Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press The enhancement of pericentromeric cohesin association by conserved kinetochore components promotes high-fidelity chromosome segregation and is sensitive to microtubule-based tension Carrie A. Eckert,1,2 Daniel J. Gravdahl,2 and Paul C. Megee1,2,3 1Program in Molecular Biology, University of Colorado Health Sciences Center, Aurora, Colorado 80045, USA; 2Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Aurora, Colorado 80045, USA Sister chromatid cohesion, conferred by the evolutionarily conserved cohesin complex, is essential for proper chromosome segregation. Cohesin binds to discrete sites along chromosome arms, and is especially enriched surrounding centromeres, but past studies have not clearly defined the roles of arm and pericentromeric cohesion in chromosome segregation. To address this issue, we developed a technique that specifically reduced pericentromeric cohesin association on a single chromosome without affecting arm cohesin binding. Under these conditions, we observed more extensive stretching of centromeric chromatin and elevated frequencies of chromosome loss, suggesting that pericentromeric cohesin enrichment is essential for high-fidelity chromosome transmission. The magnitude of pericentromeric cohesin association was negatively correlated with tension between sister kinetochores, with the highest levels of association in cells lacking kinetochore–microtubule attachments. Pericentromeric cohesin recruitment required evolutionarily conserved components of the inner and central kinetochore. Together, these observations suggest that pericentromeric cohesin levels reflect the balance of opposing forces: the kinetochore-mediated enhancement of cohesin binding and the disruption of binding by mechanical tension at kinetochores. The involvement of conserved kinetochore components suggests that this pathway for pericentromeric cohesin enrichment may have been retained in higher eukaryotes to promote chromosome biorientation and accurate sister chromatid segregation. [Keywords: Chromosome biorientation; sister chromatid cohesion; cohesin; kinetochore; mitosis; genomic integrity] Supplemental material is available at http://www.genesdev.org. Received October 2, 2006; revised version accepted December 5, 2006. To segregate correctly in mitosis, replicated sister chro- somes that are initially attached to only one pole (mono- matids must form stable attachments to microtubules oriented). Two activities are then thought to promote that emanate from opposite spindle poles of the dividing attachment of unoccupied sister kinetochores to micro- cell, a process referred to as chromosome biorientation. tubules from opposite poles. First, dynamic instability of Attachments to chromosomes are mediated by the ki- kinetochore microtubules produces oscillatory chromo- netochore, an elaborate protein complex that assembles some movements that increase the probability of micro- within centromeric DNA and mediates the capture of a tubule capture by the unattached kinetochore (Skibbens single or multiple microtubules depending on the com- et al. 1993). In addition, the “sliding” of mono-oriented plexity of the kinetochore (Bloom 1993). Kinetochore– chromosomes toward the spindle mid-zone along the ki- microtubule capture is stochastic, resulting in chromo- netochore microtubules of a neighboring bioriented chromosome may also favor the capture of microtubules originating from the opposite pole (Kapoor et al. 2006). 3Corresponding author. The arbitrary nature of these events may result in the E-MAIL [email protected]; FAX (303) 724-3215. Article published online ahead of print. Article and publication date are formation of syntelic attachments, where both kineto- online at http://www.genesdev.org/cgi/doi/10.1101/gad.1498707. chores in a sister chromatid pair are attached to micro- 278 GENES & DEVELOPMENT 21:278–291 © 2007 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/07; www.genesdev.org Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Centromeric cohesin binding regulation tubules from the same pole. The failure to eliminate syn- characteristic of higher eukaryotes as well (Fukagawa et telic attachments prior to sister chromatid separation al. 2004). may lead to the transmission of abnormal chromosome Although several factors that mediate cohesin loading numbers to daughter cells. This condition, known as an- have been identified, the mechanism of cohesin recruit- euploidy, often results in genetic disease or miscarriage ment to specific genomic locations is not well under- and is also a hallmark of tumor cells. Thus, mechanisms stood. Recent advances suggest that histone