Domain Architecture of the Catalytic Subunit in the ISW2-Nucleosome Complex^▿

Isolation of Isw2 subunits cross-linked to DNA for peptide mapping.

Synthesis of radiolabeled DNA photoaffinity probes with cross-linkers 17 to 18, 60 to 62, and 92 bp from the dyad axis and photoaffinity-labeling reactions with FLAG-purified yeast ISW2 complex were performed as described previously (15). Cross-linked Isw2 was isolated by 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), visualized by phosphorimaging, excised, electroeluted with a buffer containing 50 mM NH₄HCO₃ and 10% SDS, dried, and stored at −20°C.

Peptide mapping with NTCB digestion.

Photoaffinity-labeled Isw2 was resuspended in a freshly prepared denaturing buffer containing 8 M urea, 10 mM 2-mercaptoethanol, and 0.2 M Tris-acetate (pH 8.5) and incubated at 37°C for 2 h prior to the addition of 12.5 to 25 mM NTCB (2-nitro-5-thiocyanobenzoic acid in ethanol) and incubation for 15 min. The pH was adjusted to approximately 9.0 to 9.5 with NaOH, followed by incubation at 37°C for 1 to 16 h. The reaction was stopped by the addition of SDS sample buffer containing 250 mM Tris-HCl (pH 8.8), 10% glycerol, 2% SDS, 1 mM EDTA, 0.5 M 2-mercaptoethanol, and 0.088% Coomassie blue G250 and analyzed by 4 to 20% or 8 to 20% Tris-glycine SDS-PAGE and phosphorimaging. The apparent molecular masses of the released fragments were estimated by comparing them to the ¹²⁵I-labeled Mark12 protein standard and Isw2-NTCB markers prepared by in vitro transcription/translation.

Peptide mapping with CNBr digestion.

Photoaffinity-labeled Isw2 was resuspended in 70% formic acid, mixed with an equal volume of freshly prepared CNBr solution (125 to 500 mM cyanogen bromide in 70% formic acid), and incubated at 25°C in the dark for 5 min to 2 h. The cleavage reaction was stopped by diluting samples fivefold with water and drying by vacuum centrifugation. The dried CNBr cleavage samples were resuspended in SDS sample buffer and analyzed by 10% bis(2-hydroxyethyl)imino (BIS)-Tris SDS-PAGE (13) and phosphorimaging. The apparent molecular masses of the released fragments were estimated by comparing them to the ¹²⁵I-labeled Mark12 protein standard and Isw2-CNBr markers were prepared by in vitro transcription/translation.

Preparation and analysis of peptide-mapping markers.

Salt and detergent were removed from 50 μl (∼44 μg) of Mark12 unstained protein standard (Invitrogen) by using a Sephadex G-10 (GE Healthcare) spin column equilibrated in 200 mM sodium borate (pH 9.0). The protein standard was incubated on ice for 3 h with 0.25 mg/ml sulfo-SHPP [sulfosuccinimidyl-3-(4-hydroxyphenyl)propionate; Pierce]. Excess reagents were removed by using Sephadex G-10 spin columns equilibrated with phosphate-buffered saline (100 mM sodium phosphate, 150 mM NaCl, pH 7.2). The derivatized protein standard was transferred to an Iodo-Gen (1,3,4,6-tetrachloro-3a-6a-diphenylglycouril; Pierce)-coated tube containing 100 μCi of Na¹²⁵I (Perkin Elmer) and incubated at 25°C for 15 min. The reaction was terminated by removing the sample from the tube and adding NaI to 1 mM. Free Na¹²⁵I was removed by using Sephadex G-10 spin columns, and the radiolabeled Mark12 protein standard was mixed with SDS sample buffer (65 mM Tris-HCl [pH 6.8], 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, 0.09% bromophenol blue) and stored at −20°C.

The Isw2 fragments corresponding to both the single-hit and the complete cleavage peptides digested by using NTCB or CNBr were prepared in vitro by PCR amplification from the plasmid pET-ISW2-FLAG encoding ISW2 with a C-terminal double FLAG tag and by using a TNT quick coupled transcription/translation system (Promega) with [³⁵S]methionine (Perkin Elmer). Transcription/translation reactions were carried out at 30°C for 90 min, followed by the addition of 20 μg/ml leupeptin, and the reaction products were stored at −80°C.

