Regulation of chromatin folding by conformational variations of nucleosome linker DNA

Abstract

Linker DNA conformational variability has been proposed to direct nucleosome array folding into more or less compact chromatin fibers but direct experimental evidence for such models are lacking. Here, we tested this hypothesis by designing nucleosome arrays with A-tracts at specific locations in the nucleosome linkers to induce inward (AT-IN) and outward (AT-OUT) bending of the linker DNA. Using electron microscopy and analytical centrifugation techniques, we observed spontaneous folding of AT-IN nucleosome arrays into highly compact structures, comparable to those induced by linker histone H1. In contrast, AT-OUT nucleosome arrays formed less compact structures with decreased nucleosome interactions similar to wild-type nucleosome arrays. Adding linker histone H1 further increased compaction of the A-tract arrays while maintaining structural differences between them. Furthermore, restriction nuclease digestion revealed a strongly reduced accessibility of nucleosome linkers in the compact AT-IN arrays. Electron microscopy analysis and 3D computational Monte Carlo simulations are consistent with a profound zigzag linker DNA configuration and closer nucleosome proximity in the AT-IN arrays due to inward linker DNA bending. We propose that the evolutionary preferred positioning of A-tracts in DNA linkers may control chromatin higher-order folding and thus influence cellular processes such as gene expression, transcription and DNA repair.

Figure 1.

Design and construction of oligonucleosome arrays with directed linker DNA bending. (A and B) Frequencies of occurrence of (A) short A-tracts (A_nT_m, n+m ≥ 3) and (B) long A-tracts (A_nT_m, n+m ≥ 5) in the cores and flanking regions of chicken nucleosomes. The frequencies are ‘symmetrized’ with respect to the dyad at position 74 (data from (34)). (C) Models of WT and A-tract containing nucleosome dimers based on X-ray crystal structure of clone 601 mononucleosome with 145 bp core (3MVD, (44)) and 43 (WT) or 44 (AT-IN and AT-OUT) bp linker DNA shown in (D) and Supplementary Figure S1. (D) Linker DNA sequences of the oligonucleosome constructs. Wild-type (WT) linkers have no specific DNA sequence while AT-IN linkers have A-tracts located at −5, −15, −25 in relation to the nucleosome binding core and AT-OUT linkers have A-tracts located at 0, −10, −20 in relation to the nucleosome binding core. Sequence numbering is according to (51). The term ‘inward’ bending of the linker DNA (AT-IN) describes a trajectory toward the nucleosome core dyad axis and the ‘outward’ bending (AT-OUT)—a trajectory away from the dyad axis.

Figure 2.

Oligonucleosome array conformation is influenced by linker DNA sequence. (A) Distributions of sedimentation coefficients, c(S), for WT and A-tract core arrays in standard HNE buffer (5 mM NaCl). (B) Electron micrographs (uranyl acetate staining, dark-field imaging) of WT and A-tract core arrays fixed in HNE with 5 mM NaCl. (C) Distributions of sedimentation coefficients, c(S), for WT and A-tract core arrays in HNE with 150 mM NaCl. (D) Sedimentation coefficient averages of three independent experiments for WT and A-tract core arrays in HNE with 5 mM NaCl and 150 mM NaCl. Error bars show standard deviations (SD). P-values represent probabilities associated with Student's t-test.

Figure 3.

EMANIC analysis of internucleosome interactions in WT, AT-OUT and AT-IN 12-mer nucleosome arrays. (A) Scheme of the EMANIC procedure showing nucleosome interactions most frequently observed by EMANIC (21). (B) Bar graphs show counting of internucleosome interaction in various conditions after fixing using the EMANIC method. Internucleosome interactions of core nucleosome arrays scored either with or without cross-linking (control) or after formaldehyde cross-linking in the presence of 5 mM NaCl (HNE), 150 mM NaCl or 1 mM MgCl₂. Bar graphs on the left (Y-axes) show the total % of nucleosomes involved in no interactions (beads), doublets (i ± 1), loops (i ± 2 or more) and trans interactions (interactions between core arrays). Bar graphs on the right (Y-axes) show the fraction (percentage) of nucleosomes involved in doublet (i ± 1) and loop (i ± 2 to i ± >7) interactions.

Figure 4.

Longer oligonucleosome arrays amplify the impact of A-tract sequences in linker DNA regions. (A–C) Distributions of sedimentation coefficients, c(S), for WT and A-tract core arrays in HNE buffer with 5 mM NaCl (A), 150 mM NaCl (B) and 150 mM NaCl + 1 mM MgCl₂ (C). (D) Sedimentation coefficient averages of three independent experiments for WT and A-tract core arrays in HNE buffer with 5 mM NaCl, 150 mM NaCl and 150 mM NaCl + 1 mM MgCl₂. Error bars show SD.

Figure 5.

Electron microscopy analysis of the impact of A-tract sequences on chromatin compaction. (A) Electron micrograph (uranyl acetate staining, dark-field imaging) of WT and A-tract core arrays fixed in HNE buffer with 5 mM NaCl (left panel) and 150 mM NaCl (right panel). (B) Measurements of fiber diameter (nm) of WT and A-tract arrays under the specified conditions. (C) Number of nucleosomes per unit length (11 nm) of WT and A-tract arrays under the specified conditions. Error bars: SD.

Figure 6.

Linker histone H1 promotes compaction of 12-mer A-tract constructs. (A and B) Distributions of sedimentation coefficients, c(S), for WT and A-tract 12-nucleosome arrays in standard buffer with 5 mM NaCl (A) and 150 mM NaCl (B). (C) Averages of sedimentation experiments for WT and A-tract core arrays under 5 mM NaCl and 150 mM NaCl. (D) Distribution of sedimentation coefficients, c(S), for WT and A-tract 22–24 nucleosome arrays at 150 mM NaCl. (E–G) Electron micrographs (E) and measurements of fiber diameter (F) and number of nucleosomes per unit length (G) of WT, AT-OUT and AT-IN nucleosome arrays reconstituted with linker histone H1 and fixed at 150 mM NaCl. Error bars: SD.

Figure 7.

Linker DNA accessibility is more restricted in AT IN core arrays compared to AT OUT. (A–C) Time course of AT IN 24-mer (A) and AT OUT 22-mer (B) and 24-mer (C) digestion with MspI restriction enzyme. Digestion with this MspI cuts directly in the linker DNA region and results in 189 bp ladder fragments. Each gel shows 1 kb m.w. markers on the left, 100 bp m.w. markers on the right and DNA obtained after RE digestion of reconstituted nucleosome arrays for 0–40 min. (D) Graph showing the rate of the intact DNA band decay with linear approximation for the first-order reaction (y-axis shows logarithm (ln) of the normalized intact band concentration) per digestion time (min).

Figure 8.

Typical conformations of AT-IN and AT-OUT 12-nucleosome arrays obtained by MC simulations. Models show snapshots representing averaged MC simulations of 12-mer AT-IN (A and B) and AT-OUT (C and D) models with the energy −2.7 kT for the inter-nucleosome stacking interactions. Shown are two projections: perpendicular to the main fiber axis (A and C) and along the main fiber axis (B and D). Also shown are predicted sedimentation coefficient (s₂₀^o_,w) averages and predicted apparent outer fiber diameters calculated by fitting into minimal cylinders. The fiber snapshots shown here were selected so that their predicted sedimentation coefficients are close to the average values observed during MC simulations.