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Molecular and Cellular Biology, October 2006, p. 7388-7396, Vol. 26, No. 20
0270-7306/06/$08.00+0     doi:10.1128/MCB.01159-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Regulation of ISW2 by Concerted Action of Histone H4 Tail and Extranucleosomal DNA

Weiwei Dang ,{dagger},§ Mohamedi N. Kagalwala,{dagger},{ddagger} and Blaine Bartholomew*

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

Received 27 June 2006/ Accepted 24 July 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The stable contact of ISW2 with nucleosomal DNA ~20 bp from the dyad was shown by DNA footprinting and photoaffinity labeling using recombinant histone octamers to require the histone H4 N-terminal tail. Efficient ISW2 remodeling also required the H4 N-terminal tail, although the lack of the H4 tail can be mostly compensated for by increasing the incubation time or concentration of ISW2. Similarly, the length of extranucleosomal DNA affected the stable contact of ISW2 with this same internal nucleosomal site, with the optimal length being 70 to 85 bp. These data indicate the histone H4 tail, in concert with a favorable length of extranucleosomal DNA, recruits and properly orients ISW2 onto the nucleosome for efficient nucleosome remodeling. One consequence of this property of ISW2 is likely its previously observed nucleosome spacing activity.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ATP-dependent chromatin remodeling subfamilies ISWI, SNF2, CHD1, and INO80 have all been shown to alter the structure of chromatin to make DNA accessible for DNA-binding proteins during various regulatory processes inside the cell (10). These multiprotein complexes function in different ways to move or displace nucleosomes in order to increase the accessibility of DNA (4, 12, 14, 26, 27). Regulation of the activity of these enzymes in the cell is crucial, as perturbations can lead to neoplasias and other diseases (5).

Most ISWI complexes consist of two to four subunits (25) and are involved in chromatin assembly (17, 37), spacing of nucleosomal arrays (17, 34), and moving mononucleosomes in ~10-bp steps (20, 40). ISWI is fully stimulated only by nucleosomes with intact histone amino-terminal tails (1). Further studies have revealed that the basic patch R17H18R19 of the histone H4 tail was required for remodeling by ISWI in vitro (6, 7, 15) and in vivo (13). Acetylation of K16 on H4 was also found to impede chromatin remodeling by ISWI (8). The H4 tail has been shown to interact with nucleosomal DNA near SHL2 (superhelical location 2), two helical turns away from the dyad axis, by chemical cross-linking (11). Consistent with this finding are the recent reports showing that yeast ISW2 makes strong contacts with the SHL2 site (18) and that this contact is critical for chromatin remodeling by ISW2 (41). It has not been shown directly if the H4 tail is required for ISW2 to contact the SHL2 site or for ISW2 remodeling.

Given the recent finding that the histone H4 tail is involved in the formation of higher-order chromatin structure (31), it is important to determine how this structural role of the H4 tail may relate to its functional role in ISWI remodeling. Histone tails generally interact with linker DNA within ~25 bp from the edge of nucleosomes (38). The major binding sites for histone tails have been shown by UV cross-linking to be with linker DNA and not with the core nucleosome particle (2, 32). Specifically, extranucleosomal DNA causes a change in the contacts of the histone H2A C-terminal tail from the dyad axis to close to the entry/exit sites of nucleosomes (21, 36). The histone H3 tail has also been shown to make intranucleosomal contacts in a nucleosomal array, and acetylation of the histone H4 tail interferes with formation of a higher-order chromatin structure (31, 39). Linker DNA also has an effect on ISWI remodeling, as reflected in the ability of ISWI to equally space nucleosomes in arrays (17, 34). It has been shown that ISW2 in vitro generates nucleosomal arrays with ~50 bp of linker DNA between adjacent nucleosomes or nucleosomal repeats of ~200 bp (33). The spacing activity of ISW2 is likely affected by the interactions of the Itc1 subunit of ISW2, with linker DNA sterically blocking nucleosomes from moving together closer than ~50 bp (18). The affinity of ISW2 for nucleosomes is also reduced as the linker DNA becomes shorter, and a minimum of 20 bp of extranucleosomal DNA is needed for ISW2 to interact and remodel nucleosomes (18, 40). Experiments with DNA gaps suggest an important role of extranucleosomal DNA immediately adjacent to the nucleosome in the initial stage of nucleosome remodeling by ISW2 (40). We therefore investigated the role of the histone H4 N-terminal tail and extranucleosomal DNA length for the binding of ISW2 with nucleosomal DNA at SHL2 and for efficient remodeling.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Affinity purification of the ISW2 complex. The ISW2 complex was purified from Saccharomyces cerevisiae strain YTT480 with two copies of the FLAG epitope tag attached to the C terminus of ISW2 by immunoaffinity chromatography (18, 34).

