Nucleosomal arrays self‐assemble into supramolecular globular structures lacking 30‐nm fibers

Abstract

The existence of a 30‐nm fiber as a basic folding unit for DNA packaging has remained a topic of active discussion. Here, we characterize the supramolecular structures formed by reversible Mg2+‐dependent self‐association of linear 12‐mer nucleosomal arrays using microscopy and physicochemical approaches. These reconstituted chromatin structures, which we call “oligomers”, are globular throughout all stages of cooperative assembly and range in size from ~50 nm to a maximum diameter of ~1,000 nm. The nucleosomal arrays were packaged within the oligomers as interdigitated 10‐nm fibers, rather than folded 30‐nm structures. Linker DNA was freely accessible to micrococcal nuclease, although the oligomers remained partially intact after linker DNA digestion. The organization of chromosomal fibers in human nuclei in situ was stabilized by 1 mM MgCl2, but became disrupted in the absence of MgCl2, conditions that also dissociated the oligomers in vitro. These results indicate that a 10‐nm array of nucleosomes has the intrinsic ability to self‐assemble into large chromatin globules stabilized by nucleosome–nucleosome interactions, and suggest that the oligomers are a good in vitro model for investigating the structure and organization of interphase chromosomes.

Chromatin structure-dependent conformations of the H1 CTD

“…In this case the closer apposition of H1s its likely due to further condensation of individual arrays, perhaps to canonical 30 nm fiber-like structures (7,46) rather than inter-array interactions, as little self-association is expected at 50 mM NaCl (7). Interestingly, the H1 CTD structure appears unchanged even upon increasing the NaCl concentration to 90 mM, where array–array self-association is likely to occur, with conversion of folded arrays to interdigitated fibers (47). Thus the environment of H1 appears to remain relatively constant during formation of multiple higher order chromatin structures beyond the contacting zig-zag arrangement.…”

Budding yeast chromatin is dispersed in a crowded nucleoplasmin vivo

“…Indeed, the only two cell types shown to have 30-nm fibers-by cryo-EM-are starfish sperm and chicken erythrocytes (Woodcock, 1994;Scheffer et al, 2011), both of which are terminally differentiated cells that have minimal transcriptional activity. In vitro, 30-nm fibers can be stabilized by changing the ionic environment (Maeshima et al, 2016). What factors, then, control 30-nm-fiber formation in vivo?…”

“…In these 30-nm-fiber models, the nucleosomes pack so closely that the chromatin takes on the appearance of a discrete particle. The majority of these chromatin studies, however, were done in vitro at low ionic strength, making it unclear whether the resultant models reflect chromatin organization in the crowded, metabolically active interior of a cell's nucleus (Maeshima et al, 2010(Maeshima et al, , 2016Hansen, 2012).…”

3D genomics imposes evolution of the domain model of eukaryotic genome organization

“…This domain may appear either continuous or discontinuous on a linear chromatin fiber, depending on the pattern of DNA packaging. The shape and size of the spatial chromatin domain will be determined by characteristics of self-assembled nucleosome conglomerates (Maeshima et al 2016).…”

“…These segments are less likely to establish internucleosomal interactions since they are highly acetylated (Ulianov et al 2015a). The folding of nucleosomal arrays into secondary chromatin structures and selfassociation of arrays into higher-order tertiary structures is an inherent property of chromatin fibers (Blacketer et al 2010;Hansen 2002;Maeshima et al 2016). This follows from the ability of nucleosomes to establish contacts via interaction of positively charged tails of histones H3 and H4 with the acidic patch on the surface of a nucleosomal globule (Kalashnikova et al 2013;Pepenella et al 2014;Schalch et al 2005;Sinha and Shogren-Knaak 2010) and with negatively charged DNA (Arya and Schlick 2006).…”