The synthesized Isw2 markers were analyzed by 8 to 20% Tris-glycine SDS-PAGE and/or 4 to 12% or 10% BIS-Tris SDS-PAGE and phosphorimaging (see Fig. 3 and Table 1). The apparent molecular masses were calculated by comparing the electrophoretic mobilities of the markers to those of the ¹²⁵I-labeled Mark12 protein standard. Table 1 lists all Isw2 peptide-mapping markers with their calculated and apparent molecular masses on Tris-glycine SDS-PAGE or BIS-Tris SDS-PAGE.

Structure modeling of Isw2 ATPase and C-terminal domains.

The modeling of the Isw2 ATPase and C-terminal domains was performed with Accelrys DS modeling 1.1 with the MODELER module based on a comparative homology modeling algorithm (24). The structure of Sulfolobus solfataricus Rad54 ATPase domain in complex with 25-bp double-stranded DNA (PDB ID 1Z63) (8) and the Drosophila ISWI-C domain structure (PDB ID 1OFC) (14) were used as structural templates for the modeling of Isw2 ATPase in complex with DNA and the Isw2 C-terminal domains, respectively. The sequence alignments of Isw2 ATPase with the Sulfolobus Rad54 ATPase structure and Isw2-C with the Drosophila ISWI-C domain structure were performed with the “Align2D” command with default settings. For each structure, 200 models were built with the “refined” setting. All of the nucleotide residues in DNA strands “C” and “D” in the 1Z63 structure were selected as the ligand in building the structures for the Isw2 ATPase structure in complex with DNA. The 20 models with the lowest PDF (probability density functions) values were exported as Protein Data Bank files, and the “combined energy” values were compared by using ProSa2003 (http://www.came.sbg.ac.at/came-frames/prosa.html) (27). The structural model with the lowest “combined energy” was selected as the final model for Isw2 ATPase in complex with DNA. Since yeast Isw2-C contains two regions that are not aligned with Drosophila ISWI-C, the Isw2-C model with the lowest “combined energy” was subject to loop refinement. A total of 100 loop models were built using the “Refine Loop” command with the “refined” setting. The 10 loop models with the lowest PDF values were exported, and the model with the lowest “combined energy” was selected as the final model for Isw2-C.

Strategy for identification of Isw2 domains associated with nucleosomal and extranucleosomal DNA regions.

Previously, DNA footprinting and photochemical cross-linking had shown a tripartite architecture for Isw2. Isw2 was shown to be proximal to DNA (i) two helical turns from the dyad, (ii) near the entry/exit site, and (iii) up to 20 bp from the edge of nucleosomes with extranucleosomal DNA (15). Itc1 also contacts the same regions as Isw2, except that it binds a larger stretch of the extranucleosomal DNA from the entry/exit site, to at least 53 bp from the edge of the nucleosome. In this study, the same photo-cross-linking technique was used, in combination with peptide mapping by chemical proteolysis, in order to identify the domains of Isw2 that interact with these regions of the nucleosome. Photoreactive DNA was used to cross-link Isw2 17 to 18, 60 to 62, and 92 bp from the dyad axis (Fig. 1), corresponding to the three distinct regions protected by ISW2 binding (15). DNA was made photoreactive by the targeted incorporation of nucleotides conjugated through a short tether to a photoreactive aryl azide, as shown in Fig. 1 (18, 32). Adjacent to the photoreactive nucleotide, a ³²P-labeled nucleotide in DNA was incorporated such that, after cross-linking and DNA digestion, the radiolabel was transferred to the cross-linked protein (15). The region of Isw2 cross-linked to DNA was mapped by chemical proteolysis of the cross-linked Isw2, separation of the peptide fragments by gel electrophoresis, and visualization of the labeled peptide fragment(s) by phosphorimaging. The cleavage conditions were adjusted to examine both single-hit cutting (i.e., one cleavage per polypeptide) and extensive digestion of the labeled protein in order to determine the location of the radiolabel (i.e., the cross-linking site) based on the electrophoretic mobilities of the radiolabeled fragments. The single-hit conditions created two sets of proteolytic fragments, containing either the N or C terminus of the protein. Appropriate size markers of the different truncated forms of Isw2 containing the N or C terminus were generated by using an in vitro coupled transcription/translation system and labeled [³⁵S]methionine for comparison with the photoaffinity-labeled peptide fragments (Table 1 and Fig. 2C and 3C). Particular internal fragments were also synthesized in this way for comparison with cross-linked products that were obtained by exhaustive proteolysis.

