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Molecular and Cellular Biology, April 2007, p. 3217-3225, Vol. 27, No. 8
0270-7306/07/$08.00+0     doi:10.1128/MCB.01731-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Dependency of ISW1a Chromatin Remodeling on Extranucleosomal DNA

Vamsi K. Gangaraju and Blaine Bartholomew^*

Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, Illinois 62901-4413

Received 13 September 2006/ Returned for modification 12 November 2006/ Accepted 18 January 2007

Top Abstract Introduction Materials and Methods Results Discussion References

 
The nucleosome remodeling activity of ISW1a was dependent on whether ISW1a was bound to one or both extranucleosomal DNAs. ISW1a preferentially bound nucleosomes with an optimal length of 33 to 35 bp of extranucleosomal DNA at both the entry and exit sites over nucleosomes with extranucleosomal DNA at only one entry or exit site. Nucleosomes with extranucleosomal DNA at one of the entry/exit sites were readily remodeled by ISW1a and stimulated the ATPase activity of ISW1a, while conversely, nucleosomes with extranucleosomal DNA at both entry/exit sites were unable either to stimulate the ATPase activity of ISW1a or to be mobilized. DNA footprinting revealed that a major conformational difference between the nucleosomes was the lack of ISW1a binding to nucleosomal DNA two helical turns from the dyad axis in nucleosomes with extranucleosomal DNA at both entry/exit sites. The Ioc3 subunit of ISW1a was found to be the predominant subunit associated with extranucleosomal DNA when ISW1a is bound either to one or to both extranucleosomal DNAs. These two conformations of the ISW1a-nucleosome complex are suggested to be the molecular basis for the nucleosome spacing activity of ISW1a on nucleosomal arrays. ISW1b, the other isoform of ISW1, does not have the same dependency for extranucleosomal DNA as ISW1a and, likewise, is not able to space nucleosomes.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chromatin remodeling complexes make chromatin more accessible for various cellular processes inside the cell by either covalently modifying histones (34) or mobilizing the histone octamer along DNA in cis or trans (7). A link between these two distinct chromatin remodeling activities has been shown, and in some instances, they even reside together in the same complex (20, 44). ATP-dependent chromatin remodeling causes changes in the nucleosome translational position (12, 18, 21, 42), the exchange of histone variants (3, 9, 15, 19, 26, 29), and even the removal of nucleosomes (31).

ISWI, a class of ATP-dependent chromatin remodeling complexes (24), forms a number of distinct complexes in Saccharomyces cerevisiae (36), Drosophila melanogaster (37), and vertebrates (22). These remodeling complexes have an ATPase subunit that belongs to the SWI2/SNF2 subfamily of DEAD/H helicases (8). The ISWI family of ATPases is characterized by SANT and SANT-like domains in the catalytic subunit (11) that have been proposed to interact with histone tails (1, 2). ISWI requires histone H4 tail for stimulation of the ATPase activity, and several of the ISWI class remodelers generate regularly spaced nucleosomal arrays (13, 36, 39) and facilitate the deposition of histones onto DNA (14). In S. cerevisiae, there are two distinct ISWI genes, ISW1 and ISW2 (36). Isw1 is in two different complexes, ISW1a (Isw1 and Ioc3) and ISW1b (Isw1, Ioc2, and Ioc4), in addition to being present as a monomer (41). While no known protein motifs are evident in Ioc3, Ioc2 has a PHD finger, and Ioc4 has a PWWP motif, a putative DNA-binding (30) and chromatin-targeting domain (10). ISW2 is a four-subunit complex composed of Isw2, Itc1, Dpb4, and Dls1 (25).

ISW1a and ISW1b share the same catalytic subunit but exhibit distinct remodeling properties (41). The differences in the biochemical activities of these two complexes are probably connected to their different functional roles inside the cell and are not yet well understood. ISW1a was shown to regularly space nucleosomes in vitro every 175 bp, but not ISW1b, as suggested earlier, due to an inability to mobilize nucleosomes (41). The inability of ISW1b to mobilize nucleosomes has been attributed to its low affinity for nucleosomes. Recently, Stockdale et al. have shown that ISW1a behaves like ISW2 in mobilizing nucleosomes to their thermodynamically preferred positions along DNA (33). There are, however, conflicting data in regard to ISW2 and ISW1a about whether their direction of mobilization is dictated by the thermodynamically preferred positioning of nucleosomes. Recently, in vivo analysis of ISW2 sliding has shown that ISW2 slides nucleosomes onto unfavorable positions on DNA (43). Other studies have also shown that ISW2 and ISW1a can efficiently move nucleosomes off of a DNA sequence to which nucleosomes are bound with an affinity higher than any known natural sequence (46). Furthermore, it seems unlikely that the nucleosome-spacing activity of ISW1a can be explained as moving nucleosomes to their thermodynamically preferred positions on DNA.

The reason for ISW1a uniformly spacing nucleosomes every 175 bp is not evident, since many other complexes can mobilize nucleosomes without spacing nucleosomes. Some of the molecular details that influence nucleosome spacing were recently shown for ISW2, which spaces nucleosomes approximately every 200 bp (16). ISW2 has a built-in molecular spacer with its noncatalytic subunit, Itc1, interacting with 60 bp of extranucleosomal DNA to sterically block nucleosomes from moving too close together. As nucleosomes are moved together, ISW2 has a reduced affinity for nucleosomes and is more likely to dissociate so that it can search for nucleosomes that are farther apart to remodel. This study reveals that ISW1a remodeling activity is regulated by whether it is bound to both or just one of the extranucleosomal DNAs.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Purification of ISW1a and ISW1b. Saccharomyces cerevisiae strain YTT449, with a FLAG epitope-tagged version of ISW1, was used to purify the two complexes of Isw1. Yeast cells were grown and whole-cell extract prepared in buffer H-0.3 (300 mM NaCl, 25 mM Na-HEPES, 0.5 mM EGTA, 0.1 M EDTA, 2 mM MgCl₂, 20% glycerol, and 0.02% NP-40). The ISW1a and ISW1b complexes were separated by BioRex-70 chromatography using a linear 0.2 to 0.8 M NaCl gradient. The fractions containing Isw1 were detected by immunoblotting using FLAG M2 antibody (Sigma), and Isw1 complexes were further purified by immunoaffinity chromatography with FLAG M2 beads, as described previously (36). Elutions were analyzed on a 4 to 20% gradient of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained by colloidal blue (Pro-Blue; OWL Separation Systems) and quantified using known amounts of bovine serum albumin protein as standards.

Plasmids and DNA probes. The plasmid pGEM-3Z/601 (23) was used to obtain the nucleosome positioning sequence "601." DNA with different lengths of extranucleosomal DNA were generated by PCR (16, 45). DNA probes with labels on one end for site-directed mapping and hydroxyl radical footprinting were generated by phosphorylating the appropriate primer using Optikinase (USB) and [-³²P]ATP (6,000 Ci/mmol; Perkin-Elmer) prior to PCR. Sequences of 601 DNA and different primers used in this study are available upon request.

