Structural rearrangements of the histone octamer translocate DNA

Abstract

Nucleosomes, the basic unit of chromatin, package and regulate expression of eukaryotic genomes. Nucleosomes are highly dynamic and are remodeled with the help of ATP-dependent remodeling factors. Yet, the mechanism of DNA translocation around the histone octamer is poorly understood. In this study, we present several nucleosome structures showing histone proteins and DNA in different organizational states. We observe that the histone octamer undergoes conformational changes that distort the overall nucleosome structure. As such, rearrangements in the histone core α-helices and DNA induce strain that distorts and moves DNA at SHL 2. Distortion of the nucleosome structure detaches histone α-helices from the DNA, leading to their rearrangement and DNA translocation. Biochemical assays show that cross-linked histone octamers are immobilized on DNA, indicating that structural changes in the octamer move DNA. This intrinsic plasticity of the nucleosome is exploited by chromatin remodelers and might be used by other chromatin machineries.

Fig. 1

Structural plasticity of the nucleosome core particle (NCP). a Cryo-EM maps of the NCP in two distinct conformations. Class 1 (left, blue) resembles canonical nucleosome, whereas Class 2 (right, red) is in distorted conformation. Class 1 is resolved to 3.85 Å and Class 2 to 4.05 Å (0.143 cutoff in FSC curve). Class 1 contains 51 000 particles and Class 2 contains 58 000 particles. b Global changes in the nucleosome structure. Comparison of X-ray structure (PDB:3LZ1) and Class 2 (distorted nucleosome) models. The nucleosome core particle contracts along the symmetry axis by 8% (distance between nucleotides 2 and 38) and expands in the perpendicular direction by 5% (distance between nucleotides 17 and 58). c RMSD between the X-ray structure (PDB:3LZ1) and the Class 2 model, showing the extent of rearrangements in the NCP. The X-ray structure and the Class 2 model were superimposed and RMSD of Cα was calculated and depicted. DNA at SHL 1–2 and SHL 6–7 shows the largest movements between these two structures (> 4 Å). H3 α1, α2, and α3 show the largest rearrangements in the histone octamer

Fig. 2

Structural rearrangements in the histone core. Comparison of the X-ray structure (PDB:3LZ1, yellow) and the Class 2 model (red). Arrows depict the direction of the helix movements. The degree of movement between X-ray structure and the Class 2 model is shown, rounded to half an Å. a Conformational rearrangement of H3 α1 and DNA at SHL 2. H3 α1 moves 4.5 Å in Class 2 when compared with the X-ray structure. DNA at SHL 1.5 moves 4.5 Å towards SHL 2. b Conformational rearrangement of the H3 α2, H3 α1, and DNA at SHL 2. The H3 α2 tilts by 2 Å at its N-terminal end. The H3 α1 moves 4.5 Å. H3 α1 and H3 α2 move toward each other and push the DNA at SHL 2.5 outward. c Conformational rearrangement of H3 α2, H3 α3, and DNA at the dyad. H3 α2 tilts and moves inward by 3 Å at its C-terminal end. H3 α3 also moves inward by 3 Å. Concomitantly, the DNA at the dyad moves more than 3 Å toward the centre of the nucleosome. d Conformational rearrangement of H2B α2. H2B α2 tilts by 2 Å toward the center of the nucleosome at SHL 3.5 and 2 Å away from the center at SHL 5.5. This pushes the DNA at SHL 5.5 outward and pulls the DNA at SHL 3.5 inward

Fig. 3

Structural rearrangement of the histone core distorts the DNA at SHL 2. a Depiction of the DNA organization in the nucleosome in the X-ray structure and Class 2 cryo-EM map at SHL 2. At SHL 1.5, the DNA moves outward and toward SHL 2.5. At SHL 3.5, DNA is pulled inward and toward SHL 2.5. b Close-up view at SHL 2.5 in the Class 2 cryo-EM map. In the Class 2 map, the DNA at SHL 2.5 is less defined and does not resemble a B-DNA helix, indicating distortion in the DNA structure at SHL 2.5. At SHL 1.5 and SHL 3.5, the DNA is well resolved and resembles a B-DNA helix

