Chromatin higher-order structure studied by neutron scattering and scanning transmission electron microscopy.

Abstract

Neutron scattering in solution and scanning transmission electron microscopy were simultaneously done on chicken erythrocyte chromatin at various salt and magnesium concentrations. We show that chromatin is organized into a higher-order structure even at low ionic strength and that the mass per unit length increases continuously as a function of salt concentration, reaching a limiting value of between six and seven nucleosomes per 11 nm. There is no evidence of a transition from a 10-nm to a 30-nm fiber. Fiber diameter is correlated with mass per unit length, showing that both increase during condensation. We also find that there is no essential difference between the mass per unit length measured by scanning transmission electron microscopy and neutron scattering in solution, showing that the ordered regions seen in micrographs are representative of chromatin in solution.Nucleosomes in chromatin interact to form a higher-order structure, referred to as the 30-nm filament. Despite a large number of structural studies on chromatin, there is no unanimity on its higher-order structure. The nature and sources ofconflicting views ofthe structure ofthe 30-nm fiber have been summarized (1).Several models of the 30-nm fiber propose a helical structure for chromatin. These include the solenoidal model (2), the helical-ribbon model (3,4), the crossed-linker doublehelical model (5), and the nonsequential single-helix model (6). All of these models share certain similarities; for example, they all satisfy constraints imposed by electric dichroism measurements on the angle of the nucleosomal disk with respect to the fiber axis (7,8). Most ofthe models have similar repeat dimensions that would show up as peaks in a diffraction pattern. The helical-ribbon model predicts certain additional peaks due to the width of the ribbon. These additional peaks have not been observed in an experiment on oriented chromatin (9), but it is possible that an adjustment in the parameters of the helical-ribbon model would eliminate the additional reflections that are predicted.Despite such similarities, the models differ considerably in detail. Even at a coarse level, the various models predict different values for the mass per unit length of condensed chromatin. Thus a measurement of the physical characteristics of chromatin condensation could help to substantiate or rule out some of the existing models for chromatin structure.The mass per unit length of chromatin has been measured by small-angle scattering (10-14). The parameter has also been measured by hydrodynamic methods (7, 15) and light scattering (16). These experiments generally yield a value of 6-7 nucleosomes per 11 nm for the mass per unit length of the condensed 30-nm fiber. However, scanning transmission electron microscopy (STEM) studies on chromatin (4, 5) have yielded much higher values, of up to 11.6 nucleosomes per nm. Felsenfeld and McGhee (1) have pointed out that the discrepancy may be due to the fact that in STEM studies one selects ordered regions of chromatin, wh...

Evidence for short-range helical order in the 30-nm chromatin fibers of erythrocyte nuclei

“…3 E and F) or approximately 6.5 nucleosomes per 11 nm. This number indicates that the fibers are fully compact, because a compaction rate of 6-7 nucleosomes per 11 nm turn corresponds to the saturation point of compaction in isolated chicken erythrocyte chromatin fibers, according to measurements provided by previous studies (5,24). We have verified the reproducibility of the final subtomogram average using three individual datasets from different tomograms with independent initialization parameters and the results were very similar.…”

“…In vitro, in the presence of linker histones and a moderate concentration of monovalent cations, nucleosome arrays adopt an irregular zigzag topology (2,3). Further increase of monovalent salt and/or the presence of divalent cations leads to more compact structures, referred to as 30-nm chromatin fibers, in which nucleosomes are assumed to be helically packed with a density in the range of 6-11 nucleosomes/turn (4,5). The biological relevance of this compaction is supported by direct visibility of 30-nm fibers in situ by electron microscopy (EM) in some cell types of evolutionary distant species (6)(7)(8).…”

Mapping in Vivo Chromatin Interactions in Yeast Suggests an Extended Chromatin Fiber with Regional Variation in Compaction

“…Under conditions that are thought to mimic the in vivo milieu, i.e. 150 mM salt and 1-5 mM Mg 2ϩ , chromatin becomes compact to form more typical 30 nm fibers with a mass density of 6 nucleosomes per 11 nm (15,21). The level of compaction that is observed under these conditions further depends on the length of the linker DNA between nucleosomes and by binding of linker histone (22).…”

“…At very low salt concentrations chromatin does not form 30-nm-thick fibers, and nucleosomes appear in a zigzag arrangement to form fibers with a low mass density of around 1-2 nucleosomes per 11 nm fiber (6,12,15,21). Under conditions that are thought to mimic the in vivo milieu, i.e.…”

Structure determination of genomic domains by satisfaction of spatial restraints

“…Each particle in the system was assigned an excluded volume defined as a sphere of radius proportional to the particle's size (in base pairs). Considering the canonical 30-nm fiber, the relationship between length and base content was set to 0.01 nm per base pair (Gerchman and Ramakrishnan 1987). After the system was properly represented, a set of restraints was assigned to each particle defining their relative 3D position and thus the final spatial organization of the whole α-globin domain.…”