Glycerol gradient sedimentation of ISW1a and ISW1b. Glycerol gradients (4.4 ml) of 15 to 35% were prepared in 10 mM Na-HEPES (pH 7.8), 1 mM EDTA (pH 8.0), 0.02% Nonidet P-40, and 100 mM NaCl, with 2.5 µg of either ISW1a or ISW1b loaded per gradient. The gradient was centrifuged at 48,000 rpm in an SW55Ti (Beckman) rotor at 4°C for 20 h. After centrifugation, 100-µl fractions were collected and analyzed by immunoblotting using -FLAG antibody (Sigma). The apparent molecular masses of ISW1a and ISW1b were determined by comparing their migration with that of standard molecular mass markers (catalog no. MW-GF-1000; Sigma) ranging from 29 to 700 kDa.

Formaldehyde cross-linking. ISW1a and ISW1b were cross-linked by adding formaldehyde to a final concentration of 1% (wt/vol) in 100 mM NaCl, 25 mM Na-HEPES, 0.5 mM EGTA, 0.1 mM EDTA, 2 mM MgCl₂, 20% glycerol, 0.02% Nonidet P-40, 2 mM ß-mercaptoethanol, 0.5 mM sodium metabisulfite, 1 mM phenylmethylsulfonyl fluoride and then incubated at room temperature for 10 min. The reaction mixtures were quenched by adding glycine to a final concentration of 50 mM and analyzed on a NuPAGE Novex 3 to 8% Tris-acetate SDS midi gel (Invitrogen). The molecular mass of the cross-linked proteins was estimated by comparison with HiMark HMW molecular mass standard (40 to 500 kDa; Invitrogen) and stained with SYPRO-Ruby (Molecular Probes).

Nucleosome reconstitutions. Homogeneous reconstitutions were assembled with 10 µg (92 pmol) of recombinant histone octamers from Xenopus laevis, 100 fmol of radiolabeled DNA containing the 601 nucleosome positioning sequence, 5 µg of nonradioactive 601 DNA, and 1.8 M NaCl in a starting volume of 10 µl. Stepwise dilutions of NaCl were carried out at 37°C in three stages (1 M, 714 mM, and 270 mM) by the addition of 6.8 µl, 8.4 µl, and 42 µl of buffer D (25 mM Tris-HCl [pH 8.0], 1 mM ß-mercaptoethanol), respectively, at 10-min intervals. Nucleosome assemblies were analyzed on a 4% native polyacrylamide gel (acrylamide/bisacrylamide ratio, 35.36:1) in 0.5x Tris-borate-EDTA buffer at 4°C. Heterogeneous reconstitutions were assembled in the same fashion as homogeneous reconstitutions, except that 10 µg of sheared salmon sperm DNA was used instead of 601 DNA.

Nucleosome binding and mobilization assays. Reaction mixture conditions for ISW1a/1b binding assays were 30 mM Na-HEPES (pH 7.6), 5 mM MgCl₂, 70 mM NaCl, 0.1 mM EGTA, 0.02 mM EDTA, 10% glycerol, and 0.1 µg of bovine serum albumin/µl in a 12.5-µl volume. Four microliters of the reaction mixture was analyzed by electrophoretic mobility gel shift assay on a 4% native polyacrylamide gel (acrylamide/bisacrylamide ratio, 35:1) in 0.5x Tris-borate-EDTA at 4°C. Nucleosome mobilization assays were conducted in the same fashion, except that ATP was added to a final concentration of 400 µM and then analyzed on a 5% native polyacrylamide gel (acrylamide/bisacrylamide ratio, 60:1) in 0.2x Tris-borate-EDTA, with buffer recirculation at 4°C.

Hydroxyl radical footprinting. Standard binding reactions were set up as described above, without glycerol. Hydroxyl radical cleavage was initiated as described previously (38), except that the final concentrations of Fe(II), H₂O₂, ascorbate, and EDTA were 270 µM, 0.16%, 5.4 mM, and 215 µM, respectively. After 2 min of cleavage, glycerol was added to a final concentration of 10% to stop the reaction. The DNA was extracted using phenol-chloroform and ethanol precipitated before being analyzed on a denaturing 6.5% polyacrylamide gel (acrylamide/bisacrylamide ratio of 20:1, containing 8 M urea).

ATPase assays. All ATPase assays contained homogeneous reconstitutions, as mentioned above, for standard binding reactions. After binding for 30 min at 30°C, 1 µl of 1 mM [³²-P]ATP (0.3 to 0.5µCi/nmol) was added to a 8.5-µl sample and incubated at 30°C for 5 min. Reactions were terminated by adding SDS to a final concentration of 1%, and 1 µl of the reaction mixture was spotted on a polyethyleneimine cellulose thin layer chromatography plate (JT Baker) and separated with 0.5 M LiCl and 0.5 M formic acid.

High-resolution nucleosome mapping. End-labeled DNA was made by PCR using Hot Master Mix (Eppendorf) and 5'-labeled oligonucleotides. DNA was purified using QIAquick (QIAGEN), followed by additional purification with DEAE-Sephadex A-25. Site-directed mapping of histone-DNA contacts was done as described previously (17). Histone octamers containing cysteine at amino acid 53 of H2B (H2B53) were reconstituted into nucleosomes as described above. Nucleosome assemblies (3.0 µM) with 200 µM p-azidophenacyl bromide (APB; Fluka Chemicals) were incubated for 3 h at room temperature for coupling the photoreactive group to cysteine. Nucleosome mobilization reactions with ISW1a or ISW1b were performed as described previously. Four microliters of the reaction mixture was loaded onto a 5% polyacrylamide gel (acrylamide:bisacrylamide, 60:1), and the remainder was irradiated with a UV transilluminator (at 310 nm) for 3 min. SDS was added to a final concentration of 0.1%, heated at 70°C for 20 min, and extracted with phenol-chloroform (4:1). The organic phase and interphase were collected and washed three times with 1% SDS and 1 M Tris-HCl (pH 8.0), and the samples were ethanol precipitated. The cross-linked DNA-protein complex was resuspended in 100 µl of 2% SDS, 20 mM ammonium acetate, and 0.1 mM Na-EDTA and heated for 20 min at 90°C. The reaction mixture was heated at 90°C for 45 min after the addition of 5 µl of 2 M NaOH. Alkaline cleavage was stopped by the addition of 6.5 µl of 2 M HCl and an equal volume of 20 mM Tris-HCl (pH 8.0). The samples were ethanol precipitated and resolved by 6.5% denaturing PAGE containing 8 M urea.

Site-specific DNA photoaffinity cross-linking. A series of five site-specific photoreactive DNA probes was synthesized as described previously (32). Photoreactive nucleotide analogs (AB-dATP, AB-dCTP, or AB-dUTP) were incorporated at positions ranging from 126 bp from the dyad axis in the extranucleosomal region to 73 bp from the dyad axis at the entry/exit site. All probes were radioactively labeled with one or two -³²P-labeled nucleotides incorporated adjacent to the photoreactive nucleotide analog. Nucleosomes having photoreactive probes were reconstituted using the salt dilution method as described above. Standard binding reactions (25-µl volume) were assembled, and 4 µl of the reaction mixture was analyzed on a 4% native polyacrylamide gel (acrylamide:bisacrylamide, 38.9:1.1, in 0.5x Tris-borate-EDTA) to assess ISW1a binding. Reaction mixtures were irradiated for 2 min (at 310 nm; 2.65 mW/cm²) and then digested with 4.6 U of DNase I and 20 U of S1 nuclease, sequentially, as described previously (32). Samples were analyzed by 4 to 20% SDS-PAGE, and cross-linked ISW1a subunits were visualized by phosphorimaging.

