Irregular Orientation of Nucleosomes in the Well-Defined Chromatin Plates of Metaphase Chromosomes

Abstract

In previous studies with partially denatured metaphase chromosomes, we detected platelike structures instead of the chromatin fibers currently considered in different structural models for chromosomes. Here we have observed that dilution of compact metaphase chromosomes with hyposmotic solutions can transform whole chromatids into extended plates formed by many layers. Since this treatment is soft and it does not change the ionic conditions, these observations indicate that native chromosomes are formed by stacked plates. This strengthens our hypothesis about the multilayer structure of chromosomes, which was originally based on results obtained using stronger denaturing conditions. We have investigated the structure of plates emanated from chromosomes using electron tomography. Our three-dimensional reconstructions demonstrate conclusively that the surface of the plates is very smooth and do not show repetitive structures supporting any regular organization of nucleosomes; even the nucleosomes in plate edges show irregular orientations. Furthermore, we have used polarizing microscopy for the study of whole chromosomes in metaphase cells in aqueous solution. Our results show that condensed chromosomes are not birefringent under structuring ionic conditions similar to those used with plates. This observation is incompatible with the existence of parallel columns of nucleosomes within chromosomes. In summary, we have not detected any regular orientation of nucleosomes, but at the same time, our results indicate that the bulk of chromatin in native chromosomes is organized forming very well-defined plates, in which the nucleosomes of the successive layers are interdigitated. Presumably, this dense structure is required for safe transfer of DNA to daughter cells.

The energy components of stacked chromatin layers explain the morphology, dimensions and mechanical properties of metaphase chromosomes

“…Liquid crystals formed in vitro by parallel columns of stacked nucleosome core particles are also birefringent [56]. By contrast, metaphase chromosomes are optically isotropic [16], indicating that they do not contain parallel columns of nucleosomes. In chromatin layers nucleosomes are irregularly oriented [16], but this high degree of disorder at short distances does not preclude the formation of a well-defined multilayered structure that can be considered a lyotropic lamellar liquid crystal, in which the high water (a) Three-dimensional scheme corresponding to part of a chromatid formed by stacked planar layers; the connections between layers introduce local structural heterogeneities that could be located in internal regions or in the periphery of the chromatid (some peripheral connections are schematically indicated by short vertical lines between layers).…”

“…(b) Irregular stacking of chromatin layers destabilizes the structure because there are more nucleosomes exposed to the medium, increasing the lateral surface area. (c) Schematic representation of nucleosome organization in successive chromatin layers according to [16] (reproduced with permission from the American Chemical Society); nucleosomes are represented by two turns of DNA (black) wrapped around the core histone octamer (grey); layers are indicated by discontinuous red and green lines; the consecutive nucleosomes in each layer are connected with linker DNA (not shown in the drawing). The energy per nucleosome in bulk chromatin 1 0 has two components: the energy 1 wl due to all interactions within each layer and the energy 1 nn due to the interactions with the interdigitated nucleosomes of the two adjacent layers.…”

“…Thus, it is very likely that the energy 1 nn that stabilizes the stacking of the successive layers in chromosomes is essentially due to face-to-face interactions between nucleosomes. The energy per nucleosome in internucleosome interactions has been obtained experimentally [21,22] and in modelling studies [25 -27] in several laboratories (see Introduction); taking into account all the values obtained in these studies, it is reasonable to consider that 1 nn % 3.4 -14 k B T. On the other hand, the easy sliding between layers in chromatin plates observed in electron microscopy experiments [15,16], suggested that the forces holding the chromatin filament within a layer are higher that the interactions between adjacent layers. This is consistent with the relatively large the Young modulus [15] and the high mechanical resistance [17] found for chromatin plates in AFM experiments in aqueous media, and indicates that the energy 1 wl stabilizing the structure of layers is higher than 1 nn .…”

“…Chromosome dimensions and structural models for metaphase chromatin folding. (a) Schematic illustration of different models for chromatin structure in metaphase chromosomes: (a) chromatin fibres (grey lines) form loops that are bound to a central protein scaffold (brown) [8][9][10], (b) chromatin fibres are irregularly folded [11][12][13], and (c) chromatin has a laminar structure with many staked layers oriented perpendicular to the chromatid axis [2,[14][15][16][17][18]; parallel lines represent the side view of the stacked layers. (b) Examples showing the differences in metaphase chromosome sizes that exist among animal species: crested newt (T. cristatus) (a), human (b), and D. melanogaster (c); the three chromosome sets are represented at the same magnification; figure reproduced from [19] with permission from the Royal Society.…”

Electron microscopy and atomic force microscopy studies of chromatin and metaphase chromosome structure

“…6D). Metaphase chromosomes purified on sucrose step gradients containing Mg 2+ are very compact (Castro-Hartmann et al, 2010). Dilution with the same buffer used in the gradient but without sucrose caused a rapid influx of water into chromatids and chromosomes became denatured to different extents.…”