Plasmids and DNA probes. The nucleosome positioning sequence 601 was obtained from plasmid pGEM-3Z/601 (23). DNAs with 601 and varied extents of flanking regions (extranucleosomal DNA) were synthesized by PCR as described elsewhere (18).

Nucleosome reconstitution, binding, and sliding assays. Mononucleosomes were assembled at 37°C with 7 to 10 µg of recombinant Xenopus laevis histone octamers (wild type or with histone tail deletions), 100 to 200 fmol of radiolabeled 601 DNA, 5 to 10 µg of either sonicated salmon sperm DNA or PCR-amplified 601 DNA, and 1.8 M NaCl in a starting volume of 10 µl, followed by serial dilutions (22). Nucleosome assemblies were analyzed by 4% nondenaturing polyacrylamide gel electrophoresis (PAGE; acrylamide/bisacrylamide ratio, 36:1; 4°C) in a running buffer containing 45 mM Tris, 45 mM borate, and 1 mM EDTA (0.5x TBE).

ISW2 binding and remodeling reactions were performed as previously described (18) with nucleosomes containing either wild-type (WT) or globular H4 (gH4) and radiolabeled 601 DNA. Remodeling reaction mixtures contained 100 µM ATP, and 5-µl samples were removed from each reaction mixture at different time points and stopped with {gamma}-S-ATP (2.5 mM) and sonicated salmon sperm DNA (1 mg/ml). Binding and remodeling reactions were analyzed by nondenaturing PAGE and phosphorimaging. All lanes were normalized based on total signal, and the fraction bound or mobilized was calculated by comparing the amount of unbound or immobilized nucleosomes in each lane to nucleosomes in the control lane with no ISW2 added.

Hydroxyl radical footprinting. ISW2 binding was carried out in a 25-µl reaction mixture as described without the addition of ATP (18). The cleavage reaction was performed as described previously (35), except that the final concentrations of Fe(II), H2O2, ascorbate, and EDTA were 280 µM, 0.17%, 5.7 mM, and 220 µM, respectively, and the reaction was terminated by addition of 100 µl of 5 M ammonium acetate, 5 mM thiourea, and 10 mM EDTA. MD ImageQuant software (GE Healthcare) was used to analyze phosphorimaging scans. Lanes were selected, and the intensity of each pixel of the lanes was exported into Microsoft Excel. The intensity data were normalized based on the total signal in each lane and plotted as shown. The percent protection was calculated using the following formula, with I being the signal intensity of either nucleosome alone (IN) or bound to ISW2 (IISW2): [(IN – IISW2)/IN] x 100.

Site-specific DNA photoaffinity labeling. Biotinylated single-stranded DNA template immobilized on streptavidin-coated Dynabeads (Dynal) was used to generate DNA photoaffinity probes as previously described (19). Different probe lengths were obtained by using different restriction endonucleases to release the DNA probes from beads. Purified photoreactive DNA probes were assembled into mononucleosomes using recombinant wild-type Xenopus histone octamers or those with particular histone tail deletions. Photoreactive nucleosomes (1 pmol) were incubated with or without ISW2 (1 to 1.2 pmol) at 30°C for 30 min. After UV irradiation, samples were digested with DNase I and S1 nuclease and were analyzed with 4 to 20% sodium dodecyl sulfate-PAGE (18).