Top Abstract Introduction Materials and Methods Results Discussion References

 
ISW1a binding to nucleosomes was promoted by ISW1a association with 29 bp of extranucleosomal DNA. ISW1a and ISW1b were separated by cation exchange chromatography (BioRex-70) and then immunoaffinity purified using the two copies of the FLAG peptide fused to the C terminus of Isw1. ISW1a contained Isw1 and one additional subunit (Ioc3), and ISW1b contained two additional subunits (Ioc2 and Ioc4) (Fig. 1A). The relative affinity of ISW1a for nucleosomes was determined by electrophoretic mobility gel shift assays. The different nucleosomes were referred to by the designation "xNy," where "x" and "y" indicate the number of base pairs of extranucleosomal DNA on either side of the 147-bp core nucleosomal DNA. A high-affinity nucleosome positioning sequence termed 601 (23) was used to position nucleosomes at a single translational position (16). The nucleosome binding properties of ISW1a were examined with different extents of extranucleosomal DNA on one side of the core nucleosome particle (Fig. 1B and C). At the lowest ISW1a concentration (0.32 pmol), increasing the length of extranucleosomal DNA caused only minor increases in ISW1a affinity for nucleosomes (Fig. 1B, lanes 2, 6, 10, 14, and 18, and C). At a slightly higher ISW1a concentration (0.64 pmol), the affinity of ISW1a for nucleosomes increased dramatically as the extranucleosomal DNA length changed from 23 to 33 bp, though there was little additional increase in affinity with longer extranucleosomal DNA of 53 and 70 bp (Fig. 1B, lanes 3, 7, 11, 15, and 19, and C). This effect was modulated as the ISW1a concentration was increased further, such that the biggest difference in affinity occurred with the change in extranucleosomal DNA length from 10 to 23 bp. We concluded from these results that the optimal length of extranucleosomal DNA at only one entry/exit site for ISW1a binding to nucleosomes was 33 bp.

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FIG. 1. ISW1a binding to nucleosomes was enhanced by 33 bp of extranucleosomal DNA at one entry/exit of the nucleosome. (A) Affinity-purified ISW1a and ISW1b from strain YTT449 were resolved on 4 to 20% gradient SDS-PAGE stained with Coomassie blue. (B) Mononucleosomes (1.3 pmol) with different lengths of extranucleosomal DNA at one entry/exit site were incubated with 320 fmol (lanes 2, 6, 10, 14, and 18), 640 fmol (lanes 3, 7, 11, 15, and 19) and 1.3 pmol (lanes 4, 8, 12, 16 and 20) of ISW1a. Samples were analyzed on a 4% native polyacrylamide gel. (C) The percentage of nucleosomes bound in panel B was calculated based on the amount of nucleosomes remaining unbound, and this percentage was plotted versus the length of extranucleosomal DNA. Filled diamonds, squares, and triangles indicate values for 0.32, 0.64, and 1.3 pmol of ISW1a, respectively. (D) Nucleosomes (53N0) with DNA end-labeled on the 5' end of the bottom strand were incubated with two different concentrations of ISW1a (650 fmol, lanes 4 and 5; 1.3 pmol, lanes 6 and 7) and treated with ExoIII (2 U, lanes 2, 4, and 6; 0.2 U, lanes 3, 5, and 7 at 30°C). No ExoIII was added to the sample shown in lane 1. The site at which ExoIII digestion was stalled or halted is indicated in terms of the nucleotides away from the dyad axis.

The physical interactions of ISW1a with extranucleosomal DNA was probed by 3'5' exonuclease digestion with ExoIII. The progressive digestion of the 3' end of DNA by ExoIII revealed a strong stop site at the edge of the nucleosome that, with larger amounts of ExoIII, proceeded one helical turn of DNA toward the inside of the nucleosome (Fig. 1D, lanes 2 and 3). The strong pause/stop sites of ExoIII were not observed in the presence of free DNA (results not shown). Binding of ISW1a to nucleosomes revealed a series of new pause/stop sites that spanned a 29-bp region into the extranucleosomal DNA region (Fig. 1D, lanes 5 and 7). ExoIII mapping of ISW1a bound to the 53N0 nucleosome revealed that ISW1a interacted with 29 bp of extranucleosomal DNA immediately adjacent to the core nucleosomal particle, consistent with the 33 bp required for efficient binding (Fig. 1D).

ISW1a preferentially bound nucleosomes with 30 bp of extranucleosomal DNA at both entry/exit sites. Nucleosomes containing 53 bp of extranucleosomal DNA at both entry/exit sites (53N53) were reconstituted, and a portion was cut with NotI to remove one of the extranucleosomal DNAs (53N0) to determine if ISW1a prefers to bind nucleosomes with extranucleosomal DNA at both entry/exit sites. Nucleosomes (53N0) with extranucleosomal DNA at only one entry/exit site were bound 10-fold less efficiently than nucleosomes with extranucleosomal DNA at both entry/exit sites at the lower concentration of ISW1a (Fig. 2A, compare lanes 4 and 8). ISW1a binding was further characterized by carefully controlling the length of extranucleosomal DNA at one of the entry/exit sites of the nucleosome while maintaining the other at a constant 53 bp (Fig. 2B). Extranucleosomal DNA was incrementally added to the 53N0 nucleosome, and the affinity of ISW1a was measured by gel shift assay. The affinity of ISW1a for nucleosomes progressively increased with additional extranucleosomal DNA up to 33 bp of DNA, with no more significant increase in affinity with 43 or 53 bp of extranucleosomal DNA. These results demonstrated that ISW1a preferred to interact with both extranucleosomal DNAs and needed 33 bp of extranucleosomal DNA at both entry/exit sites for optimal binding. The length of extranucleosomal DNA required on both sides of the nucleosomes coincides remarkably well with ISW1a spacing nucleosomes approximately every 175 bp apart, with 30 bp of extranucleosomal DNA between each set of nucleosomes (36).

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FIG. 2. ISW1a preferentially bound nucleosomes with at least 33 bp of extranucleosomal DNA at both entry/exit sites. (A) Mononucleosomes reconstituted with end-labeled 253-bp 601 DNA (53N53) were incubated with (lanes 7 to 10) or without (lanes 1 and 3 to 6) 10 U of NotI. Next, nucleosomes were incubated with 0.12 (lanes 4 and 8), 0.37 (lanes 5 and 9), and 1.1 pmol (lanes 6 and 10) of ISW1a at 30°C and analyzed on a 4% native polyacrylamide gel. The * (panel A) represents ISW1a binding to a negligible amount of 53N53 nucleosomes that were not cut by NotI. (B) Mononucleosomes (1.3 pmol) having 53 bp of extranucleosomal DNA at one entry/exit site, with different lengths of extranucleosomal DNA: 0 (lanes 1 and 7), 10 (lanes 2 and 8), 20 (lanes 3 and 9), 33 (lanes 4 and 10), 40 (lanes 5 and 11), and 53 (lanes 6 and 12) bp at the other entry/exit site were incubated with 200 fmol of ISW1a at 30°C for 30 min. Samples were analyzed by 4% native gel electrophoresis. Lanes 1 to 6 are nucleosomes alone, with no ISW1a.

