The compaction level of arrays of nucleosomes may be understood in terms of the balance between the selfrepulsion of DNA (principally linker DNA) and countering factors including the ionic strength and composition of the medium, the highly basic N termini of the core histones, and linker histones. However, the structural principles that come into play during the transition from a loose chain of nucleosomes to a compact 30-nm chromatin fiber have been difficult to establish, and the arrangement of nucleosomes and linker DNA in condensed chromatin fibers has never been fully resolved. Based on images of the solution conformation of native chromatin and fully defined chromatin arrays obtained by electron cryomicroscopy, we report a linker histone-dependent architectural motif beyond the level of the nucleosome core particle that takes the form of a stem-like organization of the entering and exiting linker DNA segments. DNA completes Ϸ1.7 turns on the histone octamer in the presence and absence of linker histone. When linker histone is present, the two linker DNA segments become juxtaposed Ϸ8 nm from the nucleosome center and remain apposed for 3-5 nm before diverging. We propose that this stem motif directs the arrangement of nucleosomes and linker DNA within the chromatin fiber, establishing a unique threedimensional zigzag folding pattern that is conserved during compaction. Such an arrangement with peripherally arranged nucleosomes and internal linker DNA segments is fully consistent with observations in intact nuclei and also allows dramatic changes in compaction level to occur without a concomitant change in topology.
Despite the key role of the linker histone H1 in chromatin structure and dynamics, its location and interactions with nucleosomal DNA have not been elucidated. In this work we have used a combination of electron cryomicroscopy, hydroxyl radical footprinting, and nanoscale modeling to analyze the structure of precisely positioned mono-, di-, and trinucleosomes containing physiologically assembled full-length histone H1 or truncated mutants of this protein.Single-base resolution •OH footprinting shows that the globular domain of histone H1 (GH1) interacts with the DNA minor groove located at the center of the nucleosome and contacts a 10-bp region of DNA localized symmetrically with respect to the nucleosomal dyad. In addition, GH1 interacts with and organizes about one helical turn of DNA in each linker region of the nucleosome. We also find that a seven amino acid residue region (121-127) in the COOH terminus of histone H1 was required for the formation of the stem structure of the linker DNA. A molecular model on the basis of these data and coarse-grain DNA mechanics provides novel insights on how the different domains of H1 interact with the nucleosome and predicts a specific H1-mediated stem structure within linker DNA.nucleosome structure | chromatin higher order structure T he nucleosome is the fundamental repeating unit of chromatin in the nucleus of eukaryotic cells. The composition and the basic organization of the nucleosome is well established, and the structure of the nucleosomal core particle (NCP) has been described with nearly atomic precision by X-ray diffraction (1). However, similar information for the structure of a complete nucleosome, i.e., the NCP with associated linker DNA segments and a linker histone, is still lacking. Electron microscopy and electron cryomicroscopy (ECM) imaging have provided a relatively low-resolution picture of the complete nucleosome, both native (2) and reconstituted (3). However, important features of the structure remain obscure.Linker histones are typically ∼200 aa in length with a rather short nonstructured N terminus, followed by a ∼70-80 aa structured ("globular") domain, and a ∼100 aa long apparently unstructured C terminal domain, highly enriched in lysines. The globular domain of the linker histone appears to be internally located in the 30-nm chromatin fiber (4, 5), but its exact position within the nucleosome remains a subject of debate (for review, see ref. 6). A second question not yet resolved concerns the interactions and location of the linker histone C terminus. These issues have their origin in difficulties related to the preparation of well-defined nucleosomal samples. Indeed, direct binding of linker histone to nucleosomes in vitro is inefficient and complicated by the formation of large aggregates because of the nonspecific association of linker histones with DNA (7, 8).The situation can be considerably improved by using chaperones for linker histone deposition in vitro, a mechanism that is likely used in vivo (9). It was recently shown that NAP...
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