Nucleosomal arrays self-assemble into supramolecular globular structures lacking 30-nm fibers

Abstract

The existence of a 30-nm fiber as a basic folding unit for DNA packaging has remained a topic of active discussion. Here, we characterize the supramolecular structures formed by reversible Mg(2+)-dependent self-association of linear 12-mer nucleosomal arrays using microscopy and physicochemical approaches. These reconstituted chromatin structures, which we call "oligomers", are globular throughout all stages of cooperative assembly and range in size from ~50 nm to a maximum diameter of ~1,000 nm. The nucleosomal arrays were packaged within the oligomers as interdigitated 10-nm fibers, rather than folded 30-nm structures. Linker DNA was freely accessible to micrococcal nuclease, although the oligomers remained partially intact after linker DNA digestion. The organization of chromosomal fibers in human nuclei in situ was stabilized by 1 mM MgCl2, but became disrupted in the absence of MgCl2, conditions that also dissociated the oligomers in vitro These results indicate that a 10-nm array of nucleosomes has the intrinsic ability to self-assemble into large chromatin globules stabilized by nucleosome-nucleosome interactions, and suggest that the oligomers are a good in vitro model for investigating the structure and organization of interphase chromosomes.

Keywords: 10‐nm chromatin fiber; X‐ray scattering; analytical ultracentrifugation; microscopy; nucleosomal array.

Figure EV1. Differential centrifugation assay for the oligomer formation of 601 nucleosomal array, 5S array, and H1‐array, and fluorescence microscopy (FM) imaging of 5S array oligomers

1. The differential centrifugation assay was performed as described in Gordon et al (2005).

2. 5S array oligomers were stained with DAPI and examined using FM as described in the Materials and Methods section. Shown are representative images obtained in 5 and 10 mM MgCl₂.

3. Control FM images obtained in 0, 1, and 2.5 mM MgCl₂.

Figure 1. Nucleosomal array oligomers are globular

1. Nucleosomal array oligomers were stained with DAPI and examined using FM (fluorescence microscopy) as described in the Materials and Methods section. Shown are representative images obtained in 4.5 and 10 mM MgCl₂.

2. Control FM images obtained in 0, 1, and 2.5 mM MgCl₂.

3. Nucleosomal array oligomers were negatively stained and visualized by TEM as described under Materials and Methods. Shown in the left panels are representative images obtained in 4.5 and 10 mM MgCl₂. Shown in the right panels are images of the interior of the oligomers (white arrows, left panels) after cropping and rescaling.

Figure 2. Sedimentation velocity analysis of the salt‐dependent assembly of nucleosomal array oligomers

1. Representative experiment showing the second moment sedimentation coefficients of the oligomeric nucleosomal arrays as a function of MgCl₂. The second moment sedimentation coefficient is equivalent to the mass average sedimentation coefficient for the entire sample (see Materials and Methods). The inset shows the mean second moment sedimentation coefficient ± the standard error from three replicated experiments.

2. Analysis of the same raw data as in (A) by the time‐derivative method to yield the sedimentation coefficient distribution, g(s*).

Figure EV2. Nucleosomal array oligomerization is reversible upon the removal of salt

Sedimentation velocity analysis of reconstituted nucleosomal arrays in 10 mM Tris pH 7.8, 0.25 mM EDTA, 2.5 mM NaCl (0 mM MgCl₂ buffer). Shown is the integral distribution of sedimentation coefficients (diamonds). A portion of the same sample was then incubated in 8 mM MgCl₂ to induce oligomerization. The oligomers were pelleted, the supernatant was removed from the cell, and the pelleted oligomers were resuspended in 0 mM MgCl₂ buffer. The cell was shaken and left at room temperature for 1 h. Triangles show the integral distribution of sedimentation coefficients of the resuspended oligomers.

Figure EV3. Nucleosomal arrays fold with increasing concentrations of salt

Sedimentation velocity experiments of reconstituted nucleosomal arrays in 0 (blue), 1 (red), and 2.5 mM MgCl₂ (black) analyzed to obtain the integral distribution of sedimentation coefficients.

Figure 3. SAXS profiles of nucleosomal array oligomers and reconstructed in silico models

1. A, B

   SAXS profiles of the nucleosomal arrays in 0 (TE), 1, 2.5 mM MgCl₂ (A), and 5, 10 mM MgCl₂ (B) are shown as plots of log(I × S ²) versus 1/S [I, intensity; S, scattering vector (1/nm)].

2. C

   Two types of nucleosome positioning: face‐to‐face, at ˜6‐nm spacing, and edge‐to‐edge, at ˜11‐nm spacing. The image was made based on the structural information published in Luger et al (1997).

3. D

   The modeled scattering profile of an extended dinucleosome structure based on its atomic coordinate (for details, see Materials and Methods). Note that the modeled profile is similar to that of the nucleosomal arrays in 0 mM MgCl₂ (A).

4. E

   Two structural models of 12‐mer 30‐nm fibers: solenoid (left) and zigzag (right) as a top and side view. The models were constructed using MolScript (Kraulis, 1991).

5. F

   The scattering profiles of the solenoid (red line) and zigzag (blue line) 30‐nm fibers were made from their atomic coordinates computationally. Note that the 30‐ to 40‐nm peak is prominent in both fiber models.

