A simple model explains the cell cycle-dependent assembly of centromeric nucleosomes in holocentric species

Abstract

Centromeres are essential for chromosome movement. In independent taxa, species with holocentric chromosomes exist. In contrast to monocentric species, where no obvious dispersion of centromeres occurs during interphase, the organization of holocentromeres differs between condensed and decondensed chromosomes. During interphase, centromeres are dispersed into a large number of CENH3-positive nucleosome clusters in a number of holocentric species. With the onset of chromosome condensation, the centromeric nucleosomes join and form line-like holocentromeres. Using polymer simulations, we propose a mechanism relying on the interaction between centromeric nucleosomes and structural maintenance of chromosomes (SMC) proteins. Different sets of molecular dynamic simulations were evaluated by testing four parameters: (i) the concentration of Loop Extruders (LEs) corresponding to SMCs, (ii) the distribution and number of centromeric nucleosomes, (iii) the effect of centromeric nucleosomes on interacting LEs and (iv) the assembly of kinetochores bound to centromeric nucleosomes. We observed the formation of a line-like holocentromere, due to the aggregation of the centromeric nucleosomes when the chromosome was compacted into loops. A groove-like holocentromere structure formed after a kinetochore complex was simulated along the centromeric line. Similar mechanisms may also organize a monocentric chromosome constriction, and its regulation may cause different centromere types during evolution.

Figure 1.

Interphase and metaphase chromosomes of holocentric (Luzula elegans) and monocentric (Hordeum vulgare) species. During interphase, e.g. in L. elegans, holocentromeres disperse into a large number of CENH3-positive centromeric nucleosome clusters. With the onset of chromosome condensation, the centromeric nucleosome clusters join and form line-like structures along both chromatids. In contrast, in most monocentric species, e.g. in H. vulgare no obvious dispersion of the centromeres occurs during interphase. CENH3 (red) indicates the position of centromeres. Arrows indicate the longitudinal centromere groove in L. elegans. Chromosomes were counterstained with DAPI; bar = 5 μm. Pictures were taken from Heckmann et al. (11) with permission from S. Karger A.G., Basel.

Figure 2.

Schematic representation of the adopted chromosome and loop extrusion models. (A) The chromosomal 10 nm chromatin fibre represented as a beads-on-a-string polymer. Around ∼200 base pairs of DNA (including the linker) are wrapped around each nucleosome. (B) The loop extrusion model. The Loop Extruder (LE) is represented by a yellow ring. Nucleosomes bound by LEs are shown in yellow, the bond between them is represented as a yellow ellipsoid line, and the grey bar represents the chromatin. (C) Different examples of loops formed by two proximal LEs: (i) side-by-side loops, (ii) nested loops and (iii) combination of both. (D) Chromosome condensation by loop formation. The bases of the loops form the axis of the chromosome, and the loops are radially distributed. The degree of chromosome condensation is due to axial shortening and lateral compaction. These two parameters are functions of the number of nucleosomes and can be computed as the chromosome length and the average loop length, respectively. In this example, the chromosome length is 18 axial nucleosomes and the loop length is 23 nucleosomes. We define axial nucleosomes as bound by LEs and outside any loop.

Figure 3.

Effects of the presence of centromeric nucleosomes (in red) in the loop extrusion process. (A) Blocking and anchoring effects. In both, one centromeric nucleosome blocks the motion in its direction, but the opposite side of the LE (in yellow) continues to reel. In the blocking effect the LE interacting with the centromeric nucleosome can unbind, but in the anchoring effect the LE is permanently bound to the centromeric nucleosome. (B) For comparison, centromeric nucleosomes that do not interact with the LE can pass through it.

Figure 4.

Effects of centromeric units. (A) Chromosome length and average loop length as a function of the number of simulated LEs, and the number of nudeosomes outside chromatin loops. These parameters were calculated from simulations considering three different effects (no effect, blocking or anchoring) of centromeric units in the loop extrusion process. The grey area characterizes sparse states, when the number of LEs times the average loop length is <100 000 nucleosome. Above the grey area the chromosome is in a compacted state, with nested chromatin loops. (B–D) Final conformations of three simulations (100 000 3D steps long) with different centromeric effects (see Supplementary Movies S1–3 for simulation examples). The distribution of centromeric nucleosomes (red) and LEs (yellow) is shown in the chromatin fibre (grey), in the 3D structure (top) and sequence (bottom). For each conformation arrows indicate characteristic loop organizations. (B) With no effect, loops are observed spanning centromeric nucleosomes. (C) With the blocking effect, regions are observed outside loops as well as multiple loops between two adjacent centromeric nucleosomes. (D) With the anchoring effect, only one or two loops are observed between adjacent centromeric nucleosomes.

Figure 5.

Simulated conformations of a holocentric-like chromosome with 100 centromeric nucleosomes with anchoring effect after condensation by 1000 LEs (see Supplementary Movie S3 for the entire simulation). Centromeric nucleosomes are colored from blue to red according to the position in the linear genome. Left bar represents the chromosome length and colored lines indicate the position of centromeric nucleosomes. (A) The decondensed conformation represents an interphase with dispersed centromeric nucleosomes. (B) Condensation of a chromosome due to loop extrusion. Centromeric nucleosomes are aligned in the axis of the chromosome following the position in the chromosomal sequence. (C) Condensed holocentric chromosome colored from blue to red, as indicated by the bar at the right. The chromosome is entirely linearly arranged along the chromosome axis, following the position in the chromosomal sequence.

Figure 6.

Comparison between simulated (A) holocentric- and (B) monocentric-like chromosomes at different stages of the condensation process (Supplementary Videos S3 and 4). Bars indicate chromosome length and red lines the position of the centromeric nucleosomes. (A) The condensed holocentric-like chromosome presents an average loop size of 325 nucleosomes and a chromosome length of 374 nucleosomes. (B) The condensed monocentric-like chromosome presents loop sizes of 59 and 260 nucleosomes inside and outside the centromeric region, respectively, and a chromosome length of 650 nucleosomes. The inset shows the centromeric region, with smaller chromatin loops, resembling the centromere constriction of monocentric chromosomes.

Figure 7.

Simulation of a groove-like centromere along a holocentric condensed chromosome. (A) Representation of a simulated kinetochore arrangement as a rectangular bar formed by fixed spheres (in lilac). Red spheres represent the line of centromeric nucleosomes. Pairs of yellow spheres represent nucleosomes bound by LEs, and the chromatin fibre is shown as white beads on a string. (B) Final conformation of a simulated holocentric chromosome in the presence of the kinetochore arrangement. Components follow the same code color as in (A). The kinetochore is embedded in the chromatin fibre. On the right, the kinetochore is not shown so that the centromeric line is visible at the bottom of the groove and surrounded by LEs. Transversal (C) and longitudinal (D) cross-sections evidence the centromeric groove-like structure.