Frozen-hydrated chromatin from metaphase chromosomes has an interdigitated multilayer structure

Abstract

Cryo-electron tomography and small-angle X-ray scattering were used to investigate the chromatin folding in metaphase chromosomes. The tomographic 3D reconstructions show that frozen-hydrated chromatin emanated from chromosomes is planar and forms multilayered plates. The layer thickness was measured accounting for the contrast transfer function fringes at the plate edges, yielding a width of ~ 7.5 nm, which is compatible with the dimensions of a monolayer of nucleosomes slightly tilted with respect to the layer surface. Individual nucleosomes are visible decorating distorted plates, but typical plates are very dense and nucleosomes are not identifiable as individual units, indicating that they are tightly packed. Two layers in contact are ~ 13 nm thick, which is thinner than the sum of two independent layers, suggesting that nucleosomes in the layers interdigitate. X-ray scattering of whole chromosomes shows a main scattering peak at ~ 6 nm, which can be correlated with the distance between layers and between interdigitating nucleosomes interacting through their faces. These observations support a model where compact chromosomes are composed of many chromatin layers stacked along the chromosome axis.

Keywords: DNA packaging; chromatin higher‐order structure; cryo‐electron tomography; metaphase chromosome structure; small‐angle X‐ray scattering.

Figure 1. Examples of cryo‐tomograms containing plates emanated from metaphase chromosomes

1. A

   Whole chromosomes imaged by conventional TEM.

2. B

   Slice from a large tomographic volume; part of the carbon film surrounding a hole with vitrified ice containing the plates is indicated with an asterisk; the inset illustrates that the slice of a plate corresponds to a line in the x‐y plane (perpendicular to the direction of the electron beam).

3. C–G

   Slices from different tomograms and the corresponding 3D segmentations showing plates with different sizes and shapes. In addition to a typical slice through the x‐y plane (yellow), two slices through the x‐z (red) and y‐z (green) planes (orthogonal to the x‐y plane) are shown in (C).

Data information: Scale bars: 1 µm (A); 200 nm (B, D), 100 nm (E–G), 50 nm (C).

Figure 2. Cryo‐tomograms of compact plates and distorted plates decorated with nucleosomes

1. A

   Nucleosomes (N) decorating a relaxed plate (P1); insets in the upper right show additional examples. Nucleosomes are not visible as individual units in typical compact plates (P2). Short compact interdigitated solenoids are shown in the main image (S) and in the bottom‐left insets.

2. B–E

   Slice (B) and segmentations in three different orientations (C) of a large relaxed plate decorated with many nucleosomes on its right side. Slice (D) and segmentation (E) of a relaxed plate forming a tube decorated with nucleosomes.

3. F

   Structure of the decorative particles (like those shown in N, panel A) after subtomogram averaging. The final density map was filtered to 25 Å and fitted with the molecular structure of the nucleosome core particle (Protein Data Bank code 2CV5).

Data information: Scale bars: 50 nm (A, including the insets, B–E); 5 nm (F).

Figure EV1. Subtomogram averaging of decorative nucleosomes

Initial random reference and averaged subvolumes of decorative nucleosomes like those shown in Fig 2A (region N) through 15 reference‐free alignment iterations; the averaged maps are shown without filtering. Scale bar: 5 nm.

Figure EV2. Measurement of plate thickness, adjusted for CTF fringes

1. The discontinuous yellow lines indicate the position of the CTF fringes on a select region of a plate. These fringes (black in reverse contrast) surround both sides of the plates in the tomograms. Scale bar: 100 nm.

2. Example of an intensity profile along a vector perpendicular to a plate. Because of the CTF, the plate's intensity values do not have a hard edge, but rather gradually slope from the peak intensities of the plate to the valley of the surrounding CTF fringes. The plate thickness values presented in Table 1 correspond to the distance between the points (indicated in blue), approximately halfway between the peak and the valley. The intensities at these measurement points also roughly correspond to the background intensity of the tomogram. AU, arbitrary units.

Figure 3. Slices from tomographic volumes containing multilayered plates

1. A, B

   Plates with several layers that are not closely appressed.

2. C–E

   Large multilayer plates having the size of human metaphase chromatids [∼0.6 µm diameter (Daban, 2014)] (C); the inset schematically shows the perpendicular orientation of chromatin layers with respect to the chromatid axes proposed in the thin‐plate model (Gállego et al, 2009; Castro‐Hartmann et al, 2010). In other slices (D, E), the multilayer structures shown in (C) are more compact and the individual layers are not visible as separate elements.

Data information: Scale bars: 50 nm.

Figure EV3. Additional examples of large multilayered plates

Slices from tomographic volumes containing large multilayered plates. Scale bars: 100 nm.

Figure 4. Cryo‐tomograms of plates showing two‐layer contacts and edge‐to‐edge interactions

1. A–C

   Slices showing lateral association between two plates and the corresponding 3D segmentations.

2. D

   Edge‐to‐edge interactions form cylindrical structures.

Data information: Scale bars: 50 nm.

Figure EV4. Contact between two plates

1. Slice showing a region of a tomographic volume in which there are two independent plates (left); in another region, these two plates are in close contact (right). Scale bar: 50 nm.

2. Intensity profile (red) along a vector perpendicular to the two independent plates shown in (A), and profile (blue) of the same plates in a region in which they are in contact. AU, arbitrary units.

Figure 5. SAXS profiles of metaphase chromosomes under different conditions

1. A–C

   The cation concentrations used are indicated for each experiment; AU, arbitrary units.

Figure 6. Schematic drawing showing the main structural elements of two layers in close contact

Only a few nucleosomes are shown to illustrate the main dimensions and the interdigitation of two layers. Our results suggest that the two turns of the nucleosomal DNA are oriented slightly tilted with respect to the axis normal to the plate surface, but they may have diverse orientations (not represented in this scheme) with respect to the other two axes of the plate. The thickness of single‐ and double‐layer plates (~ 7.5 and ~ 13 nm, respectively) obtained from cryo‐tomograms (Table 1) is indicated in blue. The main scattering peak at ~ 6 nm observed in SAXS experiments with condensed chromosomes under metaphase ionic conditions (Fig 5B) is probably due to the repetitive distances between nucleosomes (face‐to‐face interactions) and between stacked layers; these distances (~ 6 nm) are indicated in red.