Research Article

ChromEMT: Visualizing 3D chromatin structure and compaction in interphase and mitotic cells

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Science  28 Jul 2017:
Vol. 357, Issue 6349, eaag0025
DOI: 10.1126/science.aag0025
  • ChromEMT reveals the in situ chromatin ultrastructure, 3D packing, and organization of DNA.

    EMT sample volume-DNA-nucleosome chains are black. Chromatin is a structurally disordered 5- to 24-nm granular chain that is packed together at different 3D CVC densities in human interphase nuclei and mitotic chromosomes (red, high density; yellow, medium density; blue, low density).

  • Fig. 1 A fluorescent DNA-binding dye that catalyzes local DAB polymerization on chromatin in the nucleus.

    (A) Hierarchical chromatin-folding model. (B) Excited fluorophores that undergo intersystem crossing generate reactive oxygen species that catalyze DAB polymerization. S0, ground state; S1, excited singlet state; T1, excited triplet state. (C) Schema for cell-based screen for DNA-binding dyes that photo-oxidize DAB. (D) U2OS cells were fixed with glutaraldehyde and stained with DRAQ5. Cells were incubated with DAB and excited by continuous epifluorescence illumination for 5 min. DAB photo-oxidation was identified by the appearance of dark DAB precipitates in the nucleus. Fluorescence (middle), transmitted-light images pre– (left panel) and post–photo-oxidation (right panel). Scale bar, 10 μm. See Movie 1 for photo-oxidation of DAB by DRAQ5.

  • Fig. 2 ChromEM: DRAQ5 excitation photo-oxidizes DAB on DNA in the nucleus and enables chromatin to be visualized by osmium staining in EM.

    (A) U2OS cells were fixed with glutaraldehyde, stained with DRAQ5, and DAB photo-oxidized for 7 min. Cells were then stained with OsO4 and thin sections prepared for TEM. EtOH, ethanol. Scale bar, 10 μm. (B to E) Magnified views of the cells in the labeled rectangles in panel (A). TEM images of a cell outside the photo-oxidation field (B), inside the photo-oxidation field (C), and bisected by the excitation field (D) and (E). Zoom-in (red box) is shown to the right. Nuclear membrane (arrow) and nucleolus (double arrow) are shown. Scale bar, 2 μm.

  • Fig. 3 ChromEMT and eight-tilt EMT enable chromatin to be visualized with high-contrast and spatial resolutions through large 3D volumes.

    SAECs were fixed with glutaraldehyde and stained with DRAQ5. Cells were either left untreated (control) or excited for 6 min to photo-oxidize DAB. Samples were stained with OsO4, cut into 250-nm sections, and imaged by EMT. (A) Single- (121 images) and eight-tilt EMT data sets (968 images) were collected by rotating the sample block from –60° to +60° at the depicted orientations (blue lines). (B) Collected data set was reconstructed into an EM tomogram of 1666 nm (x) by 1619 nm (y) by 231 nm (z). Tomographic slices (TSs) from single-tilt data sets of control (left) and photo-oxidized nuclei (right). The xy image and the respective xz and yz cross-sectional views are shown. The blue line in the xz and yz cross sections shows the z position of the xy image. Scale bar, 100 nm. (C) Same as (B) for an eight-tilt data set.

  • Fig. 4 Chromatin is a disordered chain that has diameters between 5 and 24 nm and is packed together at different concentration densities in interphase nuclei.

    (A) ChromEMT enables the ultrastructure of individual chromatin chains, megabase domains, and mitotic chromosomes to be resolved and visualized as a continuum in serial slices through large 3D volumes. (B) Reconstructed eight-tilt EMT data set (SAEC #1) of ChromEM-stained SAECs comprising 121 TSs (each 1.28 nm thick). Scale bar, 100 nm. To visualize chromatin and 3D organization as a continuum through the entire EMT data set, we compiled serial slices into a movie (Movie 2). (C) Manual measurements of chromatin diameters in a single TS. Scale bar, 50 nm. (D) The central EMT volume [red box in (B), 963 nm by 963 nm by 120 nm] was divided into an 8-by-8 grid comprising 64 subvolumes of 120-nm cubes. Chromatin volume concentrations (CVCs) are shown in the heat map. Scale bar, 100 nm. (E to G) The surface-thickness function was used to determine chromatin diameters in subvolumes with high (45%), medium (35%), and low (25%) CVCs. Irrespective of CVC, there are two major bin peak distributions for chromatin diameter: 5 to 12 nm and 12 to 24 nm. Scale bar, 20 nm. (H to J) Continuous erosion analysis to determine average chromatin diameter. The residual chromatin volume (Ve/Vtotal) is plotted against the spherical mean filter radius. The average radius of chromatin in each subvolume is the x-axis intercept of a linear fit of the first five erosion factor sizes.

