Functional role of extranucleosomal DNA and the entry site of the nucleosome in chromatin remodeling by ISW2 - PubMed (original) (raw)

Functional role of extranucleosomal DNA and the entry site of the nucleosome in chromatin remodeling by ISW2

Martin Zofall et al. Mol Cell Biol. 2004 Nov.

Abstract

A minimal amount of extranucleosomal DNA was required for nucleosome mobilization by ISW2 as shown by using a photochemical histone mapping approach to analyze nucleosome movement on a set of nucleosomes with varied lengths of extranucleosomal DNA. ISW2 was ineffective in repositioning or mobilizing nucleosomes with <or=20 bp of extranucleosomal DNA. In addition, ISW2 was able to slide nucleosomes to within only 10 to 13 bp of the edge of DNA fragments. The nucleosome mobilization was promoted by extranucleosomal single-stranded DNA with modest strand preference. Gaps (10 bp) just inside the nucleosome and in the extranucleosomal DNA showed that the transfer of torsional strain (twist) into the nucleosomal DNA region was not required for mobilizing nucleosomes. However, indications are that the extranucleosomal DNA immediately adjacent to the nucleosome has an important role in the initial stage of nucleosome movement by ISW2.

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Figures

FIG. 1.

FIG. 1.

ISW2 prefers to slide nucleosomes towards the center of DNA. Nucleosomes (125 ng) initially positioned at either the end (a) or the center (b) of DNA were mobilized with 30 ng of ISW2 and 3 mM ATP for 30 min at 30°C and then analyzed on a native 5% polyacrylamide gel.

FIG. 2.

FIG. 2.

ISW2 does not mobilize nucleosomes towards the thermodynamically preferred positions on DNA. Nucleosomes were positioned at the ends of DNA by using the nucleosome positioning sequence referred to as 601. Nucleosome samples were analyzed by gel shift on a native 5% polyacrylamide gel and quantitated as shown by phosphorimaging. Lane 1, samples with no ISW2; lane 2, samples after ISW2 remodeling; lane 3, reactions stopped after remodeling and then heated at 50°C for 30 min for equilibration to the thermodynamically preferred position(s). The samples contained 370 ng of nucleosomes and 60 ng of ISW2. After remodeling, ISW2 was inhibited by the addition of apyrase (0.1 U) and incubation for 20 min at 37°C, followed by the addition of 10 μg of DNA and 4 mM γ-S-ATP.

FIG. 3.

FIG. 3.

ISW2 requires >20 bp of extranucleosomal DNA for efficient nucleosome mobilization. Nucleosomes (370 ng) assembled at the end of DNA with 0 (A), 10 (B), 20 (C), 23 (D), 26 (E), 30 (F), or 33 (G) bp of extranucleosomal DNA were remodeled with 75 ng of ISW2 and 300 μM ATP for 30 min at 30°C. Nucleosome sliding was detected by site-directed histone-DNA cross-linking, analyzed on a 6.5% polyacrylamide gel containing 8 M urea, and visualized by phosphorimaging. Overlays of the nucleosomal cut sites before and after ISW2 remodeling are shown (black, nucleosome alone; gray, nucleosomes plus ISW2 plus 300 μM ATP).

FIG. 4.

FIG. 4.

The inability of ISW2 to slide nucleosomes with short extranucleosomal DNA is not due to an inability to bind. (a) Nucleosomes assembled with 156-, 166-, or 179-bp DNA fragments positioned towards one end were saturated by the addition of 2 μg, 900 ng, or 450 ng of ISW2, respectively, and remodeled for 10 min at 30°C. (b) ISW1a requires more than 20 bp of extranucleosomal DNA for efficient nucleosome mobilization. ISW1a was shown to slide nucleosomes with 0, 10, 20, and 33 bp of extranucleosomal DNA, as monitored by site-directed histone DNA cross-linking for ISW2 (see the legend for Fig. 3). Black lines, nucleosomes alone; gray lines, ISW1a ATP.

FIG. 5.

FIG. 5.

ISW2 and ISW1a are unable to reposition nucleosomes closer than 11 to 13 bp to the ends of DNA. (a) Nucleosomes were positioned on a 232-bp DNA such that there were 33 bp of extranucleosomal DNA on one side and 55 bp of extranucleosomal DNA on the other. After remodeling, the new translational positions closest to either end of DNA are 13 and 11/12 bp from the end. (b) Nucleosomes were mobilized with ISW2 (lane 2) and ISW1a (lane 4) and mapped by using site-directed histone-DNA cross-linking. (c) The last 13 bp of DNA on nucleosomes slid by ISW2 were not bound to the histone octamer, as shown by exonuclease III footprinting. Two discrete protections are evident when ISW2 (48 ng) and ATP (300 μM) were added to 179-bp end-positioned nucleosomes (300 ng) and incubated for 30 min at 30°C; they were not present with nucleosomes alone (compare lanes 5 and 6 to lanes 2 and 3).

