The three-dimensional architecture of chromatin in situ: electron tomography reveals fibers composed of a continuously variable zig-zag nucleosomal ribbon.

Abstract

Abstract. The three dimensional (3D) structure of chromatin fibers in sections of nuclei has been determined using electron tomography. Low temperature embedding and nucleic acid-specific staining allowed individual nucleosomes to be clearly seen, and the tomographic data collection parameters provided a reconstruction resolution of 2.5 nm. Chromatin fibers have complex 3D trajectories, with smoothly bending regions interspersed with abrupt changes in direction, and U turns. Nucleosomes are located predominantly at the fiber periphery, and linker DNA tends to project toward the fiber interior. Within the fibers, a unifying structural motif is a two nucleosome-wide ribbon that is variably bent and twisted, and in which there is little face-to-face contact between nucleosomes. It is suggested that this asymmetric 3D zig-zag of nucleosomes and linker DNA represents a basic principle of chromatin folding that is determined by the properties of the nucleosome-linker unit. This concept of chromatin fiber architecture is contrasted with helical models in which specific nucleosome-nucleosome contacts play a major role in generating a symmetrical higher order structure. The transcriptional control implications of a more open and irregular chromatin structure are discussed.

Evidence for heteromorphic chromatin fibers from analysis of nucleosome interactions

“…S6b). However, despite this broad conformational variation, the resulting nucleosome chain folds into an energetically stable fiber with uniform diameter of 31-33 nm consistent with the diameters and extended stiff conformations of long chromatin fibers revealed by ultrastructural analysis in vitro and in situ (11,17,23). Thus, our modeling and experimental approaches both converge to reveal a principle of chromatin fiber organization that reconciles the 2-start zigzag topology with linker DNA bending in one heteromorphic chromatin fiber structure.…”

“…Because DNA conformation in chromatin and nucleosome packing are intimately connected to DNA/protein recognition and gene regulation, there has been intense interest in understanding chromatin structure, energetics, and dynamics. Some experimental studies have suggested that nucleosomal arrays fold in a zigzag arrangement with relatively straight linkers and a 2-start nucleosome interaction pattern that brings each nucleosome in proximity to its second nearest neighbor (7)(8)(9)(10) consistent with chromatin fibers observed in situ (11). Other evidence suggests that chromatin condensed with linker histones and divalent cations such as Mg 2ϩ can form either zigzag structures with various nucleosome topologies (12,13) or solenoid-like arrangements.…”

“…S7 suggests how the bent linkers may destabilize i Ϯ 1 interactions in cis (within fibers) and promote nucleosome interactions in trans (between fibers). Internucleosomal interactions in trans may account for partially interdigitated 30-nm fibers in the interphase nucleus (11) and for global chromatin condensation in metaphase chromosomes where divalent cation levels are sharply increased (53) and no distinct 30 nm fibers are observed (54).…”

A metastable structure for the compact 30‐nm chromatin fibre

“…In addition to early EM images 3 a 2‐start structure is supported by Fourier analysis of images of chromatin fibres 2, chemical cross‐linking 4, the crystal structure of a tetranucleosome 5, cryo‐EM of native fibres 6, cryo‐EM structures of reconstituted fibres containing up to 24 nucleosomes 7 and cryo‐tomographic analysis of native chromatin 8, 9. Nevertheless, early studies on various native chromatin samples by photochemical dichroism 10 and X‐ray diffraction 11, 12, revealed, respectively, a small tilt of the nucleosomal disc relative to the fibre axis and narrow diffraction arcs at 110 Å.…”

The regulatory role of DNA supercoiling in nucleoprotein complex assembly and genetic activity

“…There is now compelling experimental evidence that the '30-nm' fiber can adopt a 2-start structure. In addition to early EM images (Woodcock et al 1984), a 2-start structure is supported by Fourier analysis of images of chromatin fibers (Williams et al 1986), chemical crosslinking (Dorigo et al 2004), the crystal structure of a tetranucleosome (Schalch et al 2005), cryo-EM of native fibers (Bednar et al 1998), cryo-EM structures of reconstituted fibers containing up to 24 nucleosomes (Song et al 2014), and cryo-tomographic analysis of native chromatin (Horowitz et al 1994;Scheffer et al 2011). Nevertheless, early studies on various native chromatin samples by photochemical dichroism (Sen et al 1986) and X-ray diffraction (Widom and Klug 1985; revealed, respectively, a small tilt of the nucleosomal disc relative to the fiber axis and narrow diffraction arcs at 110 Å.…”