Self-Assembly of Thin Plates from Micrococcal Nuclease-Digested Chromatin of Metaphase Chromosomes

Abstract

The three-dimensional organization of the enormously long DNA molecules packaged within metaphase chromosomes has been one of the most elusive problems in structural biology. Chromosomal DNA is associated with histones and different structural models consider that the resulting long chromatin fibers are folded forming loops or more irregular three-dimensional networks. Here, we report that fragments of chromatin fibers obtained from human metaphase chromosomes digested with micrococcal nuclease associate spontaneously forming multilaminar platelike structures. These self-assembled structures are identical to the thin plates found previously in partially denatured chromosomes. Under metaphase ionic conditions, the fragments that are initially folded forming the typical 30-nm chromatin fibers are untwisted and incorporated into growing plates. Large plates can be self-assembled from very short chromatin fragments, indicating that metaphase chromatin has a high tendency to generate plates even when there are many discontinuities in the DNA chain. Self-assembly at 37°C favors the formation of thick plates having many layers. All these results demonstrate conclusively that metaphase chromatin has the intrinsic capacity to self-organize as a multilayered planar structure. A chromosome structure consistent of many stacked layers of planar chromatin avoids random entanglement of DNA, and gives compactness and a high physical consistency to chromatids.

Structure-driven homology pairing of chromatin fibers: the role of electrostatics and protein-induced bridging

“…A recent in vitro study showed that short fiber stretches containing several nucleosomes, extracted from chromatin by mild MNase digestion, readily form ordered structures in vitro, indicating that chromatin fibers do sense the shapes of each other [103]. In addition, our modeling suggests that chromatin fibers not only sense the general shapes but can also distinguish in-and out-of-register arrangements of nucleosomes, suggesting interplay of recognition mechanisms on different length-scales.…”

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

“…In this context, recent research has revealed that multilayer plates (identical to those found in metaphase chromosomes) can be self-assembled from chromatin fragments obtained by micrococcal nuclease digestion of metaphase chromosomes [18]. This finding has reinforced the thin-plate model for metaphase chromosome structure (figure 1a,c) and suggests that the whole chromosome could be a self-organizing structure.…”

“…Typical chromatin plates can be self-assembled from chromatin fragments of metaphase chromosomes, indicating that chromatin structure can hold discontinuous DNA in the correct place within the plates [18]. However, when the relatively high lateral force of the AFM tip was applied to micrococcal nucleasedigested plates in nanotribology experiments, the structural integrity of chromatin layers was maintained only if the degree of digestion was very low [17].…”

“…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.…”

Revealing chromatin organization in metaphase chromosomes

“…In agreement with this notion, earlier results from the Daban group had shown that isolated chromatin fibers, obtained from micrococcal nucleasedigested metaphase chromosomes, can selfassociate to form chromatin plates in vitro (Milla & Daban, 2012). To this end, they carefully purified individual mitotic chromosomes from human cells.…”