Figure 2A shows a schematic diagram of the NTCB cleavage sites at cysteines in Isw2 and the fragment sizes of labeled peptides predicted for single-hit NTCB digestion of Isw2 (12). There are six cysteines in Isw2, with two pairs of cysteines either immediately adjacent to each other (Cys 463 and Cys 464 or separated by 7 amino acids (Cys 526 and Cys 534), such that NTCB cleavage creates only one of five distinct sets of radiolabeled peptide fragments, depending on the location of the radiolabeled tag. This particular type of proteolysis is done first in order to simplify the interpretation of the proteolysis because of the reduced number of cleavage sites. These results then provide an initial view of the location of the cross-link, which can be refined by using more extensive proteolysis conditions.

The DEXD domain of Isw2 was bound to DNA at 17 and 18 bp from the dyad axis.

NTCB digestion of Isw2 cross-linked to DNA 17 and 18 bp from the dyad axis generated primarily 113-, 105-, 62-, and 54-kDa radiolabeled peptides (Fig. 2B, lanes 2 to 3), consistent with cross-linking to region I (residues 1 to 463). Lesser amounts of the labeled 70- and 79-kDa proteolytic fragments were detected, consistent with minor cross-linking to regions II and III. The major cross-linking site was to region I, as shown by extensive NTCB digestion, primarily producing a 54-kDa radiolabeled proteolytic fragment, with very little of a detectable 7.8-kDa fragment (Fig. 2B, lane 5). The relative mobilities of these proteolytic fragments were confirmed by comparison with truncated versions of Isw2 that were synthesized by coupled transcription/translation production (Table 1 and Fig. 2C). The region of Isw2 cross-linked near the dyad axis includes a region having homology with the DEXD domain of the conserved ATPase domain and an N-terminal flanking region. Delineating whether the N terminus or the DEXD domain was associated with DNA was done by CNBr digestion of cross-linked Isw2 (12). There are 13 methionine, or CNBr cleavage, sites in Isw2 (Fig. 3A). Extensive CNBr digestion produced a labeled proteolytic fragment with an electrophoretic mobility corresponding to a 15.1-kDa fragment spanning residues 294 to 421 (Fig. 3A, region D, and B, lane 4). This peptide was synthesized to determine the electrophoretic mobility of this peptide region (Fig. 3C, lane 4, and Table 1). The electrophoretic mobility of this fragment was measured and compared to the predicted mobility and the mobilities of the nearby peptide fragments that were synthesized in vitro with a coupled transcription/translation system with [³⁵S]methionine. As can be seen in Table 1, there are no other proteolytic fragments generated by cleavage with CNBr from the N-terminal two-thirds of Isw2 that have an electrophoretic mobility comparable to that of this region. Consistent with this conclusion, the limited digestion generated peptide fragments with the smallest fragment corresponding to the 48.9-kDa peptide, or residues 1 to 421 of Isw2 (Fig. 3A, lane D, and B, lane 2). These results showed that the DEXD domain, and not its N-terminal flanking region, is close to SHL2 inside the nucleosome (Fig. 4A). Isw2, as part of the four-subunit ISW2 complex, has its DEXD domain close to the internal contact site at SHL2. Translocation along DNA from this same DNA site has recently been found to be essential for ISW2 remodeling, consistent with the DEXD domain of the conserved ATPase region contacting this region (34).

The HAND domain of Isw2 was bound near the entry/exit of the nucleosome.