Figure 4. Effect of MNase digestion on oligomer structure

1. 601 nucleosomal arrays were incubated in 0.5, 5 mM MgCl₂, 5 mM MgCl₂ + MNase and analyzed by the differential centrifugation assay to determine the fraction oligomeric. The amounts of DNA in the supernatant fraction were measured. Note that for the MNase‐digested oligomers, the supernatant fraction also includes the digested free linker DNA. Each value is the mean of three measurements, and the error bars represent the standard deviation.

2. Verification of complete MNase digestion. DNA was purified from the nucleosomal arrays incubated in 5 mM MgCl₂ or 5 mM MgCl₂ + MNase and then electrophoresed on agarose gel. The position of mononucleosome is marked with a star symbol.

3. Nucleosomal array oligomers without (left) or with MNase treatment (right) were stained with DAPI and examined using FM. Shown are representative images obtained. Note that the sizes of MNase‐treated oligomers are much smaller than those of the control oligomers (left and Fig 1A).

Figure 5. H1‐oligomers are globular

1. Oligomers assembled from H1‐nucleosomal arrays were stained with DAPI and examined using FM. Shown are representative images obtained in 4 and 5 mM MgCl₂.

2. Control FM images obtained in 0, 1, and 3 mM MgCl₂.

3. H1‐nucleosomal array oligomers were negatively stained and visualized by TEM. Shown in the left panel is a representative image obtained in 4 mM MgCl₂. Shown in the right panel is an image of the interior of the oligomer (white arrow, left panel) after cropping and rescaling.

Figure 6. Sedimentation analysis of salt‐dependent H1‐oligomer assembly

1. Representative experiment showing the second moment sedimentation coefficients of the H1‐oligomers as a function of MgCl₂. The dashed line indicates the upper limit of measurable sedimentation coefficients (˜10⁶ S). The white symbol is intended to show that the sedimentation coefficient of the H1‐oligomers in 5 mM MgCl₂ is beyond the detectable limit.

2. Analysis of the same raw data as in (A) by the time‐derivative method to yield the sedimentation coefficient distribution, g(s*).

3. The g(s*) profiles in 2.5, 3, and 3.5 mM from (B) are re‐plotted on a smaller scale.

Figure EV4. H1‐oligomers are smaller than nucleosomal array oligomers at equivalent extents of self‐association

1. The second moment sedimentation coefficients for oligomeric nucleosomal arrays (black) and H1‐arrays (red) are plotted against the fraction of the sample that is oligomeric.

2. The sedimentation data for nucleosomal arrays and H1‐nucleosomal arrays in 4 mM MgCl₂ were analyzed by the time‐derivative method to obtain the g(s*), which was then converted to the distribution of Mb DNA per oligomer as in Table 1.

Figure EV5. H1‐array folds extensively in the presence of salt

Shown are the integral distributions of sedimentation coefficients obtained for H1‐arrays in 0, 1, and 2.5 mM MgCl₂.

Figure 7. SAXS profiles of the H1‐oligomers, native chicken chromatin, and reconstructed in silico oligomer models

1. A, B

   SAXS profiles of the H1‐nucleosomal arrays in 0 (TE), 1, 2.5 mM MgCl₂ (A), and 5, 10 mM MgCl₂ (B) are shown as plots of log(I × S ²) versus 1/S [I, intensity; S, scattering vector (1/nm)].

2. C, D

   SAXS profiles of the native chicken chromatin in 0 (TE), 0.5, 1 mM MgCl₂ (C), and 2.5, 5 mM MgCl₂ (D) are shown as plots of log(I × S ²) versus 1/S [I, intensity; S, scattering vector (1/nm)].

3. E

   The “in silico oligomer” models were constructed in environments containing 100, 50, and 25 randomly and tightly packed 12‐mer 30‐nm fiber models (Fig 3E). The nucleosome concentration was 0.5 mM. The 100‐fiber model was drawn using MolScript (Kraulis, 1991). The broken‐lined squares show magnified regions.

4. F

   The modeled scattering profiles, yielding average among‐model values, have prominent peaks at ˜30–40, 11, and 6 nm. This shows that the in silico oligomers retain characteristics of 30‐nm fibers. Note that the modeled scattering profile is very distinct from the SAXS profiles in (B) and (D), Fig 3B, and Fig 8B (center and right).

Figure 8. Effect of MgCl₂ concentration on the chromatin structure of isolated HeLa nuclei

1. FM images of chromatin structure in the nuclei with 0 (left), 1 (center), and 5 mM MgCl₂ (right). Insets show the intensity line profiles between the two marked arrow heads in the images.

2. SAXS profiles of the isolated HeLa nuclei. SAXS profiles of the nuclei in 0 (left), 1 (center), and 5 mM MgCl₂ (right) are shown in blue. For comparison, the scattering curves of H1‐arrays from Fig 7A and B were overlaid as red lines (left, 0 mM; center and right, 10 mM MgCl₂).

3. Model scheme. The 12‐mer nucleosomal array is a well‐defined model chromatin system. In 1–2 mM Mg²⁺, the nucleosomal array folds into a folded 30‐nm chromatin fiber structure. With further increases in Mg²⁺, the nucleosome arrays assemble into supramolecular oligomers. The large oligomers are not assemblies of the 30‐nm chromatin fibers, but are proposed to be interdigitated and melted structures of 10‐nm nucleosomal arrays.