Functional Role of Extranucleosomal DNA and the Entry Site of the Nucleosome in Chromatin Remodeling by ISW2

DNA probe synthesis.

End-labeled probes for histone-DNA cross-linking were generated by PCR amplification of pGEM-3Z-601 using AmpliTaq Gold polymerase (Applied Biosystems). Oligonucleotides, appropriate for a given length and placement of the nucleosome-positioning sequence (NPS), were obtained from Integrated DNA Technologies. Upstream or downstream oligonucleotides were labeled at the 5′ end by using T4 polynucleotide kinase (New England BioLabs) and [γ-³²P]ATP (6,000 Ci/mmol) to obtain upper- or lower-strand-labeled DNA. DNA was purified with a QIAquick PCR purification kit (QIAGEN). The nine-nucleotide overhang probes were generated by digestion of PCR-generated DNA with TsrpI (3′ overhang) or NBstNB I (5′ overhang) and purified with a QIAquick PCR purification kit at 50°C to remove the smaller fragments. The probes were analyzed on a 6.5% polyacrylamide gel containing 8 M urea and were visualized by phosphorimaging.

For generation of DNA fragments containing strand breaks, a solid phase DNA synthesis on immobilized templates using M-280 magnetic beads (Dynal Biotech) was adapted. The lower strand of the DNA fragment was biotinylated and immobilized on streptavidin-coated magnetic beads. DNAs were generated by PCR amplification of pGEM-3Z-601 by using AmpliTaq Gold polymerase (Applied Biosystems) with the oligonucleotide for synthesis of the lower strand being biotinylated at the 5′ end. DNA was bound to magnetic beads, and the upper strand was removed as described previously (25). Oligonucleotides starting just downstream of the strand break were annealed to the immobilized ssDNA and extended with the Klenow fragment of DNA polymerase I (New England BioLabs). DNA upstream of the strand break was synthesized through annealing and ligation of chemically synthesized oligonucleotides, and the full-length DNA fragment was released from the beads by cutting with an appropriate restriction endonuclease and purified by phenol-chloroform extraction and ethanol precipitation.

Nucleosome reconstitution.

The end-labeled probes were assembled with recombinant octamers (20) into nucleosomes by the rapid salt dilution method. Ten micrograms of recombinant octamer was mixed with 10 μg of sheared salmon sperm DNA (100 to 700 bp) and 1 pmol of end-labeled DNA in 2 M NaCl (total volume, 10 μl; 37°C), and the salt concentration was brought down to 300 mM by the stepwise addition of 6.8, 8.4, and 42 μl of 25 mM Tris-HCl (pH 7.5) at 10-min intervals.

ISW2 and ISW1a remodeling.

ISW2 was purified as described previously (29). Remodeling assays were conducted at 30°C for 30 min and contained 25 mM HEPES-KOH (pH 7.6), 5 mM MgCl₂, 40 to 50 mM KCl, 0.1 mg of bovine serum albumin/ml, 6 mM Tris-HCl (pH 8.0), 5% glycerol, 30 mM NaCl, 300 μM ATP, 370 ng of nucleosomes, and 30 to 75 ng of ISW2 or 60 to 150 ng of ISW1a. For remodeling under ISW2-saturated conditions, nucleosomes were reconstituted with 5 pmol of a 156-, 166-, or 179-bp end-positioning DNA probe with an end-labeled upper strand and coupled with a p-azidophenacyl bromide (APB; Fluka) and purified on a 5 to 25% sucrose gradient. The amount of ISW2 needed to saturate nucleosomes was determined by gel shift assay on a 4% native polyacrylamide gel in 0.5× Tris-borate-EDTA (45 mM Tris-borate and 1 mM EDTA). Remodeling reactions were analyzed by gel shift assay on a 5% native polyacrylamide gel (acrylamide/bisacrylamide ratio, 60/1) in 0.2× Tris-borate-EDTA at 10°C.

Histone-DNA cross-linking and mapping of H2B53 contacts on DNA.

