Methyl fingerprinting of the nucleosome reveals the molecular mechanism of high-mobility group nucleosomal-2 (HMGN2) association

The nucleosome represents the basic unit of chromatin, the DNA protein complex responsible for compacting the eukaryotic genome in the nucleus. Chromatin organization and functions are largely regulated at the nucleosomal level, and thus, understanding the structure, dynamics, and interactions of the nucleosome is essential to understanding the processes of gene transcription, recombination, replication, and repair. The work by Kato et al. (1) in PNAS provides a powerful tool to investigate the structural properties and interactions of the nucleosome in solution. Kato et al. (1) report resonance assignments of the methyl groups in the context of the intact nucleosome and use this information to elucidate the molecular basis of association of the high-mobility group nucleosomal-2 (HMGN2) effector.

A high-resolution crystal structure of the nucleosome core particle, determined in 1997 (2), reveals a great deal about the core architecture. The nucleosome consists of an octamer of histone proteins (two copies each of H2A, H2B, H3, and H4) wrapped by ∼146 bp DNA. The crystal structure also illuminates the mechanisms underlying the formation of higher-order chromatin structure. However, it has proven difficult to investigate the structural basis of important cofactor interactions with the nucleosome; as of yet, only two complexes have been successfully crystallized (3, 4). Moreover, although the nucleosome itself is quite dynamic (5), to date, only one state has been captured through crystallization. NMR spectroscopy is an ideal tool to address many of these questions, because it provides solution state structural information and is very powerful in the detection and characterization of time-dependent phenomena, including intermolecular interactions and dynamics. However, until now, its use has been precluded by experimental limitations imparted by the size of the nucleosome, which at ∼206 kDa, is pushing the limits of NMR detection using traditional methods.

In PNAS, Kato et al. (1) use selective isotopic labeling strategies, mutagenesis, and nuclear Overhauser enhancements to assign the resonances of ∼90% of the Ile, Leu, and Val (ILV) methyl groups in the nucleosome in methyl-transverse relaxation optimized spectroscopy (6–8) spectra, hence generating a methylome map of the nucleosome (Fig. 1). Because the nucleosomal assembly is highly conserved, this methyl fingerprinting will be invaluable in probing interactions with various ligands, mapping the binding interface, and analyzing dynamics and orientations of the nucleosomes.

Fig. 1.

Methyl fingerprinting of the nucleosome. Histones are shown in blue, DNA is in green, and the Ile/Leu/Val methyl groups are in red.

Kato et al. (1) show the power of such an approach in characterizing the binding of HMGN2 to the nucleosome. This interaction involves the nucleosome binding domain (NBD) of HMGN2 and two binding sites on the nucleosome (9–11). Using the ILV methyl fingerprinting and a combination of chemical shift perturbation, paramagnetic relaxation enhancement, and line shape analyses, Kato et al. (1) identify the primary binding site within an acidic patch of histones H2A and H2B and determine that two HMGN2 molecules bind cooperatively to both sides of the nucleosome (Fig. 2). These findings guide mutational, isothermal titration calorimetry, and gel-shift experiments, which reveal an additional interaction with the nucleosomal DNA. Furthermore, the experimental data are used as restraints to computationally dock the HMGN2 NBD domain onto the crystal structure of the nucleosome, confirming the physical feasibility of the dual anchoring. Together, their data establish a binding mechanism in which the N-terminal region of NBD interacts with the histone core, whereas the C-terminal region associates with DNA near the DNA exit/entry point.

Fig. 2.

A model of HMGN2 action at the nucleosome. The histone core is shown in blue, and DNA is in green. Binding of the NBD domain of HMGN2 (purple) to the acidic patch of histones (outlined by the red dotted line) and DNA near the DNA exit/entry point (outlined by the black dotted line) positions the C-terminal tail of HMGN2 to block the binding of H1. On phosphorylation (yellow circle) of the histone core, HMGN2 can be displaced from the nucleosome.

What is the biological consequence of the HMGN2 association with the nucleosome? The structural results of Kato et al. (1) provide insight into the function of HMGN2 and also help to clarify some earlier observations. Binding of the NBD to DNA positions the C-terminal tail of HMGN2 to interfere with histone H1 association, thus providing a mechanism for decompacting chromatin through the displacement of the linker histone H1. Kato et al. (1) reason that this interaction may also sterically preclude recruitment of repressive nucleosome remodeling factors. Additionally, the mechanistic details of the NBD–nucleosome interaction, which can be disrupted by histone phosphorylation, suggest a molecular basis for the dissociation of HMGN2 from the nucleosome.

Altogether, the study by Kato et al. (1) reveals critical structural information on the interaction of HMGN2 with the nucleosome, which sheds light on the mechanism of action in promoting transcriptional activation. Moreover, the resonance assignments of the nucleosomal histone ILV methyl groups provide an effectual tool for the analysis of the nucleosome that could be very widely applicable. Not only will it facilitate the identification and characterization of new cofactor interactions by NMR, but it will also enable the elucidation of the nucleosome structure, dynamics, and internucleosomal contacts in the solution state. Consequently, this study by Kato et al. (1) will likely be seminal in future exploration of the structure–function relationship of the nucleosome, which is imperative to understanding the role of this fundamental particle in gene regulation and chromatin remodeling.