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.

Fig. 1.

NAP-1 facilitates binding of linker histone H1 and truncation mutants to 601 dinucleosomes. (A) Primary structure of histone H1 (Upper) and schematics of the histone H1 deletion mutants (Lower). (B) 15% SDS-PAGE of purified recombinant full-length H1, truncation mutants, and NAP-1. (C) Agarose gel electrophoresis of 601 dinucleosomes incubated with increasing amounts of either full-length histone H1 alone (lanes 7–10), NAP-1-histone H1 complex (lanes 3–6), or a complex of NAP-1 and the indicated H1 truncation mutants (lanes 11–18). Lanes 1 and 2, control dinucleosome without H1 and dinucleosomes incubated with NAP-1 only.

Fig. 2.

H1 binding to nucleosomes organizes linker DNA into a stem structure. Representative ECM images of reconstituted 601 trinucleosomes. Shown are trinucleosomes in the absence of H1 (A) and trinucleosomes assembled with full-length histone H1 (B), the 1–127 H1 truncation mutant (C), or the 35–120 truncation mutant (D). NAP-1 was present in the experiments shown in A. The arrowheads indicate selected examples of the stem. (Scale bar, 40 nm.)

Fig. 3.

Hydroxyl radical footprinting of control and H1-containing dinucleosomes. (A) Sequencing gel analysis of •OH cleavage of dinucleosomes with ³²P label incorporated at the 3′ end of the upper DNA strand. Lane 1, •OH cleavage pattern of the naked DNA template; lanes 2–5, •OH cleavage pattern of control and H1-containing dinucleosomes. The triangle and the asterisk highlight the digestion products of the central part and the ends of the linker DNA region, respectively. (B) Same as A but for dinucleosomes reconstituted with ³²P 3′-end-labeled lower DNA strand. (C) Scans of the •OH digestion pattern in the vicinity of the nucleosome dyad of control (Black) and H1-containing (Red) dinucleosomes.

Fig. 4.

Hydroxyl radical footprinting of control and H1 truncation mutants bound to dinucleosomes. The gel shows dinucleosomes in the absence of H1 (-) and in the presence of H1 or truncation mutants, as indicated on the left. Scans of the •OH cleavage patterns depicted in the gel are shown (Bottom). Cleavage products within the central part of the linker DNA are indicated by triangles, whereas asterisks indicate the 10 bp at either end of the linker DNA adjacent to the core region.

Fig. 5.

Hydroxyl radical footprinting of H1 bound to trinucleosomes. (A) •OH cleavage pattern of trinucleosomes without H1 (lanes 1–3) and with H1 (lanes 4–6). (B) shows the same samples as in A except run longer for better resolution of the linker region. (C) Scans of the •OH cleavage pattern of control, without H1 (Black) and H1-containing (Red) trinucleosomes.

Fig. 6.

Molecular models for the nucleosome particle. (A) Model of the nucleosome without H1. The model of the nucleosomal DNA alone and two views of the nucleosome with histones are shown in the top panel. The experimental •OH-accessibility profile is depicted on the three-dimensional nucleosome structure by color coding the DNA deoxyribose C5′ atoms from blue (maximal protection) to white (partial protection) to red (maximal accessibility). DNA C5′ atoms without footprinting data and all other DNA are shown in gray, the dyad in green. Protein is shown in black (omitted in the left column). Views shown, from left to right, are (i) rotated sideways and up by 30° from the NCP superhelical axis, (ii) at right angles to the superhelical and dyad axes, and (iii) along the dyad axis. The bottom panel shows plots of the experimental •OH-accessibility profile (Solid Line) and the corresponding model-derived accessibility profile (Dashed Line). Nucleosome core region is indicated by the gray oval. (B) Modeling of the nucleosome associated with the globular domain (GH1) of histone H1. The upper panel illustrates the location of GH1 in the nucleosome in the three-contact model, as described in the text. Note that GH1 protects the dyad and directly interacts with 10 bp of each linker DNA. The C terminus of GH1 is highlighted in magenta. (C) Model of the nucleosome associated with 35–127 H1 mutant. The location of the 35–127 H1 mutant and the 3D organization of the linker DNA stem obtained from constrained DNA elastic relaxation were determined as described in the text. Note the strong protection of the dyad and the presence of the 10-bp repeat within the linker DNA (Bottom). DNA within a 30 Å radius of the GH1 C terminus is colored blue. A hypothetical conformation of aa 112–127 is shown in yellow. Because the location of 5 aa (35–39) sequence of the NH₂ terminus is not known, this sequence was not shown in the model.