Histone H3 core domain in chromatin with different DNA linker lengths studied by 1H-Detected solid-state NMR spectroscopy

Abstract

Chromatin, a dynamic protein-DNA complex that regulates eukaryotic genome accessibility and essential functions, is composed of nucleosomes connected by linker DNA with each nucleosome consisting of DNA wrapped around an octamer of histones H2A, H2B, H3 and H4. Magic angle spinning solid-state nuclear magnetic resonance (NMR) spectroscopy can yield unique insights into histone structure and dynamics in condensed nucleosomes and nucleosome arrays representative of chromatin at physiological concentrations. Recently we used J-coupling-based solid-state NMR methods to investigate with residue-specific resolution the conformational dynamics of histone H3 N-terminal tails in 16-mer nucleosome arrays containing 15, 30 or 60 bp DNA linkers. Here, we probe the H3 core domain in the 16-mer arrays as a function of DNA linker length via dipolar coupling-based 1H-detected solid-state NMR techniques. Specifically, we established nearly complete assignments of backbone chemical shifts for H3 core residues in arrays with 15–60 bp DNA linkers reconstituted with 2H,13C,15N-labeled H3. Overall, these chemical shifts were similar irrespective of the DNA linker length indicating no major changes in H3 core conformation. Notably, however, multiple residues at the H3-nucleosomal DNA interface in arrays with 15 bp DNA linkers exhibited relatively pronounced differences in chemical shifts and line broadening compared to arrays with 30 and 60 bp linkers. These findings are consistent with increased heterogeneity in nucleosome packing and structural strain within arrays containing short DNA linkers that likely leads to side-chains of these interfacial residues experiencing alternate conformations or shifts in their rotamer populations relative to arrays with the longer DNA linkers.

Phosphorylated HP1α–Nucleosome Interactions in Phase Separated Environments

“…Since phase separation conditions can affect the quality of the solution NMR spectra, we used substoichiometric ratios of pHP1α to mononucleosomes, which resulted in clear samples without droplets (Figure S5) and yielded well resolved assignable spectra (Figure b). We also note that we performed all NMR structural studies (both solution and solid state) under low salt conditions to compare to previous work and to take advantage of published assignments. − …”

Conformational and Interaction Landscape of Histone H4 Tails in Nucleosomes Probed by Paramagnetic NMR Spectroscopy

“…As noted above, high-resolution structural techniques, such as X-ray crystallography or cryo-EM, are generally unable to localize the disordered histone tail domains in nucleosomes. Instead, this problem can be uniquely addressed by multidimensional NMR spectroscopy − ,− in particular, by measurements of residue-specific proton paramagnetic relaxation enhancements (PREs), which report on distances between individual backbone amides and covalent paramagnetic tags attached at specific protein sites and are significant over distances up to ∼20–30 Å. For structured biomolecules in solution, the PRE-based restraints can be used to build or refine structural models, − and indeed, studies along these lines have been reported for nucleosome particles and their complexes with CAPs. − This approach relies on the well-known Solomon–Bloembergen equation which represents PRE rates as a product of distance- and dynamics-dependent terms, where the latter can be determined from heteronuclear relaxation measurements and the former can be converted into structural restraints.…”