Fig. 4

Conformational changes in the histone octamer translocate DNA. a Cryo-EM map of the NCP Class 3 (translocated nucleosome) at 4.8 Å (0.143 FSC cutoff). Class 3 contains 39 000 particles. b Global changes in the Class 3 structure. The histone octamer in the Class 3 NCP is 62 Å wide along the symmetry axis (distance between nucleotides 2 and 38) and 70 Å in the perpendicular direction (distance between nucleotides 17 and 58). c, d Fitting of the Class 3 (purple) model into the Class 3 map. On the side A, the H3 α1 interacts with the left DNA strand of the superhelix SHL 1.5 c. On the side B, the H3 α1 interacts with the right DNA strand of SHL 1.5 d. The H4 α1 is detached from the DNA at SHL 1.5 on both sides. e Comparison of the X-ray structure (yellow), the Class 2 (red), and the Class 3 models (purple). The degree of movement between the Class 2 and the Class 3 models is shown. The H3 α1 moves back 3 Å in the Class 3 compared with the Class 2. The H3 α1 reverts to similar position that it occupies in X-ray structures, but now it interacts with another DNA strand. DNA at SHL 1.5 moves 5 Å toward SHL 2 compared with X-ray structures. The H4 α1 moves back 2 Å in the Class 3 compared with the Class 2. f The H4 α1 detached from the DNA at SHL 1.5 and interacts with the DNA at SHL 0.5. DNA at SHL 1.5 moved 4 Å toward SHL 2 compared with X-ray structures and detaches from the H4 α1. DNA at SHL 0.5 moved 3 Å toward SHL 2 compared with X-ray structures and now interacts with the H4 α1. g The model for Class LS_C1 (Class LS reconstructed with C1 symmetry) was fitted into Class LS_C1 and Class 3 cryo-EM maps. In the Class 3 map, DNA at one entry/exit site is shorter than in Class LS_C1 map, indicating that ~ 1 bp of DNA moved toward the other end

Fig. 5

Conformational rearrangement of the histone octamer is required for nucleosome sliding. a Thermal mobilization of nucleosomes on 227 bp DNA sequence containing 601 sequence in the middle. Native nuclesomes are re-positioned at 60 °C. Nucleosomes with cross-linked octamer did not move. b Quantification of three independent thermal shift assays showing that the cross-linked octamer does not move on DNA. The band intensity was quantified at starting and remodeled position at each time point. SD of three independent experiments is shown. c Salt-induced disassembly of native and cross-linked nucleosomes. Cross-linked nucleosomes disassemble at elevated salt concentration, indicating that histone octamer did not cross-link with the DNA. d Thermal mobilization of nucleosomes on 227 bp DNA sequence containing 601 sequence in the middle. NCP with the disulfide bridge between H3 F104C and H4 V43C is immobilized in the thermal shift assay. Upon addition of the reducing agent (DTT), nucleosomes are mobile again. e Model showing the conformational changes in the histone octamer that lead to nucleosome distortion. Tilting of the long α2 helices of H3, H2A, and H2B contracts the nucleosome along the symmetry axis and stretches the nucleosome in the perpendicular direction. This rearrangement of the octamer also moves the DNA. DNA gyres move by > 4 Å at SHL 1.5–2.5 and SHL 5.5–6.5. Canonical nucleosome is shown in gray, distorted in red. f Model showing DNA translocation at SHL 2. In the first step of DNA translocation, DNA and the H3/H4 α1 helices move > 4 Å, leading to nucleosome distortion (Class 2). In the next step, the H3 α1 dissociates from the DNA and translocates to DNA strand, which was previously bound by the H4 α1. The H4 α1 detaches from the DNA at SHL 1.5 and binds the DNA at SHL 0.5. This leads to translocation of the DNA by the histone octamer at SHL 2. The DNA is pushed further and moves by > 5 Å when compared with X-ray structures. Arrows indicate direction of the movement. Canonical nucleosome is shown in gray, distorted in red, and translocated in purple