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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.
,
Mohamedi N. Kagalwala,
,
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 |
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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 |
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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 |
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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
-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 |
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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.
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| DISCUSSION |
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-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).
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| FOOTNOTES |
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W.D. and M.N.K. have made equal contributions to this work. ![]()
Present address: The Wistar Institute, 3601 Spruce St., Room 201, Philadelphia, PA 19104. ![]()
Present address: Department of Molecular Genetics, Section of Cancer Genetics, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030. ![]()
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