The extent of interactions of ISW1a and ISW1b with extranucleosomal DNA at both entry/exit sites was mapped with ExoIII. ISW1a and ISW1b were bound to 70N70 nucleosomes (90% nucleosomes bound, data not shown) and digested. Nucleosomes alone produced strong stop sites close to both entry/exit sites compared to those of free DNA (Fig. 3, compare lanes 2 to 4 with 6 to 8 for the bottom strand and lanes 18 to 20 with 22 to 24 for the top strand). ISW1a protected 35 to 38 bp of extranucleosomal DNA at the two entry/exit sites of nucleosomes (Fig. 3, lanes 11, 12, 27, and 28). ISW1b interacted with less extranucleosomal DNA than ISW1a and protected only 13 and 19 bp of extranucleosomal DNA (Fig. 3, lanes 15, 16, 31 and 32).

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FIG. 3. ISW1a bound to 70N70 nucleosomes protected 35 to 38 bp of extranucleosomal DNA. Nucleosomes (70N70) with DNA end-labeled on the 5' end of the top or bottom strand were incubated with either ISW1a (lanes 10 to 12 and 26 to 28) or ISW1b (lanes 14 to 16 and 30 to 32) and treated with ExoIII (0.22 to 2 U at 30°C). No ExoIII was added to lanes 1, 5, 9, 13, 17, 21, 25, and 29. The site at which ExoIII digestion was stalled or halted is indicated in terms of the nucleotides away from the dyad axis. The nucleotide positions were determined by comparison with the DNA sequencing ladders from the same DNA.

The enhanced affinity of ISW1a for nucleosomes with extranucleosomal DNA at both entry/exit sites is a property not shared with other ISWI type complexes in yeast. First, the affinity of ISW2 for nucleosomes was found to be dependent on the length of extranucleosomal DNA at one entry/exit site and was not affected by the addition of extranucleosomal DNA at the other entry/exit site. The addition of extranucleosomal DNA to the other entry/exit site of 53N0 nucleosomes did not stimulate ISW2 binding, as was observed for ISW1a, at either of two concentrations of ISW2 (Fig. 4A and B). Second, the binding of ISW1b to nucleosomes was not affected by the presence of extranucleosomal DNA at both entry/exit sites (data not shown).

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FIG. 4. ISW2 did not preferentially bind nucleosomes with extranucleosomal DNA at both entry/exit sites. (A) Mononucleosomes, as described in the Fig. 2 legend, were incubated with either 100 (lanes 2, 4, 6, 8, 10, and 12) or 315 (lanes 3, 5, 7, 9, 11, and 13) fmol of ISW2 and analyzed as described previously. (B) The percentages of nucleosomes bound in panel A and those with 0.64 fmol of ISW1a with different lengths of extranucleosomal DNA were plotted. These results were repeated, with the standard deviations shown. The filled squares and circles indicate values for 100 and 315 fmol of ISW2, respectively, and the open circles indicate values for 640 fmol of ISW1a.

ISW1a exists as a heterodimer of Ioc3 and ISW1 and not as a heterotetramer. Recently, it has been suggested that ACF (an ISWI type of remodeling complex) exists as a heterotetramer in solution with two Acf1 and two ISWI subunits per complex (35). The researchers who made this suggestion also suggested that the complex moves mononucleosomes to the center of DNA, because each half of the complex, composed of one heterodimer of Acf1 and ISWI, pulls the nucleosome in opposing directions, creating the tendency to move mononucleosomes to the center of DNA. The stoichiometry of Isw1 and Ioc3 in ISW1a was determined by SDS-PAGE and Coomassie staining to be 1:1.2, consistent with ISW1a as either a heterodimer or a heterotetramer (Fig. 1A). The stoichiometry of the subunits in ISW1b was also observed to be 1:1.1:1.2 for Isw1:Ioc2:Ioc4, consistent with ISW1b as a heterotrimer. Two different approaches were used to determine if ISW1a, as suggested for ACF, was a heterotetramer in solution, consisting of two subunits each of Isw1 and Ioc3 or a heterodimer, with one subunit each of Isw1 and Ioc3. First, the apparent molecular weights of ISW1a and ISW1b complexes were determined by glycerol gradient sedimentation, with the underlying assumption that the overall structure of the complex was spherical and not elongated. Glycerol gradient sedimentation revealed complexes with an estimated molecular mass of 200 kDa for ISW1a and ISW1b, close to the calculated molecular masses of 222 kDa for a heterodimer for ISW1a of Isw1 and Ioc3 and 279 kDa for a heterotrimer for ISW1b of Isw1, Ioc2, and Ioc4 (data not shown).

Next, chemical cross-linking was used to determine if free ISW1a is either a heterotetramer or a heterodimer that avoids the limitation inherent in the dependency on the conformation of the complex for correctly estimating its molecular weight. If ISW1a is a heterodimer of Isw1 and Ioc3, then cross-linking should produce a single cross-linked species composed of Isw1 and Ioc3. If, instead, ISW1a is a heterotetramer, then four or more cross-linked species of higher molecular weights should be detected (i.e., Isw1-Ioc3, Isw1-Isw1, Isw1-Isw1-Ioc3, and Isw1-Isw1-Ioc3-Ioc3). In these experiments, the concentration of protein was varied to avoid random collisions of two or more complexes. Cross-linking of ISW1a produced one broad band ranging from 230 to 260 kDa, consistent with ISW1a as a heterodimer and not a heterotetramer (Fig. 5, lanes 3 to 6). The broadness of the band is likely caused by the extensive modification of amino acid side chains by formaldehyde that did not result in the formation of protein-protein cross-links. Formaldehyde cross-linking of ISW1b also produced two cross-linked species with estimated molecular masses of 235 and 402 kDa, consistent with the lower species cross-linking Isw1 to Ioc2 and the higher species cross-linking Isw1, Ioc2, and Ioc4 together (Fig. 5, lanes 8 to 11). The cross-linking pattern indicated that ISW1b was likely a heterotrimer of Isw1, Ioc2, and Ioc4.

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FIG. 5. ISW1a was shown to be a heterodimer of Isw1 and Ioc3 by cross-linking. The concentrations of ISW1a and ISW1b were varied while cross-linking with a constant final concentration of 1% formaldehyde and then separated by SDS-PAGE. Lanes 1, 2, and 7 have no formaldehyde, and the sizes of the molecular mass standards are indicated on the left in kDa.

