Poster abstracts

Poster number 32 submitted by Mohamad Zandian

Effect of Linker DNA Length on the H3 Histone Tail Flexibility in Nucleosome Arrays Studied by Magic-Angle Spinning NMR Spectroscopy

Mohamad Zandian (Department of Chemistry & Biochemistry, The Ohio State University), Matthew D. Shannon (Department of Chemistry & Biochemistry, The Ohio State University), Niki Gonzalez (Department of Chemistry & Biochemistry, The Ohio State University), Michael G. Poirier (Department of Physics), Christopher P. Jaroniec (Department of Chemistry & Biochemistry, The Ohio State University)

Abstract:
In eukaryotic cells the genome is tightly packed and regulated by chromatin, a supramolecular DNA-protein complex. The building block of chromatin is the nucleosome, composed of ~147 base pairs (bp) of DNA wrapped about twice around the histone octamer, a protein complex made up of two copies each of histones H2A, H2B, H3, and H4. In chromatin adjacent nucleosomes are separated from each other by linker DNA, which typically varies between 15 and 60 bp in length for different states of chromatin. Several studies have suggested that the dynamically disordered tail domain of histone H31-3 (residues ~1-35) may interact with the linker DNA4,5. However, the details of this interaction including possible effects of the linker DNA length have not been investigated in detail.
Here we use magic-angle spinning (MAS) NMR spectroscopy to systematically investigate the influence of the DNA linker length on histone H3 tail flexibility in large nucleosome arrays. For this purpose, eight DNA constructs were designed and prepared that contain 16 repeats of the Widom 601 nucleosome positioning sequence spaced evenly with varying lengths of linker DNA ranging between 15 and 60 bp. The quality of nucleosome array samples was verified by several assays including electrophoretic mobility shift assay and atomic force microscopy, and the nucleosome arrays were sedimented by ultracentrifugation for MAS NMR experiments. 2D 15N-1H HSQC and additional experiments that probe residue-specific 15N T1 and T2 relaxation time constants were carried out on 16-mer nucleosome arrays with 15, 30, and 60 bp DNA linker length, reconstituted with 13C,15N-labeled histone H3. These experiments enabled identification of the flexible tail residues and collectively suggest that H3 residues ~1-35 on the whole are conformationally flexible within the nucleosome arrays, irrespective of the linker DNA length in the 15-60 bp regime. Interestingly, quantitative measurements of the resonance intensities (and 15N transverse relaxation rates) suggest that certain residues (in particular aa ~1-5 and ~15-30) may be somewhat less conformationally flexible relative to the remaining amino acids.

References:
1. Luger, K., et al. 1997 Nature 389:251-60.
2. Zhou, B. R., et al. 2012 J. Mol. Biol. 421:30-7.
3. Gao, M., et al. 2013, J. Am. Chem. Soc. 135:15278-81.
4. Rhee, H. S., et al. 2014 Cell 159:1377-88.
5. Gatchalian, J., et al. 2017 Nat. Commun. 8:1489.

Keywords: Chromatin, Histone H3 Tail Domain, Magic-Angle Spinning NMR Spectroscopy