Isw2 cross-linked 60 to 62 bp from the dyad axis was digested with NTCB and generated primarily 113-, 105-, 79-, and 70-kDa radiolabeled proteolytic fragments that corresponded to cross-linking to region III—residues 534 to 903 (Fig. 2B, lane 7). More extensive digestion with NTCB enriched for a 42-kDa peptide, with no significant amounts of smaller radiolabeled peptides, consistent with cross-linking to region III (Fig. 2B, lane 10). The 42-kDa peptide has an electrophoretic mobility that is close to that of the polypeptide from residue 1 to 464 created by extensive NTCB digestion, but under our gel conditions, they are separated from each other (Table 1 and data not shown). Further confirmation of cross-linking at region III was also obtained by CNBr digestion (see below). Region III has sequence homology to a portion of the C terminus of ISWI from Drosophila and corresponds to the HAND domain. Region III also contains a part of the conserved ATPase domain observed in other Snf2 subfamily members and corresponds to the HELIC domain. Further mapping of the region of Isw2 cross-linked to DNA 60 and 62 bp from the dyad axis was done by digestion of photoaffinity-labeled Isw2 with CNBr. The smallest fragment from extensive CNBr digestion of Isw2 cross-linked to DNA had an electrophoretic mobility corresponding to that of the 26.7-kDa fragment of Isw2 from residues 669 to 897 (Fig. 3A, region J, and B, lane 8) and correlates to the region sharing homology with the HAND domain. These results demonstrated more clearly that the HAND domain of Isw2 was cross-linked to DNA near the entry/exit site and not the HELIC domain. The 26.7-kDa CNBr proteolytic fragment is distinctive because it is the largest CNBr fragment that can be obtained under exhaustive digestion conditions (Fig. 3A and C, lane 9, and Table 1). Limited CNBr digestion was consistent with this observation as the smallest radiolabeled proteolytic fragment observed, of 55.2 kDa, corresponds to a single cut at residue 668 and the radiolabel residing on the C-terminal half of this cut site (Fig. 3A and B, lane 6).

The SLIDE domain of Isw2 was associated with extranucleosomal DNA.

The peptide fragments generated by limited NTCB digestion of Isw2 cross-linked 92 bp from the dyad axis were 113, 79, 70, 28, and 19 kDa and corresponded to cross-linking to region V, residues 974 to 1120 (Fig. 2B, lane 12, and C, lane 8). Under more-exhaustive NTCB digestion conditions, less than 10% of the cross-linking was found to be at region IV (residues 903 to 973), consistent with the amount of the labeled 8.6-kDa peptide compared to the amount of 19-kDa peptide detected in lane 14. Further refinement of the region of Isw2 cross-linked to extranucleosomal DNA was done by CNBr cleavage. After extensive digestion with CNBr, Isw2 cross-linked to DNA 92 bp from the dyad axis produced a radiolabeled 8.5-kDa peptide corresponding to residues 1025 to 1099 (Fig. 3B, lanes 11 to 12, and C, lane 11), consistent with the 13.1-kDa fragment (residues 1027 to 1138) of Isw2 obtained under limited digestion conditions (Fig. 3A, region M, and B, lane 10). Peptide mapping of cross-linked Isw2 with cyanogen bromide narrowed down the region of Isw2 cross-linked to extranucleosomal DNA from residues 974 to 1138 to residues 1025 to 1099, corresponding to a region that has sequence homology with the SLIDE domain observed in the C terminus of ISWI from Drosophila. These data show that the SLIDE domain of Isw2 is directly associated with extranucleosomal DNA 92 bp from the dyad axis or 19 bp from the edge of the nucleosome.

Structural model for Isw2-nucleosome interaction.

A complete map of the conserved domains of ISW2 that were cross-linked to the three nucleosomal and extranucleosomal DNA sites is summarized in Fig. 4A. The regions of Isw2 cross-linked to nucleosomal and extranucleosomal DNA correspond to conserved regions in which crystal structure information is available from homologous proteins. In order to better visualize the domain interactions between Isw2 and the nucleosome, a structural model of Isw2-nucleosome interactions was made based on the mapping data. This overall model, for the first time, demonstrates structurally how the catalytic subunit of an ISWI chromatin remodeling complex interacts with its nucleosomal and extranucleosomal substrate and likely catalyzes the remodeling reaction as part of the ISW2 holoenzyme activity (Fig. 4B).

