Nucleosome spacing periodically modulates nucleosome chain folding and DNA topology in circular nucleosome arrays

Abstract

The length of linker DNA that separates nucleosomes is highly variable, but its mechanistic role in modulating chromatin structure and functions remains unknown. Here, we established an experimental system using circular arrays of positioned nucleosomes to investigate whether variations in nucleosome linker length could affect nucleosome folding, self-association, and interactions. We conducted EM, DNA topology, native electrophoretic assays, and Mg²⁺-dependent self-association assays to study intrinsic folding of linear and circular nucleosome arrays with linker DNA length of 36 bp and 41 bp (3.5 turns and 4 turns of DNA double helix, respectively). These experiments revealed that potential artifacts arising from open DNA ends and full DNA relaxation in the linear arrays do not significantly affect overall chromatin compaction and self-association. We observed that the 0.5 DNA helical turn difference between the two DNA linker lengths significantly affects DNA topology and nucleosome interactions. In particular, the 41-bp linkers promoted interactions between any two nucleosome beads separated by one bead as expected for a zigzag fiber, whereas the 36-bp linkers promoted interactions between two nucleosome beads separated by two other beads and also reduced negative superhelicity. Monte Carlo simulations accurately reproduce periodic modulations of chromatin compaction, DNA topology, and internucleosomal interactions with a 10-bp periodicity. We propose that the nucleosome spacing and associated chromatin structure modulations may play an important role in formation of different chromatin epigenetic states, thus suggesting implications for how chromatin accessibility to DNA-binding factors and the RNA transcription machinery is regulated.

Keywords: DNA topology; chromatin higher order structure; chromatin structure; computer modeling; conformational simulation; electron microscopy (EM); epigenetic regulation; epigenetics; gene regulation; histone; linker DNA; nucleosome.

Figure 1.

Folding of linear nucleosome arrays shows a periodic linker DNA length dependence. A and B, DNP electrophoresis of linear 183 × 12 and 188 × 12 arrays fixed by glutaraldehyde under the specified conditions. C and D, distributions of sedimentation coefficients, c(S), for 183 × 12 and 188 × 12 linear nucleosome arrays in the compact form (150 mm NaCl). E, sedimentation coefficient averages of four independent experiments for 183 × 12 and 188 × 12 arrays at 5 mm NaCl and 150 mm NaCl. F, comparison of main sedimentation coefficient peaks for 12-mer oligonucleosome linear arrays with varying NRL (165,167,169, 172, 177, 200, 205, 207, and 209 bp from Ref. and 183 and 188 bp from this work) at 5 mm NaCl, 150 mm NaCl, and 1 mm MgCl₂. G, box graphs show numbers of nucleosomes per unit length (11 nm) of 183 × 12 and 188 × 12 linear arrays at 5 mm NaCl and 150 mm NaCl. The graphs show the median (horizontal lines), the mean (X-cross), and data points (diamonds) within the standard deviations (box) as well as minimal and maximal points (whiskers). H, electron micrograph (uranyl acetate staining, dark-field imaging) of 183 × 12 and 188 × 12 arrays fixed at 5 mm NaCl, standard buffer (left panels) and 150 mm NaCl (right panels). Error bars in bar graphs show S.D. p values represent probabilities associated with Student's t test.

Figure 2.

DNA topology in circular nucleosome arrays containing 183 and 188 bp NRL nucleosome arrays depends on nucleosome spacing. A, plasmid-based circular DNA templates p-188 × 12 (lanes 2, 4, 6, 8) and p-183 × 12 (lanes 3, 5, 7, 9) were reconstituted with 0, 0.34, 0.8, and 1.0 histone octamer per nucleosome (shown on the top), treated with topoisomerase I, the circular DNA was isolated and separated on agarose gels run in the presence of 1.5 μg/ml CQ in the gel and electrode buffer. Lane 1, molecular weight markers. The numbers of superhelical turns (ΔLk) in the selected DNA topoisomers (indicated by red arrows) are given on the left and right margins of the gel. B, difference between the strongest topoisomers in the 188 and 183 bp NRL constructs, determined by gel scanning, Δ(ΔLk) = 4 sc at 100% loading of core histones. C, nucleosomes were reconstituted on the ligated DNA 188 × 12 and 183 × 12 minicircle templates, treated with topoisomerase I, and DNA separated on agarose gels run in the presence of 16 μg/ml CQ. M, molecular weight markers; sc188, supercoiled naked 188 × 12 minicircle DNA (Fig. S5); 183-NA and 188-NA, minicircle nucleosome arrays 183 × 12 and 188 × 12. The strongest topoisomers are characterized by the numbers of superhelical turns evaluated from comparison with sc188 having 12 negative supercoils (see the bands marked by asterisks and indicated by red arrows). D, Δ(ΔLk) determined by gel scanning equals 3.5 sc at 100% loading of core histones.

