The regulation of chromosome segregation via centromere loops

Abstract

Biophysical studies of the yeast centromere have shown that the organization of the centromeric chromatin plays a crucial role in maintaining proper tension between sister kinetochores during mitosis. While centromeric chromatin has traditionally been considered a simple spring, recent work reveals the centromere as a multifaceted, tunable shock absorber. Centromeres can differ from other regions of the genome in their heterochromatin state, supercoiling state, and enrichment of structural maintenance of chromosomes (SMC) protein complexes. Each of these differences can be utilized to alter the effective stiffness of centromeric chromatin. In budding yeast, the SMC protein complexes condensin and cohesin stiffen chromatin by forming and cross-linking chromatin loops, respectively, into a fibrous structure resembling a bottlebrush. The high density of the loops compacts chromatin while spatially isolating the tension from spindle pulling forces to a subset of the chromatin. Paradoxically, the molecular crowding of chromatin via cohesin and condensin also causes an outward/poleward force. The structure allows the centromere to act as a shock absorber that buffers the variable forces generated by dynamic spindle microtubules. Based on the distribution of SMCs from bacteria to human and the conserved distance between sister kinetochores in a wide variety of organisms (0.4 to 1 micron), we propose that the bottlebrush mechanism is the foundational principle for centromere function in eukaryotes.

Keywords: Centromere; DNA loops; chromosome segregation; cohesin; condensin; kinetochore; mitosis; pericentromere.

Figure 1.

Depictions of a single human centromere and yeast pericentromere. A) A schematic of 2 sister chromatids at the centromere. One pair of sisters are aligned along the vertical axis. Within the centromere multiple loops are folded intramolecularly and biorient relative to one another. The apex of a loop is positioned at the kinetochore plus-ends (red dote). The sister chromatids are in green, highlighting the proposal that 16-20 loops emanate from one pair of sister chromatids in mammals (or organisms with multiple microtubule attachment sites) (Aze et al., 2016; Bloom and Costanzo, 2017), versus the clustering of loops from individual chromosomes (16 in budding yeast) in organisms with unit attachment sites. B) A schematic of the 16 sister pericentromeres in budding yeast. Pairs of sister chromatids are aligned along the vertical axis (vertical straight lines). In the pericentromere (horizontal, bounded by red kinetochore attachment sites), each sister centromere is folded into an intramolecular C-loop (Yeh et al., 2008) that biorient relative to one another that contains several subloops (Stephens et al., 2011). The apex of each C-loop (the 125 bp centromere) is positioned at the kinetochore microtubule plus-ends (red dot). The sister chromatids are color-coded (evident in the colors at the kinetochore microtubule attachment site). The schematics scaled by centromere separation, i.e. the cluster of 16 bi-oriented kinetochores in yeast occupy roughly the same volume as one mammalian kinetochore.

Figure 2.

Positively supercoiled DNA is more resistant to extension then negatively supercoiled DNA. A) A depiction of a single molecule experiment with magnetic tweezers. Negatively supercoiled DNA is pulled with 1 pN of force and is extended. Upon extension local regions of the DNA melt/denature. B) Positively supercoiled DNA is pulled with 1 pN of force but resists extension. C) Positively supercoiled DNA is pulled with greater than 3 pN of force and is extended. Upon extension the DNA is reorganized into Pauling DNA (P-DNA).

Figure 3.

Polymer model of budding yeast pericentromere. A) A three-dimensional, polymer model of the budding yeast pericentromere. Cohesin are the white rings composed of 16 masses. The DNA is looped by the condensin complex (condensin not shown). Point centromeres are in white. The DNA is extended to its rest length to emphasize the highly looped nature of the DNA in the model. This visualization uses the simulation date from Lawrimore et al. (Lawrimore et al., 2016). B) Three-dimensional, polymer models of the budding yeast pericentromere with cohesin and condensin (Top Left), without cohesin and with condensin (Top Right), with cohesin and without condensin (Bottom Left), and without cohesin and without condensin (Bottom Right). Coloring of models is based on the average amount of force on each mass over the course of a simulation. The forces listed in the bottom right corner of the panels are the average entropic spring force on each point centromere. These are visualization of the simulations from Lawrimore et al. (Lawrimore et al., 2016) and tension data from Lawrimore et al. (Lawrimore et al., 2018).

Figure 4.

Direct comparison of simulated and experimental dicentric plasmid lengths during mitosis. A) Cartoons of the dicentric plasmid simulations without loops overlaying simulated images of the lacO/LacI-GFP array. The centromeres are in red. The unlabeled DNA is in blue. The lacO/LacI-GFP arrays are in green. The simulated images were constructed by converting the ChromoShake simulation output files (Lawrimore et al., 2016) into XML files that were in turn converted to image stacks using Microscrope Simulator 2 (Quammen et al., 2008). The simulated fluorescence images are maximum intensity projections. B) Histograms of the signal lengths of the simulated fluorescent images of the dicentric ChromoShake simulations. Each simulation condition, i.e. number and type of loops, is composed of 10 separate simulations. Each simulation either contained 0, 3, or 6 loops. These loops could be static, not increasing or decreasing in size, or dynamic, increasing or decreasing in size. The static loops were simulated by randomly joining non-consecutive bead together with a 10 nm spring. The dynamic loops were simulated using the RotoStep program (Lawrimore et al., 2017). In the RotoStep program, loops are unidirectionally extruded by simulated condensin complexes until tension on the substrate increases past a threshold causing loop dissociation, condensin translocation to a new region, and then loop extrusion resumes. The simulation data was previously published in Lawrimore et al. (Lawrimore et al., 2018). C) Histograms of experimental lacO/LacI-GFP signals on a dicentric plasmid during mitosis. The experimental data was previously published in Lawrimore et al. (Lawrimore et al., 2018).