Missing nucleoside sliding and binding interference assay. Radioactively end-labeled nucleosomes were digested with hydroxyl radicals in ISW2 buffer under conditions as described above for hydroxyl radical footprinting (18). The cleavage reaction was stopped by addition of glycerol to a final concentration of 4%. The buffer was exchanged to ISW2 buffer under conditions for remodeling or binding reactions by at least three rounds of dilution and concentration using Microcon-30 filters (Millipore). Nucleosome samples were split into four parts, and one-fourth of the reaction mixture was kept aside as unremodeled sample, whereas the other parts were each subjected to different concentrations of ISW2 for either ISW2 binding or nucleosome sliding with 500 µM ATP at 30°C for 30 min. The samples were concentrated by ultrafiltration (Microcon-30) and loaded onto a 5% native polyacrylamide gel (acrylamide/bisacrylamide ratio, 60:1) as described elsewhere (18). The bands corresponding to bound and unbound or mobile and immobile nucleosomes were excised from the gel, and DNA passively eluted from gel slices was extracted and resolved by 6.5% PAGE containing 8 M urea. Analysis was performed similarly to that described above for hydroxyl radical footprinting.

Measurement of rates of ISW2 remodeling. The rates of ISW2 remodeling with and without the histone H4 tail and different extranucleosomal DNA lengths were analyzed by nondenaturing PAGE. The rates were calculated from at least three independent time courses. The fraction of nucleosomes moved by ISW2 at each time point was determined after normalizing for the total signal in the lane.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ISW2 interaction at SHL2 is modulated by changes in extranucleosomal DNA length. Yeast ISW2 preferentially binds nucleosomes with 70 bp of extranucleosomal DNA and under these conditions interacts with nucleosomes at two disparate sites within the nucleosome, the entry/exit site and the internal SHL2 site, and extensively with extranucleosomal DNA (18). Nucleosomes containing 0 or 20 bp of extranucleosomal DNA at one entry/exit site were unable to promote stable interaction of ISW2 at SHL2, but ISW2 was able to contact SHL2 with 35 bp of extranucleosomal DNA (Fig. 1A and B and results not shown). Nucleosomes with 85 bp of extranucleosomal DNA had a strong ISW2 footprint at SHL2 (Fig. 1C), like that previously observed with 70 bp of extranucleosomal DNA (18). However, ISW2 was unable to effectively contact nucleosomes having longer extranucleosomal DNA of 138 bp at SHL2, similar to nucleosomes with short extranucleosomal DNA (Fig. 1D). The interactions of ISW2 with extranucleosomal DNA and the entry site were not dependent on the length of extranucleosomal DNA. The extent of protection at SHL2 versus extranucleosomal DNA length was plotted and revealed that optimal binding of ISW2 at this site is with 70 to 85 bp of extranucleosomal DNA and that by shortening or lengthening extranucleosomal DNA this interaction can be reduced by as much as three to nine times (Fig. 1E).


Figure 1
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  		FIG. 1. ISW2 interaction with nucleosomes at SHL2 requires an optimal length of extranucleosomal DNA. ISW2 was bound to sucrose gradient-purified wild-type nucleosomes (90 nM) assembled onto end-labeled DNA with various extranucleosomal DNA lengths under saturating conditions. After hydroxyl radical cleavage, samples were analyzed on a 6.5% polyacrylamide gel containing 8 M urea along with the sequencing ladder of the same DNA. Quantification of the hydroxyl radical protection patterns of nucleosomes with (gray) and without ISW2 (black) is shown with 20-bp extranucleosomal DNA (A), with 35-bp extranucleosomal DNA (B), with 85-bp extranucleosomal DNA (C), and with 138-bp extranucleosomal DNA on one side (D). The numbers above the peaks indicate the number of base pairs from the dyad axis, with "+" or "–" indicating two directions. The superhelical location (SHL) of the nucleosome is depicted, and the observed protection due to ISW2 binding is indicated with black (strong protection) or gray (weak protection) bars below the axis. (E) Graphical depiction of the quantification of the percent protection at SHL2 by ISW2 with respect to the length of the extranucleosomal DNA on an end-positioned nucleosome.

