The Influence of Ionic Environment and Histone Tails on Columnar Order of Nucleosome Core Particles

Abstract

The nucleosome core particle (NCP) is the basic building block of chromatin. Nucleosome-nucleosome interactions are instrumental in chromatin compaction, and understanding NCP self-assembly is important for understanding chromatin structure and dynamics. Recombinant NCPs aggregated by multivalent cations form various ordered phases that can be studied by x-ray diffraction (small-angle x-ray scattering). In this work, the effects on the supramolecular structure of aggregated NCPs due to lysine histone H4 tail acetylations, histone H2A mutations (neutralizing the acidic patch of the histone octamer), and the removal of histone tails were investigated. The formation of ordered mainly hexagonal columnar NCP phases is in agreement with earlier studies; however, the highly homogeneous recombinant NCP systems used in this work display a more compact packing. The long-range order of the NCP columnar phase was found to be abolished or reduced by acetylation of the H4 tails, acidic patch neutralization, and removal of the H3 and H2B tails. Loss of nucleosome stacking upon removal of the H3 tails in combination with other tails was observed. In the absence of the H2A tails, the formation of an unknown highly ordered phase was observed.

Frozen‐hydrated chromatin from metaphase chromosomes has an interdigitated multilayer structure

“…The intense scattering at 6 nm observed for native chromosomes under all the conditions analyzed ( Fig 5A-C and Nishino et al, 2012) indicates that face-toface association between nucleosomes ( Fig 6) is a fundamental structural element of metaphase chromosomes. This is not surprising because face-to-face interactions with different degrees of overlap between nucleosomes were observed previously in different chromatin samples (purified nucleosome cores, chromatin fibers, and nucleosome arrays) using physicochemical methods (Tatchell & Van Holde, 1978), TEM (Finch et al, 1977;Dubochet & Noll, 1978;Bartolomé et al, 1994;Robinson et al, 2006), cryo-EM (Leforestier et al, 1999;Robinson et al, 2006;Scheffer et al, 2012;Song et al, 2014;Bilokapic et al, 2018), and X-ray scattering (Mangenot et al, 2003;Bertin et al, 2007;Berezhnoy et al, 2016) and crystallography (Uberbacher & Bunick, 1985;Luger et al, 1997;Harp et al, 2000;White et al, 2001;Schalch et al, 2005;Ekundayo et al, 2017;Zhou et al, 2018); this interaction was also described in modeling studies (Stehr et al, 2010;Fan et al, 2013;Korolev et al, 2016Korolev et al, , 2018Saurabh et al, 2016;Ishida & Kono, 2017). The lateral association between two nucleosome cores involves an acidic surface formed by histones H2A and H2B and basic residues of the N-terminal tail of histone H4 (Luger et al, 1997;Harp et al, 2000;White et al, 2001;Schalch et al, 2005).…”

“…According to the X-ray diffraction patterns observed for associated nucleosome cores that form columns (Mangenot et al, 2003;Bertin et al, 2007;Berezhnoy et al, 2016), the 6-nm peak corresponds to the distance between nucleosome cores interacting face-to-face within the columns, and the 11-nm peak is related to the distance between parallel columns (i.e., the distance corresponding to edge-to-edge nucleosome contacts). Narrow diffraction peaks were found in crystalline aggregates of nucleosome columns (Mangenot et al, 2003;Berezhnoy et al, 2016). Aggregates of nucleosome columns lacking the longrange order of crystalline structures produce broad peaks (Mangenot et al, 2003).…”

Structure and Dynamics in the Nucleosome Revealed by Solid‐State NMR

“…TheNCP was precipitated with 20 mm Mg 2+ ,which yields highly ordered columnar hexagonal NCP stacking structures according to our previous study. [14] The 1 Hspectrum indicates that the precipitated NCP sample is highly hydrated ( % 50 % water content by weight) and the NCP concentration is % 500 mg mL À1 ,w hich falls in the concentration range of chromatin in vivo (50-500 mg mL À1 ). [15] Multidimensional state-of-the-art SSNMR experiments were first performed to facilitate resonance assignments for the hH4 protein in the NCP.The high hH4 conformational homogeneity is evidenced by the dipolar assisted rational resonance (DARR) [16] spectrum exhibiting outstanding spectral resolution achieved for this % 200 kDa complex ( Figure 1) where only 12 %( by weight) of the material are uniformly 13 C, 15 N-labled hH4.…”

Structure and Dynamics in the Nucleosome Revealed by Solid‐State NMR

“…The NCP was precipitated with 20 m m Mg 2+ , which yields highly ordered columnar hexagonal NCP stacking structures according to our previous study . The 1 H spectrum indicates that the precipitated NCP sample is highly hydrated (≈50 % water content by weight) and the NCP concentration is ≈500 mg mL −1 , which falls in the concentration range of chromatin in vivo (50–500 mg mL −1 ) .…”