Direct imaging of human SWI/SNF-remodeled mono- and polynucleosomes by atomic force microscopy employing carbon nanotube tips - PubMed (original) (raw)

Direct imaging of human SWI/SNF-remodeled mono- and polynucleosomes by atomic force microscopy employing carbon nanotube tips

G R Schnitzler et al. Mol Cell Biol. 2001 Dec.

Abstract

Chromatin-remodeling complexes alter chromatin structure to facilitate, or in some cases repress, gene expression. Recent studies have suggested two potential pathways by which such regulation might occur. In the first, the remodeling complex repositions nucleosomes along DNA to open or occlude regulatory sites. In the second, the remodeling complex creates an altered dimeric form of the nucleosome that has altered accessibility to transcription factors. The extent of translational repositioning, the structure of the remodeled dimer, and the presence of dimers on remodeled polynucleosomes have been difficult to gauge by biochemical assays. To address these questions, ultrahigh-resolution carbon nanotube tip atomic force microscopy was used to examine the products of remodeling reactions carried out by the human SWI/SNF (hSWI/SNF) complex. We found that mononucleosome remodeling by hSWI/SNF resulted in a dimer of mononucleosomes in which approximately 60 bp of DNA is more weakly bound than in control nucleosomes. Arrays of evenly spaced nucleosomes that were positioned by 5S rRNA gene sequences were disorganized by hSWI/SNF, and this resulted in long stretches of bare DNA, as well as clusters of nucleosomes. The formation of structurally altered nucleosomes on the array is suggested by a significant increase in the fraction of closely abutting nucleosome pairs and by a general destabilization of nucleosomes on the array. These results suggest that both the repositioning and structural alteration of nucleosomes are important aspects of hSWI/SNF action on polynucleosomes.

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Figures

FIG. 1

AFM images of SWI/SNF and altered dimers. Samples were fixed, deposited, and imaged with a nanotube tip as described in Materials and Methods. (A) Mononucleosomes on spermidine-treated mica. DNA tails, where visible, are indicated by arrows. (B) Gradient-purified hSWI/SNF on spermidine-treated mica. Multiple lobes are indicated by arrows. Small molecules are BSA from the gradient buffer. (C) hSWI/SNF-remodeled dimers on poly-

-lysine-treated mica. DNA tails are indicated by arrows.

FIG. 2

ExoIII digestion of hSWI/SNF products. 5′-end-labeled remodeled dimers (lanes 7 to 9) and control mononucleosomes (lanes 4 to 6) or bare DNA from mononucleosomes (lanes 1 to 3) were digested with ExoIII for 3 (lanes 2, 5, and 8) or 15 (lanes 3, 6, and 9) min before DNA purification and resolution by denaturing polyacrylamide gel electrophoresis. The values on the left are molecular sizes in base pairs.

FIG. 3

5S nucleosomal arrays are stably remodeled by hSWI/SNF. (A) Diagram of the 5S-G5E4 rDNA array used. White circles represent nucleosomes at preferred sites on 208-bp 5S rDNA sequences. Grey circles represent nucleosomes in the transcription template. Locations of _Sac_I and _Xba_I sites relative to inferred unremodeled nucleosome positions are indicated. For the graphs, the array was treated with hSWI/SNF without ATP (triangles), with ATP for 30 min (squares), or with ATP for 30 min, followed by apyrase for 18 min (circles), and then cut with _Sac_I (left) or _Xba_I (right) for the indicated times. The purified DNA was separated by agarose electrophoresis, and percent cutting was quantified. Similar results were obtained in three separate experiments. (B) Unlabeled arrays were incubated with both SWI/SNF (S/S) and ATP (lane 4), without ATP (lane 5), or without SWI/SNF (lane 6) for 90 min before dialysis into TE. Samples of these reaction mixtures and bare DNA (lane 2) or the untreated assembled array (lane 3) were digested with _Sac_I, purified, and separated as described above, followed by ethidium bromide staining and quantitation. Lane 1 contained uncut DNA.

FIG. 4

AFM images of fixed, hSWI/SNF (S/S)-remodeled arrays on spermidine-treated mica. (A) An array from the control reaction mixture lacking ATP (Fig. 3B, lane 5). (B) Arrays remodeled by hSWI/SNF and ATP for 90 min (Fig. 3B, lane 4). (C) Histogram of internucleosomal distances (peak to peak along the DNA contour) from two experiments. The data (23 arrays with ATP, 19 arrays without ATP) were grouped into three bins (≤14 nm, 14 to 60 nm, and >60 nm) and normalized to 100%. (D) An hSWI/SNF-bound array from the same control reaction mixture as in panel A. (E) An hSWI/SNF-bound remodeled array from the same reaction mixture as in panel B. The arrow shows potential DNA loops constrained by SWI/SNF. The height range in panels D and E is greater than in that in panels A and B to allow structural features of SWI/SNF to be evident.

FIG. 5

AFM images of unfixed remodeled arrays suggest reduced nucleosome stability. (A) Control arrays (shown fixed in Fig. 4A) were deposited on spermidine-treated mica without fixation. (B) Remodeled arrays (shown fixed in Fig. 4B) were deposited on spermidine-treated mica without fixation. The small molecules in the background are BSA.

FIG. 6

Model for SWI/SNF remodeling of arrays. Nucleosomes in initial positions (top) block some transcription factor binding sites (B and C) while leaving others open. SWI/SNF alters nucleosome positions (bottom) to uncover some sites (B) and cover others (A). In this way, SWI/SNF remodeling can facilitate both activation and repression. Nucleosome dimers are also formed, which are more accessible to some factors (site C).

References

1. Allen M J. Atomic force microscopy: a new way to look at chromatin. IEEE Eng Med Biol Mag. 1997;16:34–41. - PubMed
1. Bazett-Jones D P, Cote J, Landel C C, Peterson C L, Workman J L. The SWI/SNF complex creates loop domains in DNA and polynucleosome arrays and can disrupt DNA-histone contacts within these domains. Mol Cell Biol. 1999;19:1470–1478. - PMC - PubMed
1. Bustamante C, Rivetti C. Visualizing protein-nucleic acid interactions on a large scale with the scanning force microscope. Annu Rev Biophys Biomol Struct. 1996;25:395–429. - PubMed
1. Cheung C L, Hafner J H, Lieber C M. Carbon nanotube atomic force microscopy tips: direct growth by chemical vapor deposition and application to high-resolution imaging. Proc Natl Acad Sci USA. 2000;97:3809–3813. - PMC - PubMed
1. Cote J, Peterson C L, Workman J L. Perturbation of nucleosome core structure by the SWI/SNF complex persists after its detachment, enhancing subsequent transcription factor binding. Proc Natl Acad Sci USA. 1998;95:4947–4952. - PMC - PubMed

Direct imaging of human SWI/SNF-remodeled mono- and polynucleosomes by atomic force microscopy employing carbon nanotube tips - PubMed (original) (raw)