 
The effect of extranucleosomal DNA length on ISW2-nucleosome interactions was further explored by site-specific DNA photoaffinity labeling to determine if the subunit contacting a particular DNA site changed with reduction of the length of extranucleosomal DNA. DNA photoaffinity labeling at major ISW2 contact sites 92, 68/66, and 18/17 bp from the dyad axis were used to monitor alterations in ISW2-DNA interactions (18). The association of Isw2 and Itc1 with DNA near bp –18/–17 was reduced two- to threefold by decreasing extranucleosomal DNA length from 50 and 75 to 29 bp (Fig. 2A, lanes 13 to 18, and B). Decreasing the length of extranucleosomal DNA from 75 to 29 bp also caused a change in the interaction of ISW2 with extranucleosomal DNA at bp –92. The association of Isw2 with extranucleosomal DNA was diminished, as shown, by reduction of Isw2 cross-linking at bp –92 with shortening of the extranucleosomal DNA, while not impacting the interaction of Itc1 (lanes 1 to 6). These changes in the cross-linking pattern suggest a significant change in the conformation of the ISW2-nucleosome complex, with reduced contacts of Isw2 with extranucleosomal DNA and of Isw2 and Itc1 with nucleosomal DNA at SHL2. No change in the proximity of Itc1 or Isw2 was observed with DNA inside the nucleosome near the entry/exit site upon reduction of the length of extranucleosomal DNA (–68/–66) (lanes 7 to 12).


Figure 2
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  		FIG. 2. Interaction of Isw2 with DNA 19 bp from the edge of the nucleosome is reduced with shorter extranucleosomal DNA. (A) Schematic diagrams of the nucleosome core particle with extranucleosomal DNA indicating the positions of the photoaffinity labeling cross-linkers (denoted by four-point stars) used in this study. The dyad axis of the nucleosome is labeled as 0. (B) Effects of extranucleosomal DNA (E. DNA) length on the cross-linking of ISW2 subunits. Nucleosomes with 75, 50, or 29 bp of extranucleosomal DNA assembled with photoreactive probes were incubated with or without ISW2 (48 nM), UV irradiated, and digested with DNase I and S1 nuclease as described in the text. The probe position numbers indicate the site of incorporated photoreactive nucleotides with reference to the dyad axis. The * denotes undigested probe DNA. (C) Quantification of the relative cross-linking intensities of ISW2 subunits as affected by extranucleosomal DNA length.

 
The histone H4 N-terminal tail was required for stable interaction of ISW2 with nucleosomal DNA at SHL2. The interaction of ISW2 with nucleosomes missing one or more of the histone N-terminal tails was studied using a combination of hydroxyl radical footprinting and site-specific DNA photoaffinity labeling. The region of nucleosomes bound by ISW2 with 70 bp of extranucleosomal DNA and different histone tails deleted was determined by DNA footprinting (Fig. 3). The H4 histone tail was found to be required for binding of ISW2 to SHL2, but not for binding near the entry site or with extranucleosomal DNA (compare Fig. 3A and B). Deletion of the H3 histone tail (gH3) or H2B histone tail (gH2B) did not affect the interaction of ISW2 with DNA, as all three contacts with nucleosomes were maintained (Fig. 3C and D). Deletion of the H4 tail in combination with different lengths of extranucleosomal DNA ranging from 35 to 138 bp also abolished the internal contact at SHL2 (data not shown).