The DEXD domain is the most-conserved region in the ATPase domain of Isw2 compared to other chromatin-remodeling ATPase domains and, based on sequence homology, is located in the first lobe of the ATPase domain (8, 29). The ATP-binding site is also believed to be located within this conserved region (30). The stretch of nucleosomal DNA about 20 bp from the dyad axis was shown by two independent laboratories to be a critical point for chromatin remodelers to translocate along DNA and to generate localized torsional strains (23, 34). Our mapping results, for the first time, have demonstrated the physical interaction between the ATPase domain and this critical point on nucleosomal DNA and suggest that chromatin-remodeling events indeed are powered by the direct contact of the ATPase domain inside the nucleosome.

A model of the binding of the ATPase domain of Isw2 to nucleosomal DNA was constructed by modeling the structure based on homology to the Rad54 ATPase domain structure from Sulfolobus. The ATPase domain obtained by protein modeling was docked onto the nucleosome structure at the region found to be cross-linked with nucleosomal DNA and oriented to the nucleosomal DNA similarly to that observed in the cocrystal of the Rad54 ATPase domain with DNA. In this model, it can be clearly seen that the DEXD domain makes more-extensive contact with DNA, consistent with it being the most likely to be cross-linked to nucleosomal DNA (Fig. 5B and C, regions highlighted in green and blue).

Another outcome from this model is the observation of a moderately conserved acidic patch on the surface of the DEXD domain that would be in the correct location to make contact with the basic N-terminal tail region of histone H4 (Fig. 5C and D). Isw2 contact at SHL2 requires the presence of the basic tail region of histone H4 (6). The histone H4 tail is also required for efficient remodeling by ISW2 and other ISWI complexes (2, 3, 6, 10). These data suggest that the acidic patch may interact with the H4 tail and facilitate its recruitment of the DEXD domain of Isw2 to nucleosomal DNA at SHL2. The acidic patch, comprising residues Asp361, Asp365, Glu367, Glu374, and Glu383, is conserved in other ISWI homologs. The acidic patch seems to have a unique role in chromatin remodeling by the ISWI-type complexes as it is not found in other members of the SF2 family, such as Brg1, Swi2/Snf2, and Rad54.

Models of the C terminus of Isw2 docking to the entry site of the nucleosome and linker DNA were constructed based on sequence homology with the C terminus of ISWI from Drosophila and its known structure. Although previously the structure of the C terminus of ISWI from Drosophila was postulated to bind and bridge across two gyres of the nucleosomes, our cross-linking data were not consistent with this model (14). This elongated structure does, however, quite well accommodate having the HAND/SANT domain combination bound at the entry site, while the SLIDE domain, connected by a long, conserved, alpha-helical spacer domain, is bound to linker DNA 19 bp from the edge of the nucleosome (Fig. 4B and 6). The 30-bp distance between the nucleosome entry/exit site bound by the HAND domain and the part of extranucleosomal DNA bound by the SLIDE domain is remarkably consistent with the 100-angstrom distance observed between the SLIDE and HAND/SANT domains (14). The model of the C terminus of Isw2 docked to the nucleosome and linker DNA was such that both regions shown to be cross-linked to DNA could be simultaneously bound to DNA. The path likely traversed by DNA across the surface of the C terminus of Isw2 is strongly suggested by the relative locations of the two regions in the C terminus that are cross-linked to DNA (Fig. 6C, indicated by a dotted line). Docking was done such that the long alpha helix of the SLIDE domain was bound to the major groove of DNA at the appropriate distance from the edge of the nucleosome. Aside from the cross-linking data suggesting that this part of the SLIDE domain is in contact with linker DNA, other data indicate that the alpha helix may be involved in DNA binding and that the basic amino acids conserved in this helix are required (14). A striking feature of the path of DNA as suggested by cross-linking is the highly conserved basic amino acids that are found on this surface (Fig. 6C, shown in blue). These basic amino acids are well conserved throughout the ISWI family of chromatin-remodeling complexes, span the entire length of the postulated DNA path, and provide supporting evidence for the model of the interaction of the C terminus of Isw2 with the nucleosome and linker DNA (Fig. 6D).