Cryo-EM of nucleosome core particle interactions in trans

Abstract

Nucleosomes, the basic unit of chromatin, are repetitively spaced along DNA and regulate genome expression and maintenance. The long linear chromatin molecule is extensively condensed to fit DNA inside the nucleus. How distant nucleosomes interact to build tertiary chromatin structure remains elusive. In this study, we used cryo-EM to structurally characterize different states of long range nucleosome core particle (NCP) interactions. Our structures show that NCP pairs can adopt multiple conformations, but, commonly, two NCPs are oriented with the histone octamers facing each other. In this conformation, the dyad of both nucleosome core particles is facing the same direction, however, the NCPs are laterally shifted and tilted. The histone octamer surface and histone tails in trans NCP pairs remain accessible to regulatory proteins. The overall conformational flexibility of the NCP pair suggests that chromatin tertiary structure is dynamic and allows access of various chromatin modifying machineries to nucleosomes.

Figure 1

Nucleosome core particle dimers. (A) Representative cryo-EM raw micrographs collected on a Falcon II camera with a Titan Halo electron microscope at 300 keV. Many NCP pairs are also visible. Several NCP pairs are depicted by red circles. (B) Representative 2D class averages showing pairs of nucleosome core particles in many different orientations. Red area: particles with two histone octamers facing each other. Blue area: particles with the histone octamer facing the DNA of the second NCP. Green area: particles with the two DNA regions of the NCPs facing each. In nearly all cases, the gyre of the DNA around the histone core is visible.

Figure 2

NCP pairs can adopt multiple conformations. Cryo-EM maps of the classes of NCP pairs showing their diverse relative orientations. NCP 1, for which the orientation is fixed between classes, is shown in light blue, and NCP 2 is shown in red. The number of particles for each class is indicated. Note that the strongest contact observed in classes A1–A3 is formed proximal to the entry/exit site of NCP 1 and the DNA of NCP 2.

Figure 3

Molecular model showing long range NCP interactions. (A) Comparison of the NCP models for cryo-EM maps A1–A5. NCP 1 is shown in blue and NCP 2 is shown in red. The two NCPs are oriented with their histone octamers facing each other, but are shifted vertically to different degrees. The dyad of both NCPs is facing in the same direction. First 37 residues of the H3-tail, not observed in X-ray structure (pdb 3lz1), are indicated as a dotted line in light blue. In most classes, the histone octamer surface remains accessible to the extrinsic factors. (B) The model showing NCP orientation in the 30 nm fiber between adjacent tetra-nucleosomal units is included for comparison. The 30 nm fiber model was built using 30 nm fiber cryo-EM map.

Figure 4

H4 tail makes weak interactions with H2A/H2B or DNA. (A) A slice through the NCP pairs of classes A1-A5 showing weaker interactions at the histone octamer interface. These contacts are presumably formed by the histone H4 tail of one NCP and the H2A/H2B acidic patch or the DNA of the second NCP. The cryo-EM density for both NCPs is shown in transparent blue. The molecular model for NCP 1 is shown in blue and for NCP 2 in red. (B) The cryo-EM map of the combined reconstruction of the classes A1–A3. On the histone octamer side without the adjacent NCP 2, the strong density where the N-terminus of H4 interacts with DNA at superhelix SHL 2 is visible. On the opposite side, with the adjacent NCP 2, the density for the H4 tail is not visible. This indicates that the H4 tail dissociates from the DNA of the NCP 1 and engages in intra-nucleosomal interactions.