FIG. 6.

FIG. 6.

Single-stranded extranucleosomal DNA can promote ISW2 mobilization of nucleosomes with some strand specificity. Nucleosomes (370 ng) with 23 bp of double-stranded extranucleosomal DNA had nine nucleotides of ssDNA added onto either the 5′ or 3′ end of DNA to determine if ssDNA could substitute for dsDNA. Black lines, samples containing ISW2 (187 ng) and ATP (300 μM); gray lines, nucleosome alone; nt, nucleotides.

FIG. 7.

FIG. 7.

Gaps in extranucleosomal DNA immediately adjacent affect nucleosome sliding by ISW2. (a) Gaps of 10 bp were placed in the 3′ to 5′ strand in a region spanning from just within the nucleosomal bound region to 30 bp from the entry site. The position of the gap is indicated by the first and second numbers, indicating the start and end of the gap with regard to the edge of the nucleosome. The edge of the nucleosomeis the zero reference point; any position inside the nucleosome is negative, and any position in the extranucleosomal DNA is positive. After remodeling, nucleosomes were slid as much as 40 bp from their original positions, with the gap moving to a position inside the nucleosome as shown. (b) Mobilization of these nucleosomes was monitored by gel shift assay on a native 5% polyacrylamide gel after 5 or 15 min incubation at 30°C. The locations of free DNA and nucleosomes that were (Nuc*) and were not (Nuc) moved to the center are indicated on the left. The initial locations of the gap are indicated above the lanes, as described for panel a. (c) Movement of nucleosomes after remodeling by ISW2 with no gap or gaps at bp 10/0 and 20/10 were monitored by site-directed histone-DNA cross-linking. Black lines, original cut sites before ISW2 remodeling; gray lines, original cut sites after ISW2 remodeling.

FIG. 8.

FIG. 8.

The effect of the gap at bp 0/10 on the mobilization of nucleosomes by ISW2 is not due to reduced binding of ISW2. (A) Quantification of gel shift assays of ISW2 binding to gapped nucleosomes are shown at three different amounts of ISW2 (100, 200, and 400 ng). Binding assays were performed as described in Materials and Methods. (B) Sliding assays of nucleosomes with or without 10-bp gaps were done as described for Fig. 7b, except that saturating amounts of ISW2 (400 ng) were added and, after remodeling excess, competitor DNA was added to compete ISW2 away from the labeled nucleosomes.

FIG. 8.

FIG. 8.

The effect of the gap at bp 0/10 on the mobilization of nucleosomes by ISW2 is not due to reduced binding of ISW2. (A) Quantification of gel shift assays of ISW2 binding to gapped nucleosomes are shown at three different amounts of ISW2 (100, 200, and 400 ng). Binding assays were performed as described in Materials and Methods. (B) Sliding assays of nucleosomes with or without 10-bp gaps were done as described for Fig. 7b, except that saturating amounts of ISW2 (400 ng) were added and, after remodeling excess, competitor DNA was added to compete ISW2 away from the labeled nucleosomes.

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References

    1. Brehm, A., G. Langst, J. Kehle, C. R. Clapier, A. Imhof, A. Eberharter, J. Muller, and P. B. Becker. 2000. dMi-2 and ISWI chromatin remodelling factors have distinct nucleosome binding and mobilization properties. EMBO J. 19:4332-4341. - PMC - PubMed
    1. Cairns, B. R., Y. Lorch, Y. Li, M. Zhang, L. Lacomis, H. Erdjument-Bromage, P. Tempst, J. Du, B. Laurent, and R. D. Kornberg. 1996. RSC, an essential, abundant chromatin-remodeling complex. Cell 87:1249-1260. - PubMed
    1. Clapier, C. R., K. P. Nightingale, and P. B. Becker. 2002. A critical epitope for substrate recognition by the nucleosome remodeling ATPase ISWI. Nucleic Acids Res. 30:649-655. - PMC - PubMed
    1. Corona, D. F., G. Langst, C. R. Clapier, E. J. Bonte, S. Ferrari, J. W. Tamkun, and P. B. Becker. 1999. ISWI is an ATP-dependent nucleosome remodeling factor. Mol. Cell 3:239-245. - PubMed
    1. Dingwall, A. K., S. J. Beek, C. M. McCallum, J. W. Tamkun, G. V. Kalpana, S. P. Goff, and M. P. Scott. 1995. The Drosophila snr1 and brm proteins are related to yeast SWI/SNF proteins and are components of a large protein complex. Mol. Biol. Cell 6:777-791. - PMC - PubMed

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