  • Fig. 5 DNA and nucleosomes form disordered chromatin chains that have different particle arrangements, conformations, densities, and 3D motifs.

    (A) In interphase subvolumes that have low CVCs (<20%), chromatin chains have predominantly 5- to 12-nm diameter bin distributions (see fig. S5). Left, single TS (D6, SAEC #1); middle, segmented chromatin; right, chromatin-diameter range. Scale bar, 20 nm. (B to C) Magnified images of chromatin in volumes with low CVCs (red boxes). Chromatin chains have thin threads decorated with discrete particles (right panels: single TS, scale bar 20 nm). See movies S1 and S2. (D to I) Gallery of different structures and motifs: (D) short linear nucleosome stack, (E) helical twist, (F) and (G) two chromatin chains interact in parallel to form a hub, and (H) and (I) loops between and within chromatin chains. Scale bar, 20 nm. (J to L) Sculptor models (middle) of nucleosome–linker histone H5 (PDB 4QLC) in EMT densities of chromatin stack, helical twist, and loop (left panels). Overlap of the Sculptor models and EMT densities (right). One Sculptor solution is shown. Cross-correlation values: 82% (J), 92% (K), and 86% (L). Additional Sculptor solutions in fig. S11. Scale bar, 11 nm.

  • Fig. 6 ChromEMT enables chromatin ultrastructure and 3D organization to be visualized in situ in human mitotic chromosomes.

    (A) Mitotic U2OS cells were fixed with glutaraldehyde, stained with DRAQ5, and photo-oxidized for 3 min. Cells were stained with OsO4 and prepared for EM. Scale bar, 10 μm. (B) TEM image (250-nm section). (C) An eight-tilt EMT data set (3200 nm by 3200 nm by 138 nm) comprising 86 TSs was collected of three chromosomes [red box in (B)]. To visualize chromatin ultrastructure and 3D organization as a continuum through mitotic chromosomes, see Movie 3. TS (1.6 nm thick) image of three chromosomes (1, 2, and 3). Inset shows the microtubule structure. Microtubule (solid arrow), membrane fragments (dashed arrow). Scale bar, 100 nm. (D) A magnified image of chromatin in chromosome 2. Scale bar, 40 nm. (E) Chromatin ultrastructure and organization in chromosome 1. The xz and yz cross sections are shown. Scale bar, 100 nm. (F) TS #69 showing chromatin ultrastructure and organization in chromosome 2. The unstained space in between chromatin has a reticular pattern that permeates the 3D volume (dashed arrows). Straight chromatin chains are observed at the borders of unstained venous structures (arrowheads). Scale bar, 100 nm.

  • Fig. 7 In mitotic chromosomes, disordered 5- to 24-nm diameter chromatin chains are packed together at high 3D concentration densities.

    (A) An eight-tilt EMT data set (1024 nm by 1280 nm by 180 nm; 141 total TSs, each 1.28 nm thick) of a mitotic chromosome (white box, left panel) at 29,000× (red box, right panel). Scale bar, 100 nm. To visualize chromatin ultrastructure and 3D organization as a continuum, see Movie 4. (B) Manual measurements of chromatin diameter in a single TS. Scale bar, 50 nm. (C) The mitotic chromosome [red box in (A), 722 nm by 722 nm by 120 nm] was divided into 36 subvolumes of 120-nm cubes. CVCs are shown in a heat map. Asterisks indicate cytoplasmic fractions. Scale bar, 100 nm. (D to F) Surface-thickness estimates of chromatin diameters. There are two major bin distributions: 5 to 12 nm and 12 to 24 nm (right panel). Scale bar, 20 nm. (G to I) Continuous erosion analysis to estimate average chromatin diameter (x-axis intercept).

  • Fig. 8 Disordered 5- to 24-nm-diameter chromatin chains are flexible and can be packed together at different concentration densities in interphase nuclei and mitotic chromosomes.