H2B Ser53 was replaced with cysteine by site-directed mutagenesis and introduced into the recombinant octamer by refolding with H2A, H3, and H4 as described previously (20). The refolded octamer was reconstituted by the fast dilution method with 1 pmol of end-labeled DNA. The unique cysteine residue of H2B was coupled with APB. Two hundred micromolar APB was added to 10 μg of reconstituted nucleosomes and incubated for 3 h at room temperature in 1% dimethylformamide, 300 mM NaCl, 5% glycerol, and 10 mM Tris-HCl (pH 7.5). The excess APB was removed by dialysis against 100 mM NaCl, 5% glycerol, and 10 mM Tris-HCl (pH 7.5) or sucrose gradient centrifugation. Remodeling was conducted as described above, and cross-linking was performed by using an ultraviolet transilluminator (312 nm for 30 sec). Samples were denatured with 0.1% sodium dodecyl sulfate (SDS) and by heating for 20 min at 70°C. Histone-DNA conjugates were purified by phenol-chloroform (4/1) extraction. The organic phase was washed three times with 1% SDS and 1 M Tris-HCl (pH 8.0), precipitated with ethanol, and resuspended in 2% SDS, 20 mM NH₄CH₃COO, and 0.1 mM EDTA. The scission of DNA at the cross-linking site was triggered by incubation in 0.1 M NaOH for 45 min at 90°C. Cleaved samples were neutralized with 2 M HCl and ethanol precipitated. Samples were resuspended in formamide, resolved on a 6.5% polyacrylamide gel containing 8 M urea, and visualized by phosphorimaging. The amount of cleaved DNA present in each lane was normalized to the amount of total signal in the lane or to the amount of uncut DNA to facilitate the comparison of different lanes with similar samples before and after remodeling.

Exonuclease III mapping of nucleosome positions.

After nucleosome reconstitution and ISW2 remodeling as described above, the samples were supplemented with 0.03 or 0.1 U of exonuclease III and incubated for 10 min at 37°C. Reactions were stopped by the addition of formamide to a final concentration of 70%, resolved on a 6.5% polyacrylamide gel containing 8 M urea, and visualized by phosphorimaging.

Nucleosomes slid by ISW2 are not at their thermodynamically preferred positions.

The NPS referred to as 601 and discovered by J. Widom and colleagues has ∼150 times higher affinity for nucleosomes than sea urchin 5S ribosomal DNA (19). The 601 sequence directs nucleosomes into a single predominant translational position on DNA fragments as long as 288 bp and has been used in the present study to assemble nucleosomes on DNA fragments of variable lengths with the NPS located at the end or in the center of the DNA (Fig. 1). ISW2 can quantitatively slide nucleosomes towards the center of DNA when they initially have been positioned at the end of DNA with 67, 87, or 107 bp of extranucleosomal DNA (Fig. 1a; compare lanes 2, 4, and 6 to lanes 1, 3, and 5). ISW2 has a preference for sliding nucleosomes from the end to the center of DNA fragments, as shown by nucleosomes not being slid as efficiently from a central position on DNA as from an end (Fig. 1).

The positions to which nucleosomes were slid by ISW2 were not the thermodynamically preferred positions. After remodeling, ISW2 was stopped first by degradation of ATP with apyrase and then released from nucleosomes by competition with sonicated salmon sperm DNA (Fig. 2). Heating the nucleosomes at 50°C reversed the sliding of the nucleosome from the slid position to the starting and thermodynamically preferred position. ISW2 slides nucleosomes away from the thermodynamically preferred nucleosome position, unlike Drosophila NURF, which slides nucleosomes from the hsp70 promoter to a more thermodynamically preferred position (6).

ISW2 requires greater than 20 bp of extranucleosomal DNA for efficient nucleosome repositioning.

The minimal amount of extranucleosomal DNA required for nucleosome mobilization by ISW2 was determined by using nucleosomes assembled with DNA varying in length from 146 to 179 bp. All probes uniformly positioned the nucleosomes toward one end of the DNA and included 0 to 33 bp of extranucleosomal DNA. The extent of nucleosome sliding was determined by the photochemical mapping approach, so that even small changes in nucleosome position could be detected. The nucleosomes were constructed such that the cut site was located on the side of the dyad axis farthest in the DNA sequence from the linker DNA and was referred to as bp −55 with the dyad axis at bp 0. Nucleosomes slid 11, 12, or 20 bp towards the linker DNA when 33 bp of extranucleosomal DNA was present, as shown by the cut site moving from bp −55 to bp −35 and to bp −43 to −44, with as much as two-thirds of the nucleosomes being slid (Fig. 3G). No movement of the nucleosome was seen when no linker or 10 bp of linker DNA was present (Fig. 3A and B). Longer linker DNA initially promoted some movement of the nucleosome by ∼11 bp, as with 20 bp of linker DNA, but the majority (>95%) of the nucleosomes were still not changed (Fig. 3C). As the length of the linker DNA was increased to 23 and 26 bp, there was a gradual decrease at the original position of the nucleosome (∼70 to 80% remaining) that was associated with an increase in sliding by ∼11 bp at first and then by a total of 20 bp (Fig. 3D and E). A minimum of 30 bp of linker DNA was required for approximately two-thirds of the nucleosomes to slide from their original positions (Fig. 3F and G), whereas significant internal mobilization of the nucleosome was observed only with 26 bp of linker DNA (Fig. 3E).

