Research ArticleBIOCHEMISTRY

Uncovering the forces between nucleosomes using DNA origami

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Science Advances  23 Nov 2016:
Vol. 2, no. 11, e1600974
DOI: 10.1126/sciadv.1600974
  • Fig. 1 Studying nucleosome-nucleosome interactions with a DNA force spectrometer.

    (A) Schematic of two nucleosomes based on 3MVD.pdb (4). Yellow, DNA template; blue, histone octamer; red, N-terminal histone tails. (B) Schematic of the DNA force spectrometer featuring a spring-loaded hinge with two attached nucleosomes. The torque generated by the hinge is illustrated with a red torsional spring. Red and green spheres indicate positions of fluorescent dyes (Atto647N and Atto550) that form a FRET pair. Two nucleosomes with radially protruding DNA single strands (Fig. 2A) are attached site-specifically and in a user-defined orientation via DNA strand hybridization on the opposing faces of the beams. (C) Schematic (left) and average TEM micrographs of the spectrometer with two nucleosomes attached either 15 nm (top) or 30 nm (bottom) away from the hinge in apparent contact. Scale bar, 50 nm.

  • Fig. 2 Design, preparation, and characterization of nucleosomes with radially protruding DNA single strands.

    (A) Top left: Schematic of branched variants of the 601 nucleosome positioning sequence (33) with up to four (positions A1 to A4) protruding DNA single strands. Bottom left: Schematic of purified histone octamer. Right: Schematics of nucleosomes with radially protruding DNA single strands at positions A1 and A3 that are produced by salt gradient dialysis from the components on the left. The bottom schematic is based on 3MVD.pdb (4). (B) Native ethidium bromide–stained 4.5% polyacrylamide gel electrophoresis of various samples: lane 1, continuous template DNA; lane 2, nucleosomes assembled using continuous template DNA as in lane 1; lane 3, template DNA with four nicks at positions A1 to A4; lane 4, nucleosomes assembled using nicked template DNA as in lane 3; lane 5, template DNA with two protruding single strands at positions A1 and A3 [see (A)]; lane 6, nucleosomes assembled using template DNA with two protruding single strands as in lane 5; lane 7, template DNA with four protruding single strands at positions A1 to A4; lane 8, nucleosomes assembled using the template DNA as in lane 7. Nucleosomes were assembled by salt gradient dialysis reconstitution with Drosophila embryo histones. See note S1 for detailed protocol. (C) Representative electron micrograph of nucleosomes with two protruding DNA single strands (A1 and A3) and recombinant tailless histones from X. laevis. Scale bar, 50 nm. (D) Projection of a nucleosome crystal structure (3MVD.pdb) (top) and average electron micrograph from nucleosomes shown in (C) (bottom). See fig. S4 for additional data. Black arrowheads indicate radial intensity signatures stemming from grooves in the DNA template. Red arrowheads indicate the dyad axis in the arc segment having only one DNA turn.

  • Fig. 3 Probing nucleosome interactions with the force spectrometer using direct electron microscopy imaging and FRET.

    (A) Left: Exemplary electron micrographs of force spectrometers without attached nucleosomes. See fig. S6 for more data. Right: Statistics of opening angles Θ measured in 3091 single particles. Blue line, uniform kernel density estimation (bandwidth of 3°); black, model of spectrometer mechanics (fig. S7 and note S3). (B and C) Left: Exemplary micrographs of spectrometers with two nucleosomes with DNA single strands at A1 and A3 attached in the proximal position (B) versus distal position (C) (15 nm versus 30 nm away from hinge). See figs. S11 and S12 for more data. See note S5 for particle selection criteria and TEM image processing details. Right: Angle distribution (red) obtained from 1301 (B) and 158 (C) particles. Dashed line, distribution from (A). (D to F) Angle distributions of spectrometers with rotated (D), acetylated H4 (E), and tailless (F) nucleosomes. Particle numbers were 979, 846, and 818, respectively. In the rotated sample, the nucleosome on the long beam of the spectrometer was rotated by 180°. (G and H) FRET efficiency images computed from three-channel laser-scanned images of agarose gels in which the indicated samples were electrophoresed. See note S7 for image processing details and figs. S18 and S19 for complete gel data. Nucleosomes were prepared with recombinant Xenopus histones (wild type, tailless, or H4 acetylated, respectively). All samples were prepared in buffer containing effective MgCl2 concentrations of 10 mM (see notes S2, S5, and S7 for detailed methods). Scale bars, 30 nm.

  • Fig. 4 Energy landscapes for nucleosome pair interactions.

    Normal and uniform kernel density estimates (thick versus thin lines; kernel density bandwidth of 3°) of the nucleosome-nucleosome interaction energy landscapes plotted as a function of the distance between the nucleosome centers of mass (CM; computed from 3MVD.pdb using Cα-atoms for histones and C4′-atoms for nucleosomal DNA). Red versus dark blue lines, wild-type nucleosomes, oriented such that the dyad axis encloses an angle of 78° or 258° (Fig. 3, B and C, respectively). The dots mark the first steric clashes of the nucleosomes. Cyan lines, nucleosomes with acetylated histone H4; orange lines, nucleosomes lacking all N-terminal histone tails. Inset: The combined free-energy landscapes of nucleosome and force spectrometer (red, dark blue, cyan, and orange) plotted versus the measured opening angle, which were obtained from the angle distributions (Fig. 3) by taking the logarithm (note S8). Solid black lines, the free-energy landscape of the bare spectrometer; dashed line, free energy of the fitted force spectrometer model (note S3). Top right: Schematics indicating sample details. Border color refers to the corresponding energy landscape. Bottom right: Zoom into the average single-particle micrograph obtained from force spectrometers having vertex angles within the range of 36° to 41° and wild-type nucleosomes as in Fig. 3B. Lines schematically depict the trapezoidal shapes of the nucleosome. Red versus blue indicates samples as in the main panel. The onset of clashes depends on the relative orientation of the nucleosomes (see fig. S13 for additional average single-particle micrographs).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/11/e1600974/DC1