Although it appears that ISW1a is a heterodimer in solution, two ISW1a heterodimers could potentially bind if nucleosomes had extranucleosomal DNA at both entry/exit sites to create a situation similar to that suggested for ACF. Nucleosomes with extranucleosomal DNA at only one entry/exit site are expected to bind only one ISW1a heterodimer, because of limited extranucleosomal DNA and available space and the overall size of the 220-kDa ISW1a complex. ISW1a bound to 53N0 nucleosomes should bind only one heterodimer of ISW1a per nucleosome, since there is extranucleosomal DNA at only one entry exit site (Fig. 2A, lanes 8 to 10). The concentration of ISW1a was varied to assess the potential cooperative binding of ISW1a by comparing the electrophoretic mobilities of the ISW1a-nucleosome complex at low- and high-ISW1a occupancies. The mobility of the ISW1a-nucleosome complex was the same whether only 2% or 72% of the nucleosomes was bound, suggesting there was no cooperative binding of more than one ISW1a heterodimer to nucleosomes. The electrophoretic mobilities of ISW1a-nucleosome complexes with extranucleosomal DNA at one or both entry/exit sites were very similar (Fig. 2A, compare lanes 4 to 6 with 9 to 10), suggesting that they both had only one ISW1a heterodimer bound per nucleosome and that having two extranucleosomal DNA present does not promote the binding of a second ISW1a heterodimer. The slight shift in mobility observed between ISW1a bound to nucleosomes 53N53 and 53N0 is likely due to the inherent differences in the electrophoretic mobility of the nucleosomes (Fig. 2A, compare lanes 1 and 2).

ISW1a does not bind nucleosomal DNA two helical turns from the dyad when bound to extranucleosomal DNA at both entry/exit sites. The interactions of ISW1a with nucleosomes were further investigated by DNA footprinting to determine if there were more subtle differences in the binding of ISW1a to the two nucleosomal substrates containing extranucleosomal DNA at one or both entry/exit sites (Fig. 6). The regions of the 70N0 nucleosomes protected by ISW1a were similar to that previously observed for ISW2 (16). ISW1a contacted extranucleosomal DNA, DNA inside the nucleosome near the entry site, and an 10-bp region two helical turns from the dyad (Fig. 6A). The DNA footprint of ISW1a bound to nucleosomes with both extranucleosomal DNAs (53N53) did not have protection at the site two helical turns from the dyad, but ISW1a still bound to extranucleosomal DNA (Fig. 6B). The loss of ISW1a interaction with this internal site is likely to be important for remodeling, since the interaction of ISW2 at the same internal site is required for remodeling (46). The switch from ISW1a binding to not binding nucleosomal DNA 20 bp from the dyad could be a change from an active to an inactive conformation.

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FIG. 6. ISW1a bound to nucleosomes with both extranucleosomal DNAs did not interact with nucleosomal DNA two helical turns from the dyad axis. ISW1a bound to 70N0 or 53N53 nucleosomes was footprinted using hydroxyl radical generated from Fe-EDTA. The quantification of the cleavage pattern of nucleosomes with (N+ISW1a, gray) and without (N, black) ISW1a is shown. On the x axis is indicated the relative positions of the different nucleosomal superhelical locations (SHL).

ISW1a does not mobilize nucleosomes with 33 bp of extranucleosomal DNA at both entry/exit sites, unlike ISW1b. Gel shift and site-directed mapping were used to determine if nucleosomes with extranucleosomal DNA at both entry/exit sites were unable to be mobilized by ISW1a, as suggested by the previous DNA footprinting data. ISW1a efficiently mobilized nucleosomes with 33 bp of extranucleosomal DNA at one entry site (33N0), as shown by gel shift assay and, in earlier work, by site-directed mapping (Fig. 7A, compare lanes 2 to 5) (45). The addition of 33 bp of extranucleosomal DNA to the other entry/exit (33N33 nucleosomes) site caused ISW1a to be unable to mobilize nucleosomes under conditions comparable to that used for efficient remodeling of 33N0 nucleosomes (Fig. 7A, lanes 7 to 10). Site-directed mapping of histone-DNA contacts confirmed that the 33N33 nucleosomes were not moved by ISW1a. The vast majority of nucleosomes were found not to be moved by ISW1a, as shown when no nucleosome movement was detected on the bottom strand, while less than 3% of the total nucleosomes were moved 14 bp, as observed for the top strand (Fig. 7B). Previously, ISW1a was shown to move nucleosomes with 33 bp of extranucleosomal DNA at only one entry/exit site to within 9 to 13 bp of the edge of DNA, in contrast to that observed for 33N33 nucleosomes (45). The inability of ISW1a to remodel 33N33 nucleosomes is reminiscent of the end state of ISW1a remodeling of nucleosomal arrays, with 29 bp of extranucleosomal DNA between each nucleosome, and is consistent with the loss of ISW1a contacts 20 bp from the dyad axis (36, 40).

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FIG. 7. ISW1a was unable to mobilize 33N33 nucleosomes. ISW1a (A) and ISW1b (C) remodeling of 0N33 or 33N33 nucleosomes was monitored by native PAGE. The concentration of ISW1a or ISW1b was varied from 1 to 27 nM, and lanes 1 and 6 did not contain either ISW1a or ISW1b. Changes in nucleosome translational positions due to ISW1a (B) and ISW1b (D) remodeling of 33N33 were determined by site-directed mapping of histone-DNA interactions. Quantification of the cleaved sites in DNA is shown for nucleosomes before (black) and after (gray) remodeling. The numbering above the peaks indicates the position of the cut site relative to that of the translation position before remodeling being referred to as 0. Illustrations at the bottom of each profile show the different directions of nucleosome movement observed.

In contrast to ISW1a, ISW1b was able to efficiently remodel 33N33 nucleosomes. ISW1b equally mobilized nucleosomes 0N33 and 33N33 (Fig. 7C, compare lanes 2 to 5 with 7 to 10). ISW1b apparently has potent remodeling activity for certain nucleosomes that are intractable to ISW1a and is not less active than ISW1a due to lowered nucleosomal affinity (41). Site-directed mapping showed that ISW1b moved 33N33 nucleosomes as far as 38 to 44 bp from its original position, thereby placing the nucleosomes slightly off the edge of DNA (Fig. 7D). The ability of ISW1b to move nucleosomes regardless of the arrangement of the extranucleosomal DNA is in keeping with its lack of spacing activity, as previously observed. Nucleosomal arrays likely become intractable to ISW1a remodeling once nucleosomes are moved 30 bp apart from each other, due to ISW1a binding to both linker DNAs.

Nucleosomes with extranucleosomal DNA at both entry/exit sites were unable to stimulate the ATPase activity of ISW1a. The rate of ATP hydrolysis of ISW1a with 70N0 nucleosomes was increased threefold over that observed with DNA alone, consistent with previous data of the nucleosome-dependent stimulation of ISWI complexes (Fig. 8A) (41). The 33N33 and 70N70 nucleosomes failed to stimulate the ATPase activity of ISW1a beyond that observed for DNA alone (Fig. 8A and data not shown). Although ISW1a efficiently bound these centrally positioned nucleosomes, it seems ISW1a was unable to recognize these nucleosomes as substrates for nucleosome mobilization or for stimulation of its ATPase activity. Both 33N33 and 70N70 nucleosomes stimulated the ATPase activity of ISW1b threefold more than free DNA (Fig. 8B and data not shown). These same nucleosomes were also readily mobilized by ISW1b. Nucleosomes with one extranucleosomal DNA (70N0) stimulated the ATPase activity of ISW1b in the same manner as that of the centrally positioned nucleosomes (Fig. 8B), indicating that the position of the extranucleosomal DNA did not affect the ATPase activity of ISW1b. These data suggest that ISW1a binding to extranucleosomal DNA at both entry/exit sites inhibits the remodeling activity of ISW1a, in terms of both ATPase and nucleosome mobilization activities.