Figure 3
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  		FIG. 3. H4 tail is required for stable binding of ISW2 at SHL2. Sucrose gradient-purified nucleosomes (90 nM) assembled onto end-labeled 222-bp 601 DNA (75 bp of extranucleosomal DNA on one side of the positioning sequence) were incubated with ISW2 for complete binding. After removing glycerol by spin column purification, DNA was cleaved with hydroxyl radicals and samples were analyzed on a 6.5% polyacrylamide gel containing 8 M urea along with the sequencing ladder of the same DNA. Quantification of the hydroxyl radical protection patterns of nucleosomes with (black) and without (gray) ISW2 are shown with nucleosomes containing full-length histones (A) or missing either a histone H4 tail (B), histone H3 tail (C), or histone H2b tail (D).

 
DNA photoaffinity labeling of ISW2 showed the loss of ISW2 contact with DNA near SHL2 upon deletion of the H4 tail (Fig. 4). Consistent with the footprinting results, cross-linking of both Itc1 and Isw2 subunits at –18/–17 was diminished by 10 and 5 times, respectively (lanes 13 to 14), while no significant change was visible at any other position upon deletion of the H4 histone tail (lanes 1, 2, 5, 6, 9, and 10). Deletion of the histone H3 N-terminal tail showed only a slight reduction (twofold or less) in ISW2 cross-linking (lanes 3, 7, 11, and 15), whereas H3 and H4 tail double deletion behaved similar to H4 tail deletion (lanes 13 to 16), suggesting no additive effect of deleting both H3 and H4 tails. The cross-linking of histone H4 at bp –18/–17 was found to occur mostly to the histone tail region. Histone H4 and to a lesser extent histone H3 are both cross-linked at bp –18/–17 with nucleosomes containing all full-length histones (lane 13). Nucleosomes missing the H4 tail still cross-link H3 as efficiently, but the level of histone H4 cross-linked was significantly reduced and migrated faster than the intact H4 (lane 14).


Figure 4
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  		FIG. 4. The histone H4 tail is required for the association of Isw2 and Itc1 with nucleosomal DNA at SHL2. Nucleosomes with full-length histones (WT) or missing histone H4 tails (g4), H3 tails (g3), or both H3 and H4 tails (g34) were assembled with photoreactive DNAs that had 75 bp of extranucleosomal DNA. The probe position numbers indicate the site of incorporated photoreactive nucleotides with reference to the dyad axis. The * denotes the small amount of undigested probe DNA.

 
The histone H4 tail interaction with ISW2 contributes very little to the overall affinity of ISW2 for nucleosomes, as shown in ISW2-binding assays using nucleosomes with and without the H4 tail (Fig. 5). There is little change in affinity of ISW2 for nucleosomes with 70 bp of extranucleosomal DNA missing the H4 tail. The interactions that seem to contribute the most to the nucleosomal affinity of ISW2 are those with the extranucleosomal DNA. The interactions of ISW2 with nucleosomal DNA at SHL2 are likely to be important for the functionality and not the stability of the ISW2-nucleosome complex.


Figure 5
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  		FIG. 5. The histone H4 tail is not a major requirement for ISW2 binding to nucleosomes. The affinity of ISW2 for nucleosomes with (WT) and without (gH4) histone H4 tails were determined by gel shift as shown. The fraction of nucleosomes with 70 bp of extranucleosomal DNA bound by ISW2 is indicated below each lane. Binding reaction mixtures contained 38 nM nucleosomes with 7.3, 15, and 29 nM of affinity-purified ISW2.

