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. 2003 Nov;23(21):7767-79.
doi: 10.1128/MCB.23.21.7767-7779.2003.

Dynamic properties of nucleosomes during thermal and ATP-driven mobilization

Andrew Flaus  1 , Tom Owen-Hughes
Affiliations

Dynamic properties of nucleosomes during thermal and ATP-driven mobilization

Andrew Flaus et al. Mol Cell Biol. 2003 Nov.

Abstract

The fundamental subunit of chromatin, the nucleosome, is not a static entity but can move along DNA via either thermal or enzyme-driven movements. Here we have monitored the movements of nucleosomes following deposition at well-defined locations on mouse mammary tumor virus promoter DNA. We found that the sites to which nucleosomes are deposited during chromatin assembly differ from those favored during thermal equilibration. Taking advantage of this, we were able to track the movement of nucleosomes over 156 bp and found that this proceeds via intermediate positions spaced between 46 and 62 bp. The remodeling enzyme ISWI was found to direct the movement of nucleosomes to sites related to those observed during thermal mobilization. In contrast, nucleosome mobilization driven by the SWI/SNF and RSC complexes were found to drive nucleosomes towards sites up to 51 bp beyond DNA ends, with little respect for the sites favored during thermal repositioning. The dynamic properties of nucleosomes we describe are likely to influence their role in gene regulation.

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Figures

FIG. 1.
FIG. 1.
Increased linker DNA length reduces the temperature required for thermally driven nucleosome redistribution. (A) Native gel electrophoresis (PAGE) of nucleosomes assembled onto the 64A64 DNA fragment following incubation at 39, 41, 44, and 47°C (lanes 1 to 4, respectively). Most of the nucleosomes start as a discrete slowly migrating species, with a small proportion as a second species labeled “p.” Following thermal incubation, faster migrating species are generated. (B) Site-directed mapping of the same samples as for panel A (lanes 2 to 5, respectively). Nucleosomes generate a characteristic pattern of two cleavage sites separated by 7 bp and allow determination of nucleosome positions at base pair resolution using the G ladder in lane 1, as diagrammed to the right (nucleosomes are not to scale). A small amount of mapping is also observed at a second site labeled “p.” After thermal incubation, mapping products accumulate within 16 bp of either end of the DNA fragment. (C) Plot of the temperature required to shift 50% of nucleosomes from their initial central location on DNA fragments to the symmetric extensions of flanking DNA. Temperatures required for shifting on the MMTV NucA (open diamonds; left axis) and MMTV NucB (filled diamonds; right axis) positioning sequences are shown.
FIG. 2.
FIG. 2.
Oriented nucleosome redistribution. By creating DNA fragments with long linker DNA on one side and short linker DNA on the other, it is possible to direct thermal nucleosome redistribution onto the longer DNA arm. (A) Site-directed mapping at base pair resolution (lane 2) shows that nucleosomes are initially deposited as expected at +70 relative to the MMTV transcriptional start site, with a minor population of nucleosomes also detectable at position +53, denoted “p.” Following incubation at 47°C for 4, 8, 16, 32, 64, and 120 min (lanes 3 to 8), a loss of mapping is observed at the +70 location concurrently with an accumulation of new cleavage sites around the new major location at +22. No intermediate mapping sites are observable. (B) Native gel electrophoresis (PAGE) of the same samples is consistent with the expected migration of nucleosomes to the positions mapped in panel A. (C) A thermal shifting reaction as for panel B was performed on a preparative scale. The unshifted (+70) and major shifted (+22) species were excised. Protein was extracted from the gel slices, which were then resolved by SDS gel electrophoresis. No change in histone content was detected.
FIG. 3.
FIG. 3.
Sequential movement of nucleosomes over 154 bp. (A) Site-directed mapping on the fragment 105A18 indicates that nucleosomes are initially assembled as before, with the dyad at +70 (lane 3). Lanes 4 to 8 show that during incubation at 47°C for 4, 8, 16, 32, and 64 min, mapping is lost at +70 and transiently accumulated at +22 prior to accumulation during the last time points at −25 relative to the MMTV transcriptional start site. Lane 1, control in which the reagent-attached nucleosomal DNA was not subjected to site-directed mapping; lane 2, G ladder. (B) Tracking of the redistribution of nucleosomes assembled onto the fragment 181A18 during incubation at 47°C for 4, 8, 16, 32, 64, and 120 min (lanes 2 to 7). Nucleosomes initially accumulate at +22, subsequently at −25, and finally at −85. (C and D) Native gel electrophoresis (PAGE) of the same samples as for panels A and B, respectively. In all panels, minor amounts of nucleosomes assembled at +53 are indicated by “p.” (E and F) Quantitation of the amount of material at each position in panels B and D. These graphs illustrate how the relative amounts of nucleosomes at each position change over time.
FIG. 4.
FIG. 4.
Redistribution of nucleosomes from the MMTV NucB positioning sequence. (A) Site-directed mapping of nucleosomes assembled on the DNA fragment 18B105 results in the generation of a major signal at −127 (lane 1). Minor positions are also observed at the positions indicated by “f,” “g,” and “h.” Following thermal incubation at 62°C for 3.75, 7.5, 15, 30, and 60 min (lanes 2 to 6), new nucleosome locations are observed at −87 and later at −25. (B) Native gel electrophoresis (PAGE) results in the generation of a series of bands consistent with those observed during site-directed mapping. (C) Schematic summarizing the nucleosome movements detected in the experiments for Fig. 3 and this figure. Nucleosomes were tracked moving over the same sequences from different directions. Despite their different origins, both NucA and NucB move through the −25 and −87 positions. This suggests that DNA sequence plays an important role in determining the sites to which nucleosomes are relocated.
FIG. 5.
FIG. 5.
Nucleosome redistribution driven by ISWI protein. (A) Redistribution of nucleosomes assembled onto the DNA fragment 105A18 directed by ISWI (lanes 2 to 6) is compared to thermal redistribution (lanes 8 to 12). Nucleosomes were incubated with 0.4 pmol of ISWI for 4, 8, 16, 32, and 64 min (lanes 2 to 6). For comparison, nucleosomes were incubated at 47°C for the same times (lanes 8 to 12). (B) Native gel shift assay of the same samples as in panel A. (C) Nucleosome redistribution by thermal incubation and ISWI on the fragment 140A0 using site-directed mapping. Lanes 1 and 4 show that nucleosomes are initially located at the terminal position +70 on this DNA fragment, but following thermal incubation at 47°C (lane 2) or treatment with 0.9 pmol of ISWI (lane 4) for 60 min, nucleosomes are relocated predominantly to the −25 location. (D) Native gel electrophoresis of the samples in panel C.
FIG. 6.
FIG. 6.
Nucleosome redistribution driven by the yeast SWI/SNF and RSC complexes. (A) The fate of nucleosomes assembled onto the fragment 105A18 radiolabeled at the downstream end was monitored by site-directed mapping following incubation at 47°C for 1 h (lane 2) or treatment with 0.25 pmol of SWI/SNF complex and ATP for 4, 8, 16, 32, and 64 min (lanes 4 to 8). Nucleosomes were incubated with 0.12 pmol of RSC complex for 16, 8, and 4 min (lanes 9 to 11). (B) Native gel electrophoresis of the same samples as in panel A. (C) Extended electrophoresis was performed on the reactions from panel B to enable mapping locations towards the end of the fragment to be identified at base pair resolution.
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