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FIG. 8. The ATPase activity of ISW1a was not stimulated by 33N33 nucleosomes. The ATPase activities of ISW1a (A) and ISW1b (B) were measured in the presence of end-positioned (, 70N0) or centrally positioned (, 33N33) nucleosomes, free DNA (), and enzyme alone () with different incubation times. These assays were done in triplicate with standard deviations shown.

Ioc3 is the predominant subunit of ISW1a that associated with extranucleosomal DNA. The interactions of ISW1a with extranucleosomal DNA were investigated by site-directed photocross-linking of the subunits bound to extranucleosomal DNA. Photoreactive nucleotides were incorporated along with a radiolabeled nucleotide at five different positions, spanning 53 bp in the extranucleosomal DNA region of 75N0 nucleosomes, and four of the same positions in 28N32 nucleosomes (16, 32). In this manner, it was possible to probe the protein-DNA interactions of ISW1a bound to either 75N0 or 28N32 nucleosomes, to determine the ISW1a subunit(s) contacting extranucleosomal DNA and whether these interactions are changed dramatically in the two bound complexes. The Ioc3 subunit was most efficiently cross-linked to extranucleosomal DNA at 85 to 86 bp and slightly less efficiently at 77 and 92 bp from the dyad axis, with ISW1a bound to 75N0 nucleosomes (Fig. 9, lanes 2 to 4). The Isw1 subunit was not cross-linked efficiently at any of the positions examined, both at the edge of the nucleosome and further into the extranucleosomal region (Fig. 9, lanes 1 to 5). These results are in contrast with previous results with ISW2, where the catalytic subunit Isw2 was cross-linked well to extranucleosomal DNA at 73 to 92 bp from the dyad axis (16). Cross-linking of Ioc3 was sharply reduced at 126 bp from the dyad axis, consistent with earlier ExoIII footprinting, showing that ISW1a was not stably bound farther than 33 bp from the entry/exit site (Fig. 9, lane 1). The cross-linking efficiency of Ioc3 was also reduced at the entry/exit site (Fig. 9, lane 5). These data indicate that Ioc3 is the predominant subunit of ISW1a that interacts with extranucleosomal DNA when bound to 75N0 nucleosomes.

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FIG. 9. The Ioc3 subunit of ISW1a was associated with extranucleosomal DNA. ISW1a was DNA photoaffinity labeled at different sites in the extranucleosomal DNA while bound to either 75N0 or 28N32 nucleosomes. Nucleosomes (75N0, lanes 1 to 5; 28N32, lanes 6 to 9) assembled with a series of photoreactive DNA probes were incubated with either 1.3 pmol (lanes 1 to 5) or 430 fmol (lanes 6 to 9) of ISW1a, UV irradiated, and digested with DNase I and S1 nuclease as described previously. The probe position numbers indicate the site where photoreactive nucleotides were incorporated relative to the dyad axis.

The interaction of Ioc3 with extranucleosomal DNA was maintained when changing from the active to the inactive ISW1a-nucleosome complex, as shown by cross-linking of ISW1 bound to 28N32 nucleosomes. Ioc3 was cross-linked most efficiently at 85, 86, and 92 bp from the dyad and slightly less efficiently at 73 and 77 bp from the dyad (Fig. 9, lanes 6 to 9). The relative cross-linking efficiency between the four sites indicates a subtle change in ISW1a binding to 28N32 nucleosomes versus 75N0 nucleosomes. Yet, the primary subunit of ISW1a that still remains associated with extranucleosomal DNA is Ioc3. Likewise, the catalytic subunit Isw1 is not associated with extranucleosomal DNA when bound in the inactive conformation. The switch in ISW1a activity caused by binding to one extranucleosomal DNA versus binding to 33 bp of extranucleosomal DNA at both entry/exit sites was not due to large-scale changes in the particular subunit bound at these sites.

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In yeast, ISW1 is assembled into two complexes that both remodel chromatin by mobilizing nucleosomes, but only ISW1a, consisting of Isw1 and Ioc3, is able to position nucleosomes a constant, set distance from each other to create nucleosomal arrays with a uniform spacing of 175 bp. ISW1a was found to have two different nucleosome binding modes that regulate its nucleosome mobilization activity in a manner that is likely to account for its nucleosome-spacing activity. ISW1a bound to 33 bp of extranucleosomal DNA at both entry/exit sites was in an inactive conformation, as shown by its inability to move nucleosomes or to hydrolyze ATP in a nucleosome-dependent manner. ISW1a is active when bound to extranucleosomal DNA at only one entry/exit site. A key conformational change observed between these two nucleosomal-bound states of ISW1a is that ISW1a bound to only one extranucleosomal DNA makes a stable contact with nucleosomal DNA two helical turns from the dyad axis, and this interaction is lost when ISW1a is bound to extranucleosomal DNA at both entry/exit sites. These data show that ISW1 binding two helical turns from the dyad axis is important for ISW1a remodeling, and the loss of binding at this site is associated with the switch from ISW1a binding one to both extranucleosomal DNAs. The critical nature of the interactions at this internal site is also reflected in ISW2, as translocation by ISW2 at the same internal site is required for mobilizing nucleosomes (46). Furthermore, the histone H4 tail has been shown to be located at this region of the nucleosome and is required for the nucleosome-dependent stimulation of ISWI ATPase activity (4, 5).

The ability of ISW1a to create uniformly spaced nucleosomal arrays can be explained by the two different conformations of the ISW1a-nucleosome complex. ISW1a bound to one extranucleosomal DNA moves nucleosomes until the extranucleosomal DNAs on both sides of a nucleosome reach an optimal length to enable binding of ISW1a to both extranucleosomal DNAs and, thus, stop remodeling as a result of these new ISW1a-nucleosome contacts. The optimal length of extranucleosomal DNA on both sides of the nucleosome required for switching to the inactive conformation was 33 bp. When more than 33 bp of extranucleosomal DNA is available at one entry/exit site, ISW1a binds selectively to the longer extranucleosomal DNA and moves nucleosomes toward the extranucleosomal DNA until it becomes short enough to promote ISW1a binding to extranucleosomal DNA at both entry/exit sites. Changes in the conformation of bound ISW1a caused by this switch stop further remodeling. The uniform spacing of nucleosomal arrays by ISW1a is, therefore, the direct result of nucleosomes being moved on DNA until there is 33 bp of extranucleosomal DNA on both sides, which then prevents further remodeling by ISW1a. The regulation of ISW2 remodeling by extranucleosomal DNA has something in common with that shown here for ISW1a. It was shown recently that the length of extranucleosomal DNA can modulate the interaction of Isw2 at the internal site and, hence, regulate nucleosome movement (6). The effect of extranucleosomal DNA was found to be a progressive one: as the length of extranucleosomal DNA was reduced, the contact with the internal site was abolished and thereby stopped further remodeling. For both ISW2 and ISW1a, different types of binding to extranucleosomal DNA cause a loss of this internal contact and the ability to remodel. The main difference is that binding to only one extranucleosomal DNA is sufficient to cause this change for ISW2, whereas binding to both extranucleosomal DNAs is required for ISW1a to lose its interaction.

