Interactions of Isw2 chromatin remodeling complex with nucleosomal arrays: analyses using recombinant yeast histones and immobilized templates - PubMed (original) (raw)

Interactions of Isw2 chromatin remodeling complex with nucleosomal arrays: analyses using recombinant yeast histones and immobilized templates

M E Gelbart et al. Mol Cell Biol. 2001 Mar.

Abstract

To facilitate the biochemical characterization of chromatin-associated proteins in the budding yeast Saccharomyces cerevisiae, we have developed a system to assemble nucleosomal arrays on immobilized templates using recombinant yeast core histones. This system enabled us to analyze the interaction of Isw2 ATP-dependent chromatin remodeling complex with nucleosomal arrays. We found that Isw2 complex interacts efficiently with both naked DNA and nucleosomal arrays in an ATP-independent manner, suggesting that ATP is required at steps subsequent to this physical interaction. We identified the second subunit of Isw2 complex, encoded by open reading frame YGL 133w (herein named ITC1), and found that both subunits of the complex, Isw2p and Itc1p, are essential for efficient interaction with DNA and nucleosomal arrays. Both subunits are also required for nucleosome-stimulated ATPase activity and chromatin remodeling activity of the complex. Finally, we found that ITC1 is essential for function of Isw2 complex in vivo, since isw2 and itc1 deletion mutants exhibit virtually identical phenotypes. These results demonstrate the utility of our in vitro system in studying interactions between chromatin-associated proteins and nucleosomal arrays.

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Figures

FIG. 1

Reconstitution of recombinant yeast histone octamer. (A) Purified recombinant yeast core histones and histone octamer. Yeast core histones (H2A, H2B, H3, and H4) were expressed and purified individually from E. coli (lanes 4 to 7). Reconstituted recombinant yeast histone octamer (lane 3) is shown next to native Drosophila histone octamer (lane 2) for comparison. Proteins were separated by SDS-PAGE (15% gel) and stained with Coomassie brilliant blue R250. Lane M, size markers; ∗, minor contaminant present after purification of individual core histones. (B) Isw1 and Isw2 complexes facilitate the formation of regularly spaced nucleosomes assembled with recombinant yeast histone octamer. Nap1p-mediated nucleosome spacing assays using Drosophila (left) or yeast (right) histone octamer were performed on lambda DNA in the presence or absence of Isw1 and Isw2 complexes. All reactions contained ATP. Nucleosome spacing was analyzed by partial and extended MNase digestion. DNA was purified and separated by 1.3% agarose gel electrophoresis followed by ethidium bromide staining.

FIG. 2

Assay for protein-nucleosomal array interactions. (A) Schematic representation of the assay. Nucleosomes assembled on immobilized templates with Nap1p are washed with high salt to remove free histones and Nap1p. Nucleosomal arrays are then incubated with Isw2 complex to analyze their interactions. (B) Quantitative loading of recombinant yeast histone octamer onto immobilized templates. Increasing amounts of recombinant yeast histone octamer (0, 0.5, 1.0, 1.5, 2.0, and 3.0 μg) and Nap1p (9 μg) were mixed with a fixed amount of immobilized template (1.5 μg of DNA). Proteins were eluted from immobilized templates, assembled with no (lane 3) or increasing amounts of (lanes 4 to 7) histone octamer. Lane 2 shows elution from the beads alone. Proteins were separated by SDS-PAGE (15% gel) and silver stained. Histone H4 commonly does not stain as strongly as H2A, H2B, and H3. Lane M, size markers.

FIG. 3

Isw2 complex interacts with DNA and nucleosomal arrays in an ATP-independent manner. (A) Interaction of Isw2 complex with immobilized templates. The interaction assay was performed by incubating Isw2 complex with beads alone, immobilized DNA, or immobilized nucleosomal arrays (nucl) in the presence or absence of ATP at 95 or 135 mM salt. The supernatant was collected, beads were washed, and bound proteins were eluted from the beads in SDS-PAGE sample buffer. Equivalent amounts of unbound (U) and bound (B) fractions were separated by SDS-PAGE, and Isw2p was detected by Western blotting. “Input” indicates the amount of the fractions used in each reaction. (B) Quantitative interaction of Isw2 complex with templates. Threefold serial dilutions of Isw2 complex were used in the interaction assay in the presence and absence of ATP. x1/3, x1, and x3 correspond to 15, 45, and 135 fmol of Isw2 complex. For Western blotting, the samples from x1 and x3 reactions were diluted three- and ninefold, respectively. The standard reaction uses 45 fmol of Isw2 complex. In this experiment, Isw2 complex with FLAG-tagged Isw2p and Itc1p was used. (C) Interactions of wild-type and catalytically inactive (Isw2-K214R) Isw2 complexes with immobilized templates. The experiment was performed under standard conditions.

