Single-base resolution mapping of H1–nucleosome interactions and 3D organization of the nucleosome

Abstract

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...

A metastable structure for the compact 30‐nm chromatin fibre

“…Packing was permitted to ≥ 12 Å between helical axes of neighbouring DNA duplexes in crossovers 18, 19. We assumed that the H5 linker histone stabilises a left‐handed crossing of linker DNAs 20, 21.…”

Ubiquitous nucleosome crowding in the yeast genome

“…We obtain f = 0:5, which implies that after averaging over hydroxyl cleavage preferences, interdyad distances are predicted to be 5 bp longer than the distances between hydroxyl cleavage sites marking neighboring nucleosomes (SI Appendix). With this correction, 25.8% of all interdyad distances are less than 147 bp. Our model reproduces both the overall shape and the fine oscillatory structure of the observed histogram of DNA fragment lengths ( Fig.…”

“…1B describes both DNA attachment to the histone octamer (up to 73 bp from the dyad) and the effects of higher-order chromatin structure, including binding of Hho1p, the H1 linker histone of S. cerevisiae (24)(25)(26), or histone tails (27) to the DNA immediately outside the nucleosome core. Although Hho1p is less abundant in yeast than in higher eukaryotes, it is involved in higher-order chromatin organization, including chromatin compaction in stationary phase (26,28).…”

“…about the molecular mechanism of H1 binding, including the length and symmetry of its DNA footprint (24,25). H1 binding and other factors that mediate chromatin folding into higherorder structures cause linker lengths to be discretized (1,29).…”

Rapid Deamination of Cyclobutane Pyrimidine Dimer Photoproducts at TCG Sites in a Translationally and Rotationally Positioned Nucleosome in Vivo

“…In addition, chromatin may also contain histone H1 that might affect photoproduct formation and deamination at the dyad and the linkers. H1 has been found to suppress hydroxyl radical cleavage of a 10-bp region at the dyad of a nucleosome in vitro as well as extend the characteristic 10-base repeat in the cleavage pattern up to 30 bp beyond the edge of the nucleosome core into the linker DNA (21).…”