The property of ISW1a to form an inactive complex with nucleosomes is not inherent to the catalytic subunit Isw1, as ISW1b containing Isw1, Ioc2, and Ioc4 does not display this activity. Instead, ISW1b is able to move nucleosomes with extranucleosomal DNA lengths of 33, 53, and 70 bp at both entry/exit sites, and ISW1b can move nucleosomes toward DNA ends rather than, like ISW1a, strongly preferring to move nucleosomes to the center of DNA. ISW1b contacts only 13 to 19 bp of extranucleosomal DNA, significantly less than the 35 to 38 bp of extranucleosomal DNA contacted by ISW1a. ISW1b was shown not to bind nucleosomes in a stable fashion but was able to push nucleosomes off the ends of DNA by as much as 5 to 28 bp. Loss of histone-DNA contacts beyond the edge of the extranucleosomal DNA can destabilize the H2A/H2B dimer. Consistent with our data, ISW1b has been shown to transfer H2A onto a new DNA fragment, while ISW1a could not do the same (3). Clearly, the auxiliary subunits Ioc2 to Ioc4 modulate the activity of Isw1 and can account for the different functional roles of ISW1a and ISW1b, as shown recently in vivo (27, 28).

 
This work was supported by Public Health Service grant GM 48413 from the National Institutes of Health.

We thank Toshio Tsukiyama for providing yeast strains YTT449, YTT2094, and YTT2092. We thank members of the Bartholomew laboratory for their suggestions and comments.

 
* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, 1245 Lincoln Dr., Neckers Bldg. Room 229, Carbondale, IL 62901-4413. Phone: (618) 453-6437. Fax: (618) 453-6440. E-mail: bbartholomew{at}siumed.edu

Published ahead of print on 5 February 2007.