It was possible that the inability of ISW2 to change the translational position of nucleosomes with less than 26 to 30 bp of extranucleosomal DNA might have been due to inefficient binding of ISW2 (14, 33). Nucleosome sliding was done with saturating amounts of ISW2 to determine if the differences in nucleosome mobilization with shorter linker DNA were due to differences in ISW2 binding to nucleosomes. Nucleosomes with shorter or no linker DNA had 2 or 4.4 times more ISW2 added to compensate for differences in affinity (Fig. 4A). Although there was some increase of the 10-bp movement within nucleosomes containing 20 bp of extranucleosomal DNA when using more ISW2, over three-fourths of the nucleosomes still remained in the original position (Fig. 4A). A slight but detectable amount of a 10-bp movement was observed with 10 bp of extranucleosomal DNA, although no loss in the amount of nucleosomes at the original position was detected. The inability of ISW2 to slide nucleosomes with 10 and 20 bp of extranucleosomal DNA was thus shown not to be caused by an inability to bind nucleosomes.

In order to determine if the properties of ISW2 are general for yeast ISWI complexes, the ability of ISW1a (29, 31) to slide nucleosomes was also examined. Extranucleosomal DNA of 20 bp or less was not sufficient to promote efficient displacement of nucleosomes from their original positions (Fig. 4B), the same result as with ISW2 (Fig. 3B). Similar to results with ISW2, ISW1a repositioned nucleosomes by 11, 12, and 20 bp increments when there was sufficient length of DNA (33 bp); although most of the nucleosomes remained at the original position at the intermediate length of 20 bp, some nucleosomes were moved by 11 to 12 bp. ISW1a was shown to have requirements similar to those of ISW2 for extranucleosomal DNA for nucleosome mobilization; the presence of extranucleosomal DNA is likely to be a general requirement for most or all of the ISWI-containing remodeling complexes.

ISW2 prefers not to slide nucleosomes to the very end of DNA fragment.

Although ISW2 preferentially slides nucleosomes towards the center of DNA fragments, a smaller percentage of nucleosomes are slid from the center towards the ends (Fig. 5a). Presumably because of the requirement for extranucleosomal DNA for nucleosome mobilization, nucleosomes were slid to within only 11 to 13 bp from DNA ends by ISW2 (Fig. 5b, lane 2) and ISW1a (lane 4). Earlier results had also shown that nucleosome repositioning on 183 bp DNA generated a single translational position with 15 and 16 bp of extranucleosomal DNA (15). All of these results demonstrate the mechanistic inability of ISW2 to slide nucleosomes to the very end of DNA.

It might be that ISW2 cannot slide nucleosomes over the last ∼10 bp of DNA because the DNA path in the remodeled nucleosome is such that this DNA region may already be associated with the nucleosome. Exonuclease footprinting of the edge of the nucleosome showed that as the nucleosome moved toward the DNA end, the terminal 13 bp of DNA remained accessible to exonuclease cleavage. Before remodeling, exonuclease digestion generates a single pause site 33 bp from the end of the DNA that corresponds well with the edge of the end-positioned nucleosome, as expected based on the previous site-directed mapping (Fig. 5C, lanes 2 and 3). ISW2 remodeling caused the appearance of two additional pause sites, located 11 and 20 bp from the original position (Fig. 5C, lanes 5 and 6). The ability of exonuclease to digest the DNA region in question verifies its expected accessibility and potential ability of ISW2 to bind to this region.

Strand-specific translocation of ISW2 along extranucleosomal DNA.

Single-stranded DNA was added in place of some of the extranucleosomal DNA to determine if only dsDNA would suffice for ISW2. A construct was made in which nine nucleotides of ssDNA was added onto 23 bp of extranucleosomal dsDNA to examine whether such extension would promote nucleosome mobilization by ISW2. A small amount of nucleosomes were slid 11 and 12 bp with 23 bp of extranucleosomal dsDNA, with 63% of the nucleosomes remaining in their original position, whereas the addition of nine nucleotides of ssDNA increased the efficiency and extent of nucleosome sliding (Fig. 6). The addition of nine nucleotides of ssDNA to the 5′ end enhanced movement away from the original position, with only 46% remaining and with a significant increase in the amount of nucleosomes moved 11, 12, and 20 bp. The addition of ssDNA to the 3′ end was even more stimulatory, with only 26% of the nucleosomes remaining in the original position and a larger number of nucleosomes moving 11, 12, and 20 bp away. The efficiency of nucleosome mobilization was comparable to that seen with 30 and 33 bp of extranucleosomal dsDNA (compare Fig. 6 with Fig. 3F and G). These results showed that the essential ISW2-extranucleosomal DNA interactions can be supported by ssDNA for nucleosome mobilization and demonstrate a modest strand preference for 3′ single-stranded extension. Recent results from others showed strand-specific translocation of ISWI by assaying for triplex displacement that was specifically blocked by placing 5- or 10-bp gaps in DNA in one strand and not the other (33). ISWI translocated along DNA away from the nucleosome in a 3′→5′ direction, as shown by gaps in the 3′→5′ strand preferentially interfering with triplex displacement by ISWI.

