase motif, a chromodomain (Stokes et al., 1996; Woodage et al., 1997). The human autoantigen Mi-2, a SWI2/SNF2 family member containing two chromodomains, resides in a highly modular multienzyme complex (NuRD, Zhang et al., 1998; Mi-2 complex, Wade et al., 1998), integrating ATP-dependent nucleosome remodeling (presumably by Mi-2) and regulators of histone acetylation. Importantly, ATPases of the SWI2/SNF2 superfamily are not only associated with transcriptional regulation in chromatin, but several subfamilies contain ATPases known to have a role in various forms of DNA damage repair, such as the yeast proteins RAD16, RAD26, and RAD54 and mammalian homologs (Eisen et al., 1995). Since the repair of damaged DNA must occur in the context of chromatin, it is possible that those ATPases integrate a chromatin remodeling function into the repair process. The analysis of individual ATPases in assays that monitor dynamic transitions in nucleosome structure should reveal their role as nucleosome remodeling modules in various physiological contexts. Experimental Procedures Expression of rISWI in E. coli The D. melanogaster ISWI protein containing an N-terminal flag tag (rISWI) was produced in E. coli using the Impact System and vector pMYB4 according to the manufacturer’s specifications (NEBiolabs). The entire ISWI coding sequence, as well as the newly generated junction, was confirmed by sequencing. Elution of ISWI by inteincatalyzed self cleavage occurred overnight at 48C in the presence of 1 ml/ml beads of Cleavage Buffer (20 mM Tris-HCl [pH 8.0]; 50 mM NaCl; 0.1 mM EDTA; 13 Complete protease inhibitor [Boehringer, Mannheim]; 30 mM DTT). Eluted protein was dialyzed overnight at 48C with Spectra/Por (MWCO: 6–8000) against 1 liter of 20 mM TrisHCl [pH 8.0], 50 mM NaCl, 0.1 mM EDTA, and 50% Glycerol and stored at 2208C. Activity of full-length rISWI was only observed if N-terminal degradation products were removed by gel filtration on a Superdex-200 column (Pharmacia). Since the fraction of active recombinant ATPase is not known, it remains uncertain whether in the remodeling reactions ISWI functioned catalytically.
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ATPase Assay The standard reaction (10 ml) contained 6.6 mM HEPES (pH 7.6), 0.66 mM EDTA, 0.66 mM 2-mercaptoethanol, 0.033% N-P40, 1.1 mM MgCl2, 33 mM ATP, 5 mCi [g-32P]ATP (3000 Ci mmol21, NEN), 1.3 pmol (160 ng) of recombinant ISWI, and various substrates as specified in figure legends. Unreacted ATP and free phosphate were separated by thin layer chromatography using TLC cellulose Ready-foils (Schleicher and Schu¨ll). Spots were quantified by PhosphoImager and Imagequant software. Arbitrary units given in Figure 1 represent the percentage of ATP hydrolyzed in the reaction.
Potential Substrates Included in ATPase Assays Mononucleosomes, a kind gift of Drs. G. Mu¨ller and W. Waldeck (DKFZ, Heidelberg), were purified from Micrococcal Nucleasetreated chicken blood cell chromatin by sucrose gradient sedimentation according to standard procedures and used as nucleosome substrates. Identical results were obtained with sucrose gradient– purified oligonucleosomes reconstituted from Drosophila histones by standard salt gradient dialysis. The synthetic four-way junction DNA was prepared as described earlier (Ferrari et al., 1992). Histone octamers were purified to homogeneity from Drosophila embryos by hydroxylapatite-fractionation of chromatin (Simon and Felsenfeld, 1979). Histone octamers were eluted from the HAP column with a buffer containing 2 M NaCl in phosphate buffer. Glycerol (50% v/v) was directly added to the histone-containing fraction before storage at 2208C. The Drosophila histone tail–GST fusion proteins were expressed in E. coli and purified as described (Georgel et al., 1997). In general, the reagents that were included in ATPase reactions had very low intrinsic ATPase activity themselves. However, all four histone tail–GST fusion proteins (but not the GST preparation) were contaminated with significant ATPase activity from E. coli (about 15% of ATP hydrolyzed in a standard reaction), which was subtracted from the total ATPase activity of reactions that contained ISWI and the tail fusions Figure 1D. The amount of individual histone tail–GST fusions that was assayed in competition with nucleosomes was a 50-fold molar excess over the number of nucleosome particles (a 6-fold excess over the total number of histone N termini or a 25fold excess over each individual histone tail in those nucleosomes).
Nucleosome Remodeling Reactions Chromatin assembly and Sarkosyl treatment followed published procedures (Varga-Weisz et al., 1997, 1999) except that the chromatin was treated with 0.1% Sarkosyl for 10 min at room temperature to inactivate the remodeling factors prior to purification over a Sephacryl 300 HR spin column. The remodeling reactions shown in Figure 2A were as described (Wall et al., 1995). The reaction contained recombinant GAGA factor (Wall et al., 1995) and one of the following: 0.13 pmol (16 ng) rISWI, the equivalent amount of rISWI-K159R, 0.15 pmol purified CHRAC, or 4 ml Drosophila embryo extract (Becker and Wu, 1992). The reaction was allowed to proceed for 30 min at 268C before MNase analysis, Southern blotting, and oligonucleotide probing as described (Wall et al., 1995). TTF-1-mediated remodeling of the rDNA promoter was essentially assayed as published previously (La¨ngst et al., 1997, 1998). Chromatin was assembled on plasmid pMrWT, containing the rDNA promoter from position 2170 to 1155 and the TTF-I binding site T0 at position 2169. Chromatin was stripped with Sarkosyl, purified, and supplied with fresh McNAP buffer. Samples (30 ml) were incubated with or without TTF-I (50 ng), 0.3 pmol CHRAC, and 0.33 pmol rISWI or rISWI-K159R for 30 min at 268C. Chromatin assembly with polyglutamic acid (Laybourn and Kadonaga, 1991) was done by mixing polyglutamic acid (PGA; Sigma, P4886) and histones at a ratio of 1:2 and a final concentration of 20 ng/ml histones in 0.1 M NaCl, 10 mM Tris-Cl (pH 7.6), and 1 mM EDTA. The reaction was incubated for 1 hr at room temperature, precipitates were removed by centrifugation, and the supernatant was called HPmix. Chromatin assembly was performed with 800 ng plasmid pMrWT and 60 ml HP mix in a final volume of 100 ml 0.1 M NaCl, 10 mM Tris-Cl (pH 7.6), and 1 mM EDTA for 2 hr at 378C. Remodeling reactions (60 min at 308C) contained 80 ng chromatin, 50 ng TTF-I, 0.3 pmol CHRAC, and 0.3 pmol rISWI or rISWI-K195R as indicated.
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