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Domain architecture of the catalytic subunit in the ISW2-nucleosome complex - PubMed

Domain architecture of the catalytic subunit in the ISW2-nucleosome complex

Weiwei Dang et al. Mol Cell Biol. 2007 Dec.

Abstract

ATP-dependent chromatin remodeling has an important role in the regulation of cellular differentiation and development. For the first time, a topological view of one of these complexes has been revealed, by mapping the interactions of the catalytic subunit Isw2 with nucleosomal and extranucleosomal DNA in the complex with all four subunits of ISW2 bound to nucleosomes. Different domains of Isw2 were shown to interact with the nucleosome near the dyad axis, another near the entry site of the nucleosome, and another with extranucleosomal DNA. The conserved DEXD or ATPase domain was found to contact the superhelical location 2 (SHL2) of the nucleosome, providing a direct physical connection of ATP hydrolysis with this region of nucleosomes. The C terminus of Isw2, comprising the SLIDE (SANT-like domain) and HAND domains, was found to be associated with extranucleosomal DNA and the entry site of nucleosomes. It is thus proposed that the C-terminal domains of Isw2 are involved in anchoring the complex to nucleosomes through their interactions with linker DNA and that they facilitate the movement of DNA along the surface of nucleosomes.

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Figures

FIG. 1.
FIG. 1.

Site-specific cross-linking of DNA to Isw2. In the left panel, the structure of the photoreactive nucleotide AB-dUMP is shown as incorporated into DNA. The distance from the cross-linking site (N3) to the C-5 position of deoxyuridine is 9 to 10 Å, as indicated. The location of the 32P radiolabel is boxed in the structure to illustrate its general position. In the right panel, a model of the nucleosome is shown with the sites that are targeted for modification and photo-cross-linking to Isw2 highlighted in black; the numbering refers to the number of base pairs from the dyad axis.

FIG. 2.
FIG. 2.

Mapping the regions of Isw2 cross-linked to three sites in the nucleosome by NTCB digestion. (A) A schematic diagram of Isw2 is shown, with residue numbers for the different cysteine cut sites indicated. The locations of the conserved Isw2 domains are indicated, along with the size of each proteolytic fragment (kDa). The set of fragments obtained with single-hit NTCB digestion are shown below the Isw2 schematic, with two fragments created from each single hit depicted as either a shaded or open box. The predicted sets of radiolabeled single-hit proteolytic fragments that can be obtained depending on the site of Isw2 that is cross-linked to DNA are shown to the right. (B) Photoaffinity-labeled Isw2 was digested with the indicated concentrations of NTCB and incubation times, followed by analysis by 4 to 20% Tris-glycine SDS-PAGE. The apparent molecular masses of the radiolabeled bands were calculated as described in Materials and Methods and are shown in kDa. (C) Fragments of Isw2 were synthesized in vitro that correspond to fragments that would be obtained by NTCB digestion (Table 1) and contain either the N terminus (NN1, NN2, NN3, and NN4), the C terminus (NC1, NC2, NC3, and NC4), or an internal fragment (NI2). These truncated proteins were analyzed by 4 to 20% Tris-glycine SDS-PAGE. The molecular masses of the 125I-labeled Mark12 protein standards are indicated to the right. The slowest-migrating major bands were fully translated products and were used in calculating the apparent molecular masses.

FIG. 3.
FIG. 3.

The DEXD, HAND, and SLIDE domains were cross-linked to nucleosomal and extranucleosomal DNA. (A) The methionine sites (numbers below the schematic) and predicted molecular masses (numbers above the schematic) of the proteolytic fragments are shown for Isw2. The set of fragments obtained with single-hit CNBr digestion are shown below the Isw2 schematic on the left, with two fragments created from each single hit depicted as either a shaded or an open box. Below on the right are shown the predicted radiolabeled fragments as resolved by SDS-PAGE and obtained from cross-linking in three different regions of Isw2, labeled D, J, and M. (B) Photoaffinity-labeled Isw2 was digested with CNBr at the indicated concentrations (mM) and incubation times, followed by analysis by 10% BIS-Tris SDS-PAGE. The apparent molecular masses, in kDa, of the released bands were calculated as described in Materials and Methods. The molecular masses for the smallest bands released by limited and extensive CNBr digestion are indicated by arrowheads. (C) Peptides corresponding to fragments of Isw2 that could be expected from complete CNBr digestion (Table 1) were synthesized and were analyzed by 10% BIS-Tris SDS-PAGE. Arrowheads highlight the markers that show migrations similar to those of the bands observed in CNBr peptide-mapping experiments. The molecular masses in kDa of 125I-labeled Mark12 protein standards (Std) are indicated.