 
Nucleosome mobilization by ISW2 is affected by both H4 tail and extranucleosomal DNA length. Previous studies with Drosophila melanogaster ISWI complexes (CHRAC and NURF) have shown that the ATPase activity of these is not stimulated in the absence of the H4 tail, and nucleosome movement is totally abolished without the critical H4 tail (6, 7, 15). The effect of extranucleosomal DNA length and the histone H4 N-terminal tail on the ability of ISW2 to mobilize nucleosomes was examined. A time course was performed to study the efficiency of nucleosome mobilization by ISW2 with a combination of different lengths of extranucleosomal DNA and deletion of the H4 tail. Consistent with Drosophila ISWI complexes, yeast ISW2 showed a ~20-fold reduction in the rate of movement for nucleosomes missing H4 N-terminal tails (gH4) compared to wild type with 70 bp of extranucleosomal DNA (Fig. 6B). A reduction in the rate of remodeling was observed for all lengths of extranucleosomal DNA when the H4 tail was missing (Fig. 6B). The decreased efficiency of nucleosome movement is not because of an overall reduction of ISW2 affinity for nucleosomes missing H4 tails (Fig. 5), but rather is correlated with the selective loss of contact with nucleosomal DNA at SHL2. This conclusion is consistent with observations that the SHL2 contact is required for remodeling by ISW2 as well as for other chromatin remodeling enzymes (29, 30, 41). The deletion of H3 and H2B tails did not inhibit nucleosome mobilization with the varied extranucleosomal DNA lengths (data not shown). Although the rates of nucleosome movement were severely affected, more than 50% of the nucleosomes were moved after 20 to 60 min in the absence of H4 tails when they contained an optimal length of extranucleosomal DNA (Fig. 6A, compare lanes 15 and 16 to lanes 11 and 12). This same effect can also be seen by adding higher amounts of ISW2 and collectively suggest that the H4 tail-ISW2 interactions facilitate a step in the remodeling process that would be otherwise rate limiting. The combination of nonoptimal length of extranucleosomal DNA of 35 and 138 bp and the absence of H4 tails more severely impaired the remodeling activity of ISW2 than the lack of H4 tails alone (Fig. 6A, lanes 7, 8, 23, and 24). These results suggest that extranucleosomal DNA and the histone H4 tail act in a coordinate, but nonoverlapping, way to recruit ISW2 to SHL2 and facilitate remodeling by ISW2.


Figure 6
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  		FIG. 6. Loss of the H4 tail greatly reduces the rate of ISW2 remodeling. (A) The rate of ISW2 remodeling was determined with 5.8 nM ISW2, 38 nM nucleosomes, and 100 µM ATP. The numbers of base pairs indicate the lengths of extranucleosomal DNA located at the one entry/exit site of nucleosomes. Reaction times were 20 s, 1 min, and 5 min for wild-type nucleosomes or 5 min, 20 min, and 1 h for nucleosomes without the H4 tail; analysis was by nondenaturing PAGE. (B) The rates of ISW2 remodeling with and without the histone H4 tail and different extranucleosomal DNA lengths are expressed as nanomolars of substrate per minute and plotted with respect to various extranucleosomal DNA lengths. Results are shown for wild-type nucleosomes (WT) and nucleosomes missing the H4 tail (gH4).

 
Transient interaction of ISW2 at the internal site is likely required for moving nucleosomes missing the H4 tail. Interference assays were performed with DNAs missing single nucleosides to determine if ISW2 interactions were required at SHL2 for nucleosome movement with nucleosomes missing H4 tails, even though no stable interactions at SHL2 were observed. The approach was to determine if DNA gaps at or near the SHL2 region interfered with ISW2 remodeling as observed for wild-type nucleosomes because of blocking binding or translocation at this site (41). Nucleosomes with single nucleoside gaps were created by hydroxyl radical cleavage and remodeled with ISW2 (9, 30). DNA from the slid and unslid nucleosomes was isolated and analyzed on a denaturing polyacrylamide gel to determine the gaps that interfered with ISW2 remodeling. The profiles of the distribution of gapped DNA present in the slid and unslid nucleosomes were overlaid, as shown in Fig. 7A and B, to highlight the gaps that were selectively enriched in the immobile nucleosome fraction and interfered with ISW2 remodeling. Even though there is no stable interaction of ISW2 with nucleosomal DNA at SHL2 in the absence of the H4 tail (Fig. 3 and 4), there is still a critical role for this region in nucleosome mobilization, as shown by 1-nucleoside gaps interfering with sliding from nucleotide –17 to –24 (Fig. 7B, upper strand) and –16 to –26 (lower strand [data not shown]). As expected, this same effect was also observed for wild-type nucleosomes (Fig. 7A). DNA gaps at SHL2 interfered with nucleosome movement without affecting the binding of ISW2 to gH4 nucleosomes as shown in Fig. 7D (results not shown for the lower strand). These data imply that even in the absence of the H4 tail, ISW2 still has to contact DNA at SHL2 in order to mobilize the nucleosome. These contacts occur less efficiently or frequently without the H4 tail to recruit it to DNA at SHL2, causing the much slower rate of remodeling.


