Condensin Regulates the Stiffness of Vertebrate Centromeres

Abstract

When chromosomes are aligned and bioriented at metaphase, the elastic stretch of centromeric chromatin opposes pulling forces exerted on sister kinetochores by the mitotic spindle. Here we show that condensin ATPase activity is an important regulator of centromere stiffness and function. Condensin depletion decreases the stiffness of centromeric chromatin by 50% when pulling forces are applied to kinetochores. However, condensin is dispensable for the normal level of compaction (rest length) of centromeres, which probably depends on other factors that control higher-order chromatin folding. Kinetochores also do not require condensin for their structure or motility. Loss of stiffness caused by condensindepletion produces abnormal uncoordinated sister kinetochore movements, leads to an increase in Mad2(؉) kinetochores near the metaphase plate and delays anaphase onset. INTRODUCTIONCentromeric chromatin is a special region of chromosomes that has important mechanical and signaling functions in mitosis (Pidoux and Allshire, 2005;Ekwall, 2007;Cheeseman and Desai, 2008;Vagnarelli et al., 2008). In metaphase, pulling forces generated by interactions between spindle microtubules (MTs) and kinetochores are opposed by tension produced by centromeric chromatin stretch. Centromere and kinetochore tension and stretch are important for maintaining chromosome alignment (McIntosh et al., 2002), stabilizing kinetochore microtubule (kMT) attachments (Nicklas and Koch, 1969), spindle checkpoint signaling (Musacchio and Salmon, 2007;McEwen and Dong, 2009), and also for the back-to-back orientation of sister kinetochores (Loncarek et al., 2007). At least three independent factors have roles in the establishment of centromeric tension in metaphase: sister chromatid cohesion (Yeh et al., 2008), the elastic properties of chromatin (Houchmandzadeh et al., 1997;Almagro et al., 2004;Marko, 2008), and the higher order structure of the centromeric chromatin.Condensin is important for the architecture of mitotic chromosome arms (Coelho et al., 2003;Hudson et al., 2003;Hirota et al., 2004;Hirano, 2006), but it also localizes to centromeres (Saitoh et al., 1994;Gerlich et al., 2006), where condensin I, but not condensin II was reported to have a role in stabilizing the structure (Gerlich et al., 2006). It has recently been suggested that condensin could have a role in regulating the elastic behavior of centromeric chromatin. One study found that condensin I-depleted Drosophila chromosomes were unable to align at a metaphase plate, had distorted kinetochore structures, and lost elasticity of their centromeric chromatin (Oliveira et al., 2005). However a similar study in human cells reported that although loss of condensin I caused kinetochores to undergo abnormal movements, these movements were bidirectional (e.g., reversible; Gerlich et al., 2006).Even after the publication of those results, the regulation and functional significance of centromere stretch remained unknown. An elegant study in budding yeast went on to find that chromatin struct...

Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function

“…S1A-C), indicating resistance of the CENP-A domain to large-scale chromatin unfolding in vivo. This is consistent with the increased resistance of CENP-A chromatin to low-salt-induced unfolding observed in our previous studies (Ribeiro et al, 2009). It remains to be determined whether this difference can be ascribed to a higher rigidity for CENP-A nucleosomes, as revealed by deuterium exchange experiments (Black et al, 2004;Black et al, 2007).…”

A super-resolution map of the vertebrate kinetochore

“…Centromeres are usually embedded in the condensed chromatin of the primary constriction. However, in condensin-depleted SMC2 OFF metaphase cells, kinetochores undergo dramatic poleward "excursions" (15,21), trailing behind them a thread of extended pericentromeric chromatin (Fig. 1B).…”

“…1B). Importantly, these kinetochores are functional, and after a slight delay, will subsequently direct sister chromatid segregation (15,22). We used this system to map the distribution of histone modifications in the pericentromeric chromatin by indirect immunofluorescence and deconvolution microscopy.…”

“…Here, we have used DT40 cells genetically depleted of condensin (15) and several constitutive centromere-associated network (CCAN) proteins (16)(17)(18)(19) to map the pericentromeric region and to study chromatin organization in unfolded kinetochore fibers formed in vitro under conditions that preserve nonhistone protein attachments to chromatin (20). Examination of these unfolded fibers using super-resolution fluorescence microscopy with single molecule sensitivity allowed us to map the distribution of CENP-A relative to several histone modifications and other kinetochore proteins along the chromatin fiber with unprecedented resolution.…”

PR65, the HEAT-repeat scaffold of phosphatase PP2A, is an elastic connector that links force and catalysis

“…For example, cohesin and condensin complexes, both of which include HEAT-repeat-containing subunits, link pairs of chromatin fibers, in chromatin, or in complex arrays along chromosome axes. Involvement of HEATrepeat domains will give such networks greater elastic coherence than would be conferred by linkages involving rigid tethers connecting highly extensible chromatin/DNA segments (e.g., [61][62][63].…”