    (A) Global average of chromatin diameters in interphase and mitotic EMT data sets using continuous erosion analysis. (B) Histogram of subvolume CVC frequencies in interphase and mitotic cells. (C) In mitotic chromosomes, an individual chromatin chain is traced, which bends back on itself at short intervals along its length, forming a compact 3D structure. Left, single TS (1.6 nm thick); right, rendered chromatin surface; middle, overlay. Scale bar, 40 nm. (D) In interphase cells, an individual chromatin chain is traced, which has a more extended curvilinear structure than in mitotic chromosomes. Left, single TS (1.28 nm thick); right, rendered chromatin surface; middle, overlay. Scale bar, 40 nm. (E) Higher-disorder 3D chromatin packing. Chromatin is a flexible disordered 5- to 24-nm-diameter granular chain that is packed together at different 3D volume concentration density distributions in interphase nuclei and mitotic chromosomes.

  • Movie 1 Time-lapse imaging of DAB photo-oxidation upon excitation of DRAQ5-labeled DNA.

    U2OS cells were fixed with glutaraldehyde, DNA-stained with DRAQ5, bathed in DAB, and excited by continuous illumination for 6 min (light flash) under the microscope. The movie shows the live transmitted-light images of DRAQ5-induced DAB photo-oxidation on chromatin in the nucleus. DAB polymerization can be visualized by the appearance of dark precipitates in cell nuclei.

  • Movie 2 The chromatin ultrastructure and 3D organization of the human genome in the nucleus.

    ChromEM staining and multitilt EMT enable the ultrastructure of individual chromatin chains to be resolved and their 3D organization to be visualized as a continuum in the context of the nuclear architecture. Resting human SAECs were fixed, stained with ChromEM, and an eight-tilt EM tomogram (29,000×) was collected of a 250-nm-thick section (SAEC #1 from Fig. 4B). The reconstructed EMT volume is 1206 nm (x) by 1418 nm (y) by 155 nm (z) and comprises 121 TSs (each 1.28 nm thick). We compiled serial TS slices (TS #0 to TS #120) into a movie that enables chromatin to be visualized as a continuum from the top to the bottom of the nuclear volume. Chromatin is a disordered particulate chain that is packed together at different concentrations in the nucleus, with higher densities at the nuclear lamina. The gaps in the nuclear membrane correspond to the insertion sites of nuclear pores.

  • Movie 3 ChromEM and multitilt EMT enable chromatin structure and organization to be visualized in mitotic chromosomes in situ.

    Mitotic U2OS cells (anaphase) were fixed, stained with ChromEM (Fig. 6C), and an eight-tilt EM tomogram (11,000×) was collected of three chromosomes (1, 2, and 3). The reconstructed EMT volume is 3200 nm (x) by 3200 nm (y) by 138 nm (z) comprising 86 TSs (each 1.6 nm thick). We compiled serial TS slices (TS #0 to TS #85) into a movie, enabling chromatin ultrastructure and organization to be visualized as a continuum through large 3D volumes of mitotic chromosomes in situ. Disordered chromatin chains (darkly stained polymers) are packed together at high uniform densities in mitotic chromosome scaffolds. OsO4-stained membrane fragments and microtubule surfaces (parallel lines) can also be seen.

  • Movie 4 The chromatin ultrastructure and 3D organization of mitotic chromosomes.

    A 29,000× reconstructed eight-tilt EMT data set of ChromEM-stained chromatin in a human mitotic chromosome (1024 nm by 1280 nm by 180 nm; 141 total TSs, each 1.28 nm thick) from Fig. 7A. We compiled serial TS slices (TS #0 to TS #140) into a movie, enabling chromatin ultrastructure and organization to be visualized as a continuum.

Supplementary Materials

  • ChromEMT: Visualizing 3D chromatin structure and compaction in interphase and mitotic cells

    Horng D. Ou, Sébastien Phan, Thomas J. Deerinck, Andrea Thor, Mark H. Ellisman, Clodagh C. O'Shea

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

    Download Supplement
    • Materials and Methods
    • Figs. S1 to S13
    • Tables S1 and S2
    • Captions for Movies S1 and S2
    • References

    Images, Video, and Other Media

    Movie S1
    EMT densities of discrete particles in chromatin chains are consistent with nucleosome volumes. In regions with low CVC% (15-17%), we observe chromatin chains that have thin threads decorated with discrete particles (Fig. 5B). Using Chimera we show that DNA/nucleosome particle (PDB: 3W96) dimensions are consistent with chromatin chain particle EMT volumes.
    Movie S2
    EMT densities of discrete particles in chromatin chains are consistent with nucleosome volumes. In regions with low CVC% (14-15%), we observe chromatin chains that have thin threads decorated with discrete particles (Fig. 5C). Using Chimera we show that DNA/nucleosome particle (PDB: 3W96) dimensions are consistent with chromatin chain particle EMT volumes. 28

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