Figure 7
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  		FIG. 7. ISW2 remodeling in the absence of the H4 tail still requires the interaction of ISW2 at SHL2. Wild-type (A) and H4 tail-deleted (B) nucleosomes (80 to 100 nM) with 70 bp of extranucleosomal DNA were incubated with ISW2 (3 nM) and ATP (400 µM) before separation on a 5% native polyacrylamide gel. DNA contained single-nucleoside gaps that were generated by hydroxyl radical cleavage, and DNA was eluted from the slid and unslid nucleosomes for analysis on a denaturing 6.5% polyacrylamide gel. The profiles of the slid (gray) and unslid (black) lanes were overlaid as shown. Only the plots with labeled upper strands are shown. The numbers on top of the peaks indicate the distance from the dyad axis. (C and D) Samples were the same as in panels A and B, except for the omission of ATP and a higher concentration of ISW2 (35 nM). The ISW2-bound and free nucleosomes were excised and eluted from native PAGE gels and analyzed by denaturing 6.5% PAGE. The overlaid plots of the bound (gray) and unbound (black) nucleosomes are shown.

 
DNA gaps from bp –17 to –25 did, however, block ISW2 binding to wild-type nucleosomes, and the results suggest that the gaps in this case may block nucleosome movement by preventing the binding of ISW2 (Fig. 7C). This observation is contradictory to what has been observed with Drosophila NURF, where no interference with binding was observed (30). Noticeably, this interference of ISW2 binding is only observed when the whole nucleoside including both the deoxyribose ring and the base is removed by a hydroxyl radical, in contrast to no detectable binding interference when just nicking by DNase I or by base excision using uracil-containing DNA treated with uracil DNA glycosylase and endonuclease III (41).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we have shown that the histone H4 N-terminal tail and the length of extranucleosomal DNA affect the conformation of the ISW2-nucleosome complex and are required for the stable interaction of ISW2 with nucleosomal DNA at SHL2. As interactions of ISW2, NURF, SWI/SNF, and RSC with nucleosomes at this region are required for remodeling (17, 28, 34), we favor a model of the histone H4 tail and extranucleosomal DNA acting in concert to direct ISW2 to the critical SHL2 site of nucleosomes for efficient nucleosome mobilization (Fig. 8).


Figure 8
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  		FIG. 8. The histone H4 tail and extranucleosomal DNA act in concert to direct ISW2 to the critical SHL2 site of nucleosomes for efficient nucleosome mobilization. The critical ISW2 contact at SHL2 of nucleosomes is maintained only when the optimal extranucleosomal DNA (E. DNA) and the histone H4 N-terminal tail are both present (left). Either the presence of a suboptimal length of extranucleosomal DNA (middle) or the deletion of the H4 tail (right) disrupts the ISW2-nucleosome contact at the SHL2 site. The ISW2 complex and the nucleosome are depicted schematically, highlighting the three contact sites with the nucleosome and extranucleosomal DNA. The dyad axis of the nucleosome is denoted by 0.