Present address: Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, NS287, New Haven, CT 06520.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Boyer, L. A., M. R. Langer, K. A. Crowley, S. Tan, J. M. Denu, and C. L. Peterson. 2002. Essential role for the SANT domain in the functioning of multiple chromatin remodeling enzymes. Mol. Cell 10:935-942.[CrossRef][Medline]
2
2. Boyer, L. A., R. R. Latek, and C. L. Peterson. 2004. The SANT domain: a unique histone-tail-binding module? Nat. Rev. Mol. Cell Biol. 5:158-163.[CrossRef][Medline]
3
3. Bruno, M., A. Flaus, C. Stockdale, C. Rencurel, H. Ferreira, and T. Owen-Hughes. 2003. Histone H2A/H2B dimer exchange by ATP-dependent chromatin remodeling activities. Mol. Cell 12:1599-1606.[CrossRef][Medline]
4
4. Clapier, C. R., G. Langst, D. F. Corona, P. B. Becker, and K. P. Nightingale. 2001. Critical role for the histone H4 N terminus in nucleosome remodeling by ISWI. Mol. Cell. Biol. 21:875-883.[Abstract/Free Full Text]
5
5. Clapier, C. R., K. P. Nightingale, and P. B. Becker. 2002. A critical epitope for substrate recognition by the nucleosome remodeling ATPase ISWI. Nucleic Acids Res. 30:649-655.[Abstract/Free Full Text]
6
6. Dang, W., M. N. Kagalwala, and B. Bartholomew. 2006. Regulation of ISW2 by concerted action of histone H4 tail and extranucleosomal DNA. Mol. Cell. Biol. 26:7388-7396.[Abstract/Free Full Text]
7
7. Eberharter, A., and P. B. Becker. 2004. ATP-dependent nucleosome remodelling: factors and functions. J. Cell Sci. 117:3707-3711.[Free Full Text]
8
8. Eisen, J. A., K. S. Sweder, and P. C. Hanawalt. 1995. Evolution of the SNF2 family of proteins: subfamilies with distinct sequences and functions. Nucleic Acids Res. 23:2715-2723.[Abstract/Free Full Text]
9
9. Farris, S. D., E. D. Rubio, J. J. Moon, W. M. Gombert, B. H. Nelson, and A. Krumm. 2005. Transcription-induced chromatin remodeling at the c-myc gene involves the local exchange of histone H2A.Z. J. Biol. Chem. 280:25298-25303.[Abstract/Free Full Text]
10
10. Ge, Y. Z., M. T. Pu, H. Gowher, H. P. Wu, J. P. Ding, A. Jeltsch, and G. L. Xu. 2004. Chromatin targeting of de novo DNA methyltransferases by the PWWP domain. J. Biol. Chem. 279:25447-25454.[Abstract/Free Full Text]
11
11. Grune, T., J. Brzeski, A. Eberharter, C. R. Clapier, D. F. Corona, P. B. Becker, and C. W. Muller. 2003. Crystal structure and functional analysis of a nucleosome recognition module of the remodeling factor ISWI. Mol. Cell 12:449-460.[CrossRef][Medline]
12
12. Hamiche, A., R. Sandaltzopoulos, D. A. Gdula, and C. Wu. 1999. ATP-dependent histone octamer sliding mediated by the chromatin remodeling complex NURF. Cell 97:833-842.[CrossRef][Medline]
13
13. Ito, T., M. Bulger, M. J. Pazin, R. Kobayashi, and J. T. Kadonaga. 1997. ACF, an ISWI-containing and ATP-utilizing chromatin assembly and remodeling factor. Cell 90:145-155.[CrossRef][Medline]
14
14. Ito, T., M. E. Levenstein, D. V. Fyodorov, A. K. Kutach, R. Kobayashi, and J. T. Kadonaga. 1999. ACF consists of two subunits, Acf1 and ISWI, that function cooperatively in the ATP-dependent catalysis of chromatin assembly. Genes Dev. 13:1529-1539.[Abstract/Free Full Text]
15
15. Jin, J., Y. Cai, B. Li, R. C. Conaway, J. L. Workman, J. W. Conaway, and T. Kusch. 2005. In and out: histone variant exchange in chromatin. Trends Biochem. Sci. 30:680-687.[Medline]
16
16. Kagalwala, M. N., B. J. Glaus, W. Dang, M. Zofall, and B. Bartholomew. 2004. Topography of the ISW2-nucleosome complex: insights into nucleosome spacing and chromatin remodeling. EMBO J. 23:2092-2104.[CrossRef][Medline]
17
17. Kassabov, S. R., and B. Bartholomew. 2004. Site-directed histone-DNA contact mapping for analysis of nucleosome dynamics. Methods Enzymol. 375:193-210.[Medline]
18
18. Kassabov, S. R., N. M. Henry, M. Zofall, T. Tsukiyama, and B. Bartholomew. 2002. High-resolution mapping of changes in histone-DNA contacts of nucleosomes remodeled by ISW2. Mol. Cell. Biol. 22:7524-7534.[Abstract/Free Full Text]
19
19. Korber, P., and W. Horz. 2004. SWRred not shaken; mixing the histones. Cell 117:5-7.[CrossRef][Medline]
20
20. Krebs, J. E., C. J. Fry, M. L. Samuels, and C. L. Peterson. 2000. Global role for chromatin remodeling enzymes in mitotic gene expression. Cell 102:587-598.[CrossRef][Medline]
21
21. Langst, G., E. J. Bonte, D. F. Corona, and P. B. Becker. 1999. Nucleosome movement by CHRAC and ISWI without disruption or trans-displacement of the histone octamer. Cell 97:843-852.[CrossRef][Medline]
22
22. LeRoy, G., G. Orphanides, W. S. Lane, and D. Reinberg. 1998. Requirement of RSF and FACT for transcription of chromatin templates in vitro. Science 282:1900-1904.[Abstract/Free Full Text]
23
23. Lowary, P. T., and J. Widom. 1998. New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning. J. Mol. Biol. 276:19-42.[CrossRef][Medline]
24
24. Lusser, A., and J. T. Kadonaga. 2003. Chromatin remodeling by ATP-dependent molecular machines. Bioessays 25:1192-1200.[CrossRef][Medline]
25
25. McConnell, A. D., M. E. Gelbart, and T. Tsukiyama. 2004. Histone fold protein Dls1p is required for Isw2-dependent chromatin remodeling in vivo. Mol. Cell. Biol. 24:2605-2613.[Abstract/Free Full Text]
26
26. Mizuguchi, G., X. Shen, J. Landry, W. H. Wu, S. Sen, and C. Wu. 2004. ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex. Science 303:343-348.[Abstract/Free Full Text]
27
27. Morillon, A., N. Karabetsou, A. Nair, and J. Mellor. 2005. Dynamic lysine methylation on histone H3 defines the regulatory phase of gene transcription. Mol. Cell 18:723-734.[CrossRef][Medline]
28
28. Morillon, A., N. Karabetsou, J. O'Sullivan, N. Kent, N. Proudfoot, and J. Mellor. 2003. Isw1 chromatin remodeling ATPase coordinates transcription elongation and termination by RNA polymerase II. Cell 115:425-435.[CrossRef][Medline]
29
29. Park, Y. J., J. V. Chodaparambil, Y. Bao, S. J. McBryant, and K. Luger. 2005. Nucleosome assembly protein 1 exchanges histone H2A-H2B dimers and assists nucleosome sliding. J. Biol. Chem. 280:1817-1825.[Abstract/Free Full Text]
30
30. Qiu, C., K. Sawada, X. Zhang, and X. Cheng. 2002. The PWWP domain of mammalian DNA methyltransferase Dnmt3b defines a new family of DNA-binding folds. Nat. Struct. Biol. 9:217-224.[Medline]
31
31. Reinke, H., and W. Horz. 2003. Histones are first hyperacetylated and then lose contact with the activated PHO5 promoter. Mol. Cell 11:1599-1607.[CrossRef][Medline]
32
32. Sengupta, S. M., J. Persinger, B. Bartholomew, and C. L. Peterson. 1999. Use of DNA photoaffinity labeling to study nucleosome remodeling by SWI/SNF. Methods 19:434-446.[CrossRef][Medline]
33
33. Stockdale, C., A. Flaus, H. Ferreira, and T. Owen-Hughes. 2006. Analysis of nucleosome repositioning by yeast ISWI and Chd1 chromatin remodeling complexes. J. Biol. Chem. 281:16279-16288.[Abstract/Free Full Text]
34
34. Strahl, B. D., and C. D. Allis. 2000. The language of covalent histone modifications. Nature 403:41-45.[CrossRef][Medline]
35
35. Strohner, R., M. Wachsmuth, K. Dachauer, J. Mazurkiewicz, J. Hochstatter, K. Rippe, and G. Langst. 2005. A "loop recapture" mechanism for ACF-dependent nucleosome remodeling. Nat. Struct. Mol. Biol. 12:732-733.[CrossRef][Medline]
36
36. Tsukiyama, T., J. Palmer, C. C. Landel, J. Shiloach, and C. Wu. 1999. Characterization of the imitation switch subfamily of ATP-dependent chromatin-remodeling factors in Saccharomyces cerevisiae. Genes Dev. 13:686-697.[Abstract/Free Full Text]
37
37. Tsukiyama, T., and C. Wu. 1995. Purification and properties of an ATP-dependent nucleosome remodeling factor. Cell 83:1011-1020.[CrossRef][Medline]
38
38. Tullius, T. D., B. A. Dombroski, M. E. Churchill, and L. Kam. 1987. Hydroxyl radical footprinting: a high-resolution method for mapping protein-DNA contacts. Methods Enzymol. 155:537-558.[Medline]
39
39. Varga-Weisz, P. D., M. Wilm, E. Bonte, K. Dumas, M. Mann, and P. B. Becker. 1997. Chromatin-remodelling factor CHRAC contains the ATPases ISWI and topoisomerase II. Nature 388:598-602.[CrossRef][Medline]
40
40. Vary, J. C., Jr., T. G. Fazzio, and T. Tsukiyama. 2004. Assembly of yeast chromatin using ISWI complexes. Methods Enzymol. 375:88-102.[CrossRef][Medline]
41
41. Vary, J. C., Jr., V. K. Gangaraju, J. Qin, C. C. Landel, C. Kooperberg, B. Bartholomew, and T. Tsukiyama. 2003. Yeast Isw1p forms two separable complexes in vivo. Mol. Cell. Biol. 23:80-91.[Abstract/Free Full Text]
42
42. Whitehouse, I., C. Stockdale, A. Flaus, M. D. Szczelkun, and T. Owen-Hughes. 2003. Evidence for DNA translocation by the ISWI chromatin-remodeling enzyme. Mol. Cell. Biol. 23:1935-1945.[Abstract/Free Full Text]
43
43. Whitehouse, I., and T. Tsukiyama. 2006. Antagonistic forces that position nucleosomes in vivo. Nat. Struct. Mol. Biol. 13:633-640.[CrossRef][Medline]
44
44. Xue, Y., J. Wong, G. T. Moreno, M. K. Young, J. Cote, and W. Wang. 1998. NURD, a novel complex with both ATP-dependent chromatin-remodeling and histone deacetylase activities. Mol. Cell 2:851-861.[CrossRef][Medline]
45
45. Zofall, M., J. Persinger, and B. Bartholomew. 2004. Functional role of extranucleosomal DNA and the entry site of the nucleosome in chromatin remodeling by ISW2. Mol. Cell. Biol. 24:10047-10057.[Abstract/Free Full Text]
46
46. Zofall, M., J. Persinger, S. R. Kassabov, and B. Bartholomew. 2006. Chromatin remodeling by ISW2 and SWI/SNF requires DNA translocation inside the nucleosome. Nat. Struct. Mol. Biol. 13:339-346.[CrossRef][Medline]

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Molecular and Cellular Biology, April 2007, p. 3217-3225, Vol. 27, No. 8
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