post-trans- that promote chromosome biorientation and correct ab- lational modifications mediate cohesin recruitment to errant microtubule attachments are essential for the pre- sites of DNA damage and also to the pericentromeric vention of genomic instability. domains of regional centromeres. For example, cohesin’s The robust association, or cohesion, of sister chroma- interaction with phosphorylated histone H2A (␥-H2AX tids in centromeric regions is likely to promote chromo- in metazoans), which accumulates throughout broad re- some biorientation, possibly by constraining the micro- gions flanking a DNA double-strand break, is responsible tubule-binding sites on sister kinetochore pairs in oppo- for its recruitment to sites of DNA damage (Ünal et al. site directions. This arrangement theoretically favors the 2004). Furthermore, the association of cohesin with capture of microtubules emanating from opposite poles. Swi6, the Schizosaccharomyces pombe ortholog of het- In support of this model, the elimination of cohesion in erochromatin protein 1 (HP1), contributes to cohesin re- budding yeast increases syntelic attachments (Tanaka et cruitment to pericentromeric heterochromatin in fission al. 2000). Sister chromatid cohesion is also instrumental yeast (Bernard et al. 2001; Nonaka et al. 2002). Swi6 is for the generation of tension between sisters, which is essential for heterochromatin formation, and is recruited thought to play important roles in stabilizing amphitelic to centromeric regions by the binding of its chromodo- (bipolar) attachments and also in resisting poleward mi- main to methylated Lys 9 residues of histone H3, a modi- crotubule forces until all chromosomes have achieved fication commonly found in heterochromatin. Budding biorientation (Nicklas and Ward 1994). Interestingly, the yeast pericentromeric cohesin enrichment must occur tension exerted on bioriented chromosomes is capable of through an alternative mechanism, however, given that separating sister chromatids in centromere-proximal re- this organism lacks an HP1 homolog, H3 Lys 9 methyl- gions despite robust centromeric cohesion (Waters et al. ation, and repetitive heterochromatic DNA flanking 1996; Goshima and Yanagida 2000; He et al. 2000; centromeres. Accordingly, we have recently demon- Tanaka et al. 2000; Sonoda et al. 2001). However, this strated that the budding yeast centromere/kinetochore preanaphase separation is transient, as sister chromatid complex functions as an enhancer of cohesin recruit- centromeric regions frequently reestablish associations ment, generating large ∼20- to 50-kb pericentromeric do- prior to anaphase onset. mains that are highly enriched for cohesin binding (We- Sister chromatid cohesion is mediated by a multisub- ber et al. 2004). Whether the kinetochore-mediated en- unit complex, called cohesin, whose constituents have hancement of cohesin association in budding yeast also been conserved in organisms spanning the yeasts to ver- involves histone post-translational modifications re- tebrates (Nasmyth and Haering 2005). Cohesin is com- mains to be determined. posed of four subunits: two members of the SMC family The apparent conservation of the pericentromeric co- of chromosomal ATPases (Smc1 and Smc3), as well as hesin enrichment in eukaryotes and the possible exis- two non-SMC subunits (Scc3 and Mcd1/Scc1). Biophysi- tence of multiple pathways for pericentromeric cohesin cal studies have shown that the cohesin subunits form a recruitment suggest that this enrichment plays an im- ring-shaped complex, leading to the suggestion that co- portant role in chromosome segregation and the mainte- hesins may topologically encircle sister chromatids nance of genomic integrity. To distinguish between the (Haering et al. 2002; Gruber et al. 2003; Ivanov and Nas- relative contributions of arm and pericentromeric cohe- myth 2005). However, the precise mechanism by which sion in chromosome segregation, we have specifically cohesin rings interact with chromatin to mediate cohe- reduced pericentromeric cohesin association on a single sion remains unknown (Huang et al. 2005). Genome- chromosome without affecting arm cohesion, which had wide maps of cohesin-binding sites have also contributed not been possible using previous experimental ap- to our understanding of sister chromatid cohesion. Co- proaches. We find that chromosomes with reduced peri- hesin and Pds5, another mediator of cohesion, associate centromeric cohesin association exhibit elevated levels with discrete sites along budding and fission yeast chro- of pericentromeric sister chromatid separation and chro- mosomes (Blat and Kleckner 1999; Hartman et al. 2000; mosome loss. Using a collection