 
Histone H4 tails project out of the nucleosome at about two helical turns from the dyad axis (SHL2) and probably assume an {alpha}-helical structure (16, 24) when bound to DNA (3). Our data have demonstrated a strong correlation between H4 tail deletion and the loss of the critical SHL2 contact by ISW2, suggesting that the H4 tail physically recruits Isw2 and Itc1 subunits to this internal site of nucleosomes. In other words, ISW2 has to interact with the H4 tail before it can make stable contact with the critical internal site of the nucleosome. As expected, the loss of this internal contact upon deletion of the H4 tail does not significantly change the binding affinity of ISW2 to the nucleosome, since other contacts of ISW2 with extranucleosomal DNA and the nucleosome entry/exit site are maintained. We have shown that the recruitment of ISW2 to the nucleosome SHL2 site by the H4 tail is, however, critical for efficient remodeling. Optimal binding of ISW2 to SHL2 also required 70 to 85 bp of extranucleosomal DNA, as shown by hydroxyl radical footprinting and photo-cross-linking. In addition, by photo-cross-linking, other conformational changes were observed with extranucleosomal DNA 19 bp from the edge of the nucleosome. Shortened extranucleosomal DNA caused conformational changes as well as reduced binding affinity, whereas extranucleosomal DNA that is too long is unable to properly position ISW2, presumably due to the potential multiplicity of binding sites available. These unfavorable lengths of extranucleosomal DNA reduced nucleosome mobilization by ISW2, consistent with their loss of the critical SHL2 contact.

We have found that optimal extranucleosomal DNA length can partially compensate for the loss of the H4 tail, but the rate of nucleosome movement remains low in the absence of the H4 tail. Nonoptimal extranucleosomal DNA length, in combination with loss of the H4 tail, is so severely affected that longer incubation or more ISW2 cannot compensate for the loss of the H4 tail. Previously, CHRAC from Drosophila was shown to be completely unable to remodel nucleosomes missing the H4 tail (6, 7), suggesting a difference between these two homologous complexes in their histone H4 tail dependency. These differences could also be due to differences in the reaction conditions, such as the lower CHRAC concentrations (0.5 to 3 fmol CHRAC with 60 fmol of nucleosomes) or shorter incubation times (5 min). We showed that the H4 tail likely facilitates a rate-limiting step in the ISW2 remodeling reaction, which could be either the binding of ISW2 to nucleosomes at SHL2 and/or the remodeling reaction being made more processive. Even in the absence of the H4 tail it appears from the gap interference experiments that ISW2 still must interact with the SHL2 region for remodeling to occur. Since no stable footprint is detected at this region in the absence of the H4 tail, it appears that ISW2 must transiently interact with this region. Extranucleosomal DNA was also indicated to contribute to the interaction of ISW2 with the SHL2 nucleosomal DNA in two ways. First, if extranucleosomal DNA was either too long or too short, then the interaction of ISW2 with SHL2 was negatively affected even in the presence of the histone H4 tail. Second, the putative transient interaction of ISW2 with SHL2 in the absence of the H4 tail required for remodeling appeared to be negatively affected by nonoptimal extranucleosomal DNA length. These effects demonstrate that optimal extranucleosomal DNA length and the H4 tail act in parallel to direct ISW2 to the nucleosome SHL2 site in the proper and active conformation for efficient nucleosome mobilization (Fig. 8).

Earlier results had suggested that the nucleosome spacing activity of ISW2 could be due to steric hindrance caused by binding of the largest subunit, Itc1, to linker DNA and/or to a reduced binding affinity of ISW2 to nucleosomes with linker DNA shorter than 50 bp (18). The results in this report show that maybe even more important for the observed nucleosome spacing activity of ISW2 is that, as the linker DNA becomes shorter, the conformation of the ISW2-nucleosome complex changes such that it loses its critical contact inside the nucleosome and thereby becomes unable to move the nucleosome further. This effect is likely to be a graded one that progressively occurs as the linker DNA becomes shorter, consistent with our data, and would account for the observation that ISW2 spacing of nucleosomes is not as periodic or consistently spaced, for example, as seen for ISW1a (34).

.


   FOOTNOTES
 
* 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. Back

{dagger} W.D. and M.N.K. have made equal contributions to this work. Back

§ Present address: The Wistar Institute, 3601 Spruce St., Room 201, Philadelphia, PA 19104. Back

{ddagger} Present address: Department of Molecular Genetics, Section of Cancer Genetics, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030. Back


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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Molecular and Cellular Biology, October 2006, p. 7388-7396, Vol. 26, No. 20
0270-7306/06/$08.00+0     doi:10.1128/MCB.01159-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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