Generation of torsional strain in extranucleosomal DNA is not required for nucleosome mobilization.

Strand-specific translocation of the remodeling complex away from the nucleosome was proposed in order to create torsional strain at the entry site of the nucleosome, which might cause nucleosome movement either by twist propagation through the DNA or by displacing DNA from the entry site to create a bulge on the surface of the nucleosome that can move around the surface in a wave-like manner (24). The need for torsional strain in the extranucleosomal DNA for nucleosomes to move was tested in a manner similar to that of Whitehouse et al. (33), by placement of 10-bp gaps in the 3′→5′ strand at locations throughout the extranucleosomal DNA and just within the nucleosomal bound region (Fig. 7a). The gaps were placed in 10-bp increments starting from just within the nucleosome to 30 bp from the edge of the nucleosome. These nucleosome constructs are referred to as −10/0 to 30/40, with the numbers referring to the numbers of base pairs from the edge of the nucleosome; the first and second numbers are the start and end positions of the gap (Fig. 7a). A negative number indicates that the gap is within the nucleosomal bound region.

The sliding of nucleosomes positioned towards the far end of a 204-bp DNA strand was monitored by gel shift assays (Fig. 7b). Most of the nucleosomes without gaps were slid from the end to a central position on DNA within 5 min (compare lanes 17 and 18). Gaps either just inside the nucleosome or 20 bp or more from the edge did not interfere with the translocation of nucleosomes (Fig. 7b, lanes 2 to 4 and 11 to 16). These nucleosomes moved 30 and 40 bp from their original positions as determined by site-directed mapping (Fig. 7c and results not shown), with the gap being slid inside the nucleosome two to three helical turns away from the dyad axis to just within the nucleosome (Fig. 7a). Gaps in the extranucleosomal DNA closest to the nucleosome (0 to 20 bp) did affect nucleosome mobilization either by slowing down (Fig. 7b, compare lanes 9 and 10) or by blocking their movement (Fig. 7b, lanes 5 to 7). The DNA gap immediately adjacent to the edge of the nucleosome showed the most severe effects on nucleosome sliding.

High-resolution mapping of the position of the 0/10- and 10/20-bp gapped nucleosomes showed that there was indeed a significant amount of the original nucleosome position present after remodeling when the gap was immediately adjacent to the edge of the nucleosome (Fig. 7c). Whereas 75% of the nucleosomes were slid from their original position when there was no gap, only 26 and 45% of nucleosomes were slid when the gap was at bp 0/10 and 10/20, respectively. The overall change in nucleosome position mapped in this manner was consistent with that observed by gel shift assay. However, the lack of movement from the original nucleosome position did not appear to be due to an inability to mobilize nucleosomes, as a significant amount of nucleosomes were shifted by 30 and 40 bp. These data suggest that ISW2, with the placement of these gaps, was unable to stably push the nucleosome in one direction and instead shuttles nucleosomes rapidly back and forth between the original and slid positions. Potentially, the effects of DNA gaps on nucleosome mobilization may be caused by effects on ISW2 binding to nucleosomes. ISW2 was found to bind generally less well to gapped nucleosomes than to normal nucleosomes and, in particular, the 0/10 gap in the extranucleosomal DNA interfered the most with ISW2 binding (by factors of as much as approximately four to six) (Fig. 8A). The reduced affinity of ISW2 for the gapped nucleosomes was, however, readily compensated for at higher ISW2 concentrations. Since the previous nucleosome mobilization assays were done at low, substoichiometric amounts of ISW2, the reduced sliding could have been due to reduced binding of ISW2. The nucleosome mobilization experiments were repeated with saturating amounts of ISW2 to determine if the inability to slide persisted even when reduced binding of ISW2 was compensated for. After remodeling, ISW2 was stripped off the nucleosomes by using excess competitor DNA and nucleosome movement was determined by gel shift assay. As before, when the gap was located at bp 0/10, nucleosome mobilization was severely affected (Fig. 8B; compare lane 4 to lanes 2, 3, and 5 to 8). The gap at bp 10/20 did not show much of an effect, likely because the previously observed lower rate of sliding was compensated for due to the saturating amounts of ISW2 and/or the time of incubation. These results show that defects in nucleosome mobilization with nucleosomes containing gaps at bp 0/10 are not due to an overall reduced binding of ISW2 to nucleosomes.