    note S1. Reconstitution of nucleosomes with single-strand branches

    note S2. Design and assembly of the force spectrometer

    note S3. Calibration of the force spectrometer

    note S4. Attachment of nucleosomes to the force spectrometer

    note S5. TEM imaging and particle selection

    note S6. Comparison of negative staining versus cryo-EM

    note S7. Gel-based measurements of ensemble FRET

    note S8. Calculation of nucleosome-nucleosome energy landscapes

    note S9. Geometric nucleosome arrangement on the force spectrometer

    note S10. Gay-Berne potentials fitted to energy landscapes

    fig. S1. Assembly of wild-type, tailless, and acetylated NCPs with single-strand branches.

    fig. S2. Salt stability of NCPs without or with nicks.

    fig. S3. Sequences of single-stranded DNA handles protruding from the nucleosome.

    fig. S4. Direct imaging of nucleosomes.

    fig. S5. Design diagram of the force spectrometer generated with caDNAno v0.1 (57).

    fig. S6. Exemplary particles of the force spectrometer.

    fig. S7. Characterization of the force spectrometer.

    fig. S8. Attachment of nucleosomes to the force spectrometer.

    fig. S9. Orientation of nucleosomes on the force spectrometer.

    fig. S10. Rules for particle selection.

    fig. S11. Exemplary particles of the force spectrometer with two bound NCPs (wild-type and X. laevis) at the proximal position.

    fig. S12. Exemplary particles of the force spectrometer with two bound NCPs (wild-type and X. laevis) at the distal position.

    fig. S13. Average micrographs of force spectrometers with attached NCPs (wild-type and X. laevis) at the proximal position.

    fig. S14. Negative staining versus cryo-EM micrographs.

    fig. S15. Comparison of single particles from negative staining versus cryo-EM.

    fig. S16. Quantitative comparison of distributions and energy landscapes from negative staining versus cryo-EM.

    fig. S17. Calculation of FRET efficiencies and depiction of laser-scanned agarose gels.

    fig. S18. Nucleosome-nucleosome interaction observed using gel-based ensemble FRET measurements.

    fig. S19. Gel-based ensemble FRET measurements of nucleosome variants and orientations.

    fig. S20. Calculation of free-energy landscapes.

    fig. S21. Configuration of nucleosomes on the force spectrometer.

    fig. S22. Gay-Berne potential fits.

  • Supplementary Materials

    This PDF file includes:

    • note S1. Reconstitution of nucleosomes with single-strand branches
    • note S2. Design and assembly of the force spectrometer
    • note S3. Calibration of the force spectrometer
    • note S4. Attachment of nucleosomes to the force spectrometer
    • note S5. TEM imaging and particle selection
    • note S6. Comparison of negative staining versus cryo-EM
    • note S7. Gel-based measurements of ensemble FRET
    • note S8. Calculation of nucleosome-nucleosome energy landscapes
    • note S9. Geometric nucleosome arrangement on the force spectrometer
    • note S10. Gay-Berne potentials fitted to energy landscapes
    • fig. S1. Assembly of wild-type, tailless, and acetylated NCPs with single-strand branches.
    • fig. S2. Salt stability of NCPs without or with nicks.
    • fig. S3. Sequences of single-stranded DNA handles protruding from the nucleosome.
    • fig. S4. Direct imaging of nucleosomes.
    • fig. S5. Design diagram of the force spectrometer generated with caDNAno v0.1 (57).
    • fig. S6. Exemplary particles of the force spectrometer.
    • fig. S7. Characterization of the force spectrometer.
    • fig. S8. Attachment of nucleosomes to the force spectrometer.
    • fig. S9. Orientation of nucleosomes on the force spectrometer.
    • fig. S10. Rules for particle selection.
    • fig. S11. Exemplary particles of the force spectrometer with two bound NCPs (wild-type and X. laevis) at the proximal position.
    • fig. S12. Exemplary particles of the force spectrometer with two bound NCPs (wild-type and X. laevis) at the distal position.
    • fig. S13. Average micrographs of force spectrometers with attached NCPs (wild-type and X. laevis) at the proximal position.
    • fig. S14. Negative staining versus cryo-EM micrographs.
    • fig. S15. Comparison of single particles from negative staining versus cryo-EM.
    • fig. S16. Quantitative comparison of distributions and energy landscapes from negative staining versus cryo-EM.
    • fig. S17. Calculation of FRET efficiencies and depiction of laser-scanned agarose gels.
    • fig. S18. Nucleosome-nucleosome interaction observed using gel-based ensemble FRET measurements.
    • fig. S19. Gel-based ensemble FRET measurements of nucleosome variants and orientations.
    • fig. S20. Calculation of free-energy landscapes.
    • fig. S21. Configuration of nucleosomes on the force spectrometer.
    • fig. S22. Gay-Berne potential fits.

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