Figure 3.

Circular nucleosome arrays with 183- and 188-bp NRLs show similar degree of salt compaction independent of linker DNA length and topology. A, electron micrographs (uranyl acetate staining, dark-field imaging) of circular p-183 × 12 and p-188 × 12 arrays fixed at 5 mm NaCl, standard buffer (left panels) and 150 mm NaCl (supercoiled, middle and topoisomerase-relaxed, right panels). B, box graphs show numbers of nucleosomes per unit length (11 nm) of topoisomerase-relaxed and supercoiled circular p-183 × 12 and p-188 × 12 arrays at 5 mm NaCl and 150 mm NaCl. Elements of the graph are as described in the legend for Fig. 1G. C, DNP electrophoresis of supercoiled and relaxed circular p-183 × 12 and p-188 × 12 arrays cross-linked under the specified conditions.

Figure 4.

Chromatin Mg²⁺-dependent self-association is independent of the NRL, DNA topology, and unconstrained supercoiling. A, DNA electrophoresis showing separation of covalently closed (longer gray arrow) and nicked (shorter white arrow) DNA from circular plasmids. B, DNA from a covalently closed (cc) band (gray arrow) and nicked band (white arrow) was extracted from the gel (A) and separated on an agarose gels run in the presence of 1.5 μg/ml CQ in the gel and electrode buffer. C, rates of Mg²⁺-dependent self-association in linear and circular (relaxed and supercoiled) nucleosome arrays. D, graphs showing the concentration of Mg²⁺ at which 50% of the material is precipitated. Error bars, S.D.

Figure 5.

EMANIC analysis of internucleosomal interactions within the linear and circular nucleosome arrays. A, representative TEM images of the folded and unfolded formaldehyde–cross-linked p-183 × 12 nucleosome arrays and scheme of EMANIC scoring showing some frequently observed internucleosomal interactions. B–E, box graphs show percentage of individual loops (i ± 1 to i ± 7), and combined larger loops (i ± 8 to i ± 11) within linear and circular nucleosome arrays scored either without cross-linking (control) or after formaldehyde cross-linking in the presence of 150 mm NaCl. Elements of the graph are as described in the legend for Fig. 1G. F and G, bar graphs show difference in percentage of cross-linked interactions minus non–cross-linked controls for individual loops (i ± 1 to i ± 7), and combined larger loops (i ± 8 to i ± 11). H and I, bar graphs show Student's t test p values for the difference in percent of cross-linked interactions minus non–cross-linked controls.

Figure 6.

3D computational modeling of 12-mer oligonucleosome arrays with varying linker DNA lengths. A and B, images of typical conformers obtained during MC simulations of 183 × 12 (A) and 188 × 12 (B) models. Shown are the projections perpendicular to the main fiber axis. Note that the 183 × 12 (A) structure is less compact than the 188 × 12 (B) structure. Cross-linkable contacts between nucleosomes are indicated by black arrows (i ± 2) and red arrows (i ± 3). C, FINI calculated for the MC ensembles of 183 × 12 (red) and 188 × 12 (blue) nucleosome arrays for E = 5 kT, with dynamic linker DNA unwrapping (see “Experimental procedures”). D, predicted sedimentation coefficient (s_20,w⁰) calculated for MC ensembles of 12-mer arrays with NRL varying between 162 and 212 bp with stacking interaction E = −5.0 kT and dynamic linker DNA unwrapping. S.D.s are shown as vertical bars. Experimental s_20,w⁰ values obtained at 1 mm MgCl₂ (blue squares) or 150 mm NaCl (yellow triangles) were taken from Fig. 1F.