FIG. 4

Both Isw2p and Itc1p of Isw2 complex are required for efficient interactions. (A) Purified Isw2 subunits used in analyses. Lanes 2 and 5 show a silver stain of wild-type (WT) Isw2 complex, purified from strains in which only Isw2p and both subunits, respectively, were FLAG tagged. Lane 3 shows a silver stain of the complex from an itc1 deletion background in which Isw2p was FLAG tagged. Lane 4 shows protein purified from an isw2 deletion background in which Itc1p was FLAG tagged. Lane M, size markers; ∗, minor contaminant present in some but not all preparations. (B) Interaction assay using native Isw2 complex, monomeric Isw2p, and monomeric Itc1p. Monomeric Isw2p and Itc1p were purified from itc1 and isw2 deletion mutants, respectively. Unbound (U) and bound (B) proteins were separated by SDS-PAGE and detected by Western blotting. (C) Interaction of reconstituted Isw2 complex with immobilized nucleosomal arrays. Unbound (U) and bound (B) proteins were separated by SDS-PAGE and detected by Western blotting. “Isw2p + Itc1p” denotes Isw2 complex reconstituted in vitro by mixing monomeric Isw2p and Itc1p at equimolar ratios and incubating the mixture for 30 min on ice. In this experiment, Isw2 complex with FLAG-tagged Isw2p and Itc1p was used.

FIG. 5

Both Isw2p and Itc1p are required for biochemical activities of Isw2 complex. (A) ATPase assays using native Isw2 complex, monomeric Isw2p, monomeric Itc1p, and reconstituted Isw2 complex. Assays were done in the presence of buffer (B) or immobilized nucleosomal arrays (N). (B) Chromatin remodeling assay performed on preassembled nucleosomal arrays on immobilized templates. After incubation of the templates with ATP and the fractions indicated above, limited (lanes 1, 3, 5, 7, and 9) and extensive (lanes 2, 4, 6, 8, and 10) MNase digestion was performed. Nucleosomal arrays containing 25 ng of DNA and 45 fmol of Isw2 complex, Isw2p, or Itc1p were used. Arrowheads denote mononucleosome signals increased by native and reconstituted Isw2 complex. The arrow on the right indicates a dinucleosome signal that is shifted upward by the native Isw2 complex (lane 3). DNA was visualized by Southern blotting.

FIG. 6

Isw2 and Itc1 function in the same pathways. (A) Deletion of ISW2 or ITC1 results in derepression of meiotic genes. Northern blot analysis was performed to compare expression levels of meiotic genes in vegetatively growing cells. The genotype of cells used is listed above each lane; the gene probed is indicated at the right. Numbers below the lanes represent levels of transcription in mutants relative to wild-type cells. ACT1 was used as a loading control. WT, wild type. (B) isw2 and itc1 mutations confer temperature-sensitive growth defects in combination with isw1 and chd1 mutations. Tenfold serial dilutions of saturated wild-type and mutant liquid cultures were spotted onto rich medium (YEPD) and incubated at 30, 37, or 38.5°C.

References

1. Almouzni G, Mechali M. Assembly of spaced chromatin promoted by DNA synthesis in extracts from Xenopus eggs. EMBO J. 1988;7:665–672. - PMC - PubMed
1. Becker P B, Tsukiyama T, Wu C. Chromatin assembly extracts from Drosophila embryos. Methods Cell Biol. 1994;44:207–223. - PubMed
1. Belotserkovskaya R, Berger S L. Interplay between chromatin modifying and remodeling complexes in transcriptional regulation. Crit Rev Eukaryot Gene Expr. 1999;9:221–230. - PubMed
1. Bochar D A, Savard J, Wang W, Lafleur D W, Moore P, Cote J, Shiekhattar R. A family of chromatin remodeling factors related to Williams syndrome transcription factor. Proc Natl Acad Sci USA. 2000;97:1038–1043. - PMC - PubMed
1. Brehm A, Langst G, Kehle J, Clapier C R, Imhof A, Eberharter A, Muller J, Becker P B. dMi-2 and ISWI chromatin remodelling factors have distinct nucleosome binding and mobilization properties. EMBO J. 2000;19:4332–4341. - PMC - PubMed

Interactions of Isw2 chromatin remodeling complex with nucleosomal arrays: analyses using recombinant yeast histones and immobilized templates - PubMed (original) (raw)