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DNA topology in chromatin is defined by nucleosome spacing

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Science Advances  27 Oct 2017:
Vol. 3, no. 10, e1700957
DOI: 10.1126/sciadv.1700957
  • Fig. 1 Models of two-start chromatin fibers with NRLs = 167 and 172 bp.

    (A and B) Energy-optimized regular fibers (43, 44) containing 12 nucleosomes, with NRL = 167 bp (A) and NRL = 172 bp (B). The DNA linking number per nucleosome, ΔLk = −1.37 and −0.93, respectively. The DNA is shown in alternating blue and orange colors to emphasize the two stacks of nucleosomes; the DNA “entry” points are shown as red balls. The histone cores are shown in two colors—The entry sides are in yellow, and the “exit” sides are in white. In this manner, it is easier to distinguish the fiber configurations. In addition, the green arrows indicate different DNA folding pathways in the two topoisomers. (C and D) Representative configurations of the 167x11 and 172x11 circular NAs obtained during Monte Carlo simulations (see main text and Fig. 3 for details). Note that the circular topoisomers (C and D) are significantly distorted and extended compared to the regular conformers (A and B). The nucleosome-free DNA fragments are shown as white tubes.

  • Fig. 2 Topological difference between NAs with 167- and 172-bp NRLs reconstituted on plasmid DNA.

    Plasmid-based circular DNA templates p-167x12 (lanes 1, 4, 6, 8, and 10) and p-172x12 (lanes 2, 5, 7, 9, and 11) were reconstituted with 0, 25, 50, 75, and 100% core histones (top), treated with Topo I, and the circular DNA was isolated and separated on agarose gels run in the absence (A) and presence of CQ (1.5 μg/ml) (B) and CQ (4.0 μg/ml) (C) in the gel and TAE buffer. Lanes 3 and 12 show molecular weight markers. The strongest bands are indicated by blue circles for the 167-bp NRL and by red circles for the 172-bp NRL. The numbers of superhelical turns in the topoisomers corresponding to these bands are given in the same colors (B). In particular, for the bands denoted +8 and +9 (lanes 4 and 5), the shifts from the top of the gel (nicked circles, form II) are counted directly. For the bands denoted +14 (lanes 1 and 2), the shift from the top is evaluated from the relative shift of six supercoils between these bands and the band +8 (lane 4). Difference in DNA linking number between the 167- and 172-bp NRL constructs, or Δ(ΔLk), equals 1 at 25%, 5 at 50%, 6 at 75%, and 7 at 100% loading of core histones. Note that the presence of intercalator CQ introduces additional positive supercoils in DNA and changes its electrophoretic mobility. For example, the DNA circles relaxed in the absence of histones (lanes 1 and 2) become strongly supercoiled and run at the bottom of the gel. By contrast, the strong negative superhelical density of DNA obtained at 100% loading of histones (lanes 10 and 11) is partially compensated by CQ, and thus, its mobility is decreased (see fig. S3 for details).

  • Fig. 3 Topological difference between 167x12 and 172x12 NAs reconstituted on minicircular DNA.

    Nucleosomes were reconstituted on the scDNA templates prepared in the presence of EtBr (2 μg/ml) (A to D) and EtBr (4 μg/ml) (E to H). (A and E) Scans of gels in (B) and (F). The strongest bands are marked with “+”. (B and F) Electrophoretic mobility of the DNA topoisomers extracted from NAs was compared with the mobility of scDNA templates obtained at [EtBr] = 2 μg/ml (lanes 1 and 2). Gels were run in the presence of CQ (8 μg/ml) (B) and CQ (32 μg/ml) (F). Lane 5 shows molecular weight markers. Note that the scDNAs with 167-bp (lane 1) and 172-bp (lane 2) NRLs have similar distributions of topoisomers, with the strongest bands corresponding to ΔLk = −12 (fig. S5D). NAs reconstituted on these scDNAs (obtained at [EtBr] = 2 μg/ml) have ΔLk = −11 and −8 (lanes 3 and 4) (B). Arrays reconstituted on scDNA obtained at [EtBr] = 4 μg/ml have ΔLk = −15 and −10 (lanes 3 and 4 ) (F). (C and G) Graphs showing the average number of nucleosomes (10.9) on 167x12 and 172x12 arrays and distributions of the number of nucleosomes per one minicircle (calculated from EM_images_interactive PDF file). Normalization of the values ΔLk = −15 and −10 [shown in (F)] gives the DNA linking number per nucleosome, ΔLk = −1.38 and −0.92 for the 167x12 and 172x12 arrays, respectively. (D and H) Representative transmission EM images of relaxed minicircular 167x12 and 172x12 arrays reconstituted on scDNA obtained at [EtBr] = 2 μg/ml (D) and [EtBr] = 4 μg/ml (H). Scale bars, 50 nm.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/10/e1700957/DC1

    fig. S1. Analysis of circular nucleosome array reconstitution by electrophoretic mobility shift assay.

    fig. S2. Analysis of circular nucleosome array reconstitution by restriction enzyme digestion and electrophoretic DNA band-shift assay.

    fig. S3. Binding of intercalator CQ to supercoiled DNA changes its electrophoretic mobility.

    fig. S4. 2D gel electrophoresis of DNA extracted from NAs with 167- and 172-bp NRLs.

    fig. S5. Modulating negative supercoiling in DNA minicircles in the presence of various concentrations of EtBr.

    fig. S6. Preparation of supercoiled DNA constructs 167x12 and 172x12 relaxed by Topo I in the presence of EtBr (4.0 μg/ml).

    fig. S7. Negative supercoiling of DNA minicircles with 207-bp NRL in the presence of EtBr.

    fig. S8. Negative supercoiling of circular NAs with 207-bp NRL.

    fig. S9. Linking number per nucleosome, ΔLk, in regular fibers with various linker lengths.

    EM data set file (pdf)

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Analysis of circular nucleosome array reconstitution by electrophoretic mobility shift assay.
    • fig. S2. Analysis of circular nucleosome array reconstitution by restriction enzyme digestion and electrophoretic DNA band-shift assay.
    • fig. S3. Binding of intercalator CQ to supercoiled DNA changes its electrophoretic mobility.
    • fig. S4. 2D gel electrophoresis of DNA extracted from NAs with 167- and 172-bp NRLs.
    • fig. S5. Modulating negative supercoiling in DNA minicircles in the presence of various concentrations of EtBr.
    • fig. S6. Preparation of supercoiled DNA constructs 167x12 and 172x12 relaxed by Topo I in the presence of EtBr (4.0 μg/ml).
    • fig. S7. Negative supercoiling of DNA minicircles with 207-bp NRL in the presence of EtBr.
    • fig. S8. Negative supercoiling of circular NAs with 207-bp NRL.
    • fig. S9. Linking number per nucleosome, ΔLk, in regular fibers with various linker lengths.

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