FIG. 4.
FIG. 4.

Model of Isw2 docking to the nucleosome. (A) A summary of the mapping results for regions of Isw2 cross-linked to the nucleosomal and extranucleosomal DNA. Isw2 is schematically shown as a gray bar with conserved domains boxed in various colors. The fragments cross-linked to indicated DNA sites (17 to 18, 60 to 62, and 92 bp from the dyad axis of the nucleosome) are shown as color-coded narrow bars above the Isw2 schematic and are denoted by mapping agent, NTCB or CNBr. Beginning and ending amino acid positions for each domain and mapped fragment are given. (B) Structural model of Isw2 interacting with the nucleosomal core particle. The regions of Isw2 that interact with the SHL2 site, the entry/exit site, and extranucleosomal DNA are colored green, brown, and cyan, respectively, as in panel A and are modeled based on homology and peptide mapping data. The domains of Isw2 are labeled, and their proposed functional roles in chromatin remodeling by ISW2 are given.

FIG. 5.
FIG. 5.

Model for the binding of the ATPase domain of Isw2 to nucleosomal DNA. (A) The locations of the α/β helicase lobes (Lobe 1 and Lobe 2) in the primary amino acid structure of the Isw2 ATPase domain, based on sequence alignment with Sulfolobus Rad54, are shown, along with the relationship to the region of Isw2 cross-linked to DNA 17 and 18 bp from the dyad axis. (B) The Isw2 ATPase domain model was generated as described in Materials and Methods, based on homology modeling and the structure of Sulfolobus Rad54 bound to DNA. The first helicase lobe is depicted in blue and the second in red, with the region between the lobes shown in gray. (C) The region of Isw2 cross-linked to the SHL2 site is highlighted in green in the same model that is shown in panel B. The amino acids contacting DNA as suggested by the structure of Rad54 bound to DNA are highlighted in blue and displayed in a space fill format. A putative acidic patch is highlighted in red with a space fill format on the surface of the first α/β-helicase lobe. (D) An acidic patch on the surface of Isw2 is moderately conserved in the ISWI subfamily. A sequence alignment of SNF2 family ATPases is shown. Helicase- and SNF2-specific motifs are boxed and labeled as indicated. The fragment identified as cross-linking DNA at SHL2 is boxed in green.

FIG. 6.
FIG. 6.

Model for the C terminus of Isw2 and its interaction with the nucleosome. (A) The positions of the HAND, SANT, and SLIDE domains in Isw2 are shown and correlated with the regions of Isw2 that were cross-linked to nucleosomal and extranucleosomal DNA 60 to 62 and 92 bp from the dyad axis. (B) A model of a portion of the C terminus of Isw2 is shown, with the HAND domain in purple, the SANT domain in light green, the SLIDE domain in yellow, and the spacer helix in gray. (C) A DNA binding path is suggested by the surface charge distribution of the Isw2 C-terminal domain model and the regions found to be cross-linked to DNA. The same model that is shown in panel B is depicted with the regions cross-linked to the entry/exit site in brown and extranucleosomal DNA in cyan (left panel). The distribution of the surface charges (positive in blue, negative in red) of the Isw2 C-terminal domain model suggests a putative path of DNA as indicated by the dashed line (right panel). (D) A sequence alignment of the C-terminal domains of ISWI subfamily ATPases illustrates the conserved, positively charged residues (highlighted by blue dots) that are likely to interact with DNA. Conserved domains are indicated by colored bars below the sequence alignment. The regions that cross-link to the entry/exit site of the nucleosome (positions 60 to 62) and the extranucleosomal DNA (position 92) are boxed in brown and cyan, respectively.

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