INVESTIGATION Centromeric Barrier Disruption Leads to Mitotic Defects in Schizosaccharomyces pombe Terilyn L. Gaither,* Stephanie L. Merrett, 1, * Matthew J. Pun,* and Kristin C. Scott* ,†,2 *Institute for Genome Sciences and Policy, Duke University, DUMC 3382, Durham, North Carolina 27708, and † Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710 ABSTRACT Centromeres are cis-acting chromosomal domains that direct kinetochore formation, enabling faithful chromosome segregation and preserving genome stability. The centromeres of most eukaryotic organisms are structurally complex, composed of nonoverlapping, structurally and functionally distinct chromatin subdomains, including the specialized core chromatin that underlies the kinetochore and peri- centromeric heterochromatin. The genomic and epigenetic features that specify and preserve the adjacent chromatin subdomains critical to centromere identity are currently unknown. Here we demonstrate that chromatin barriers regulate this process in Schizosaccharomyces pombe. Reduced fitness and mitotic chro- mosome segregation defects occur in strains that carry exogenous DNA inserted at centromere 1 chromatin barriers. Abnormal phenotypes are accompanied by changes in the structural integrity of both the centro- meric core chromatin domain, containing the conserved CENP-A Cnp1 protein, and the flanking pericentric heterochromatin domain. Barrier mutant cells can revert to wild-type growth and centromere structure at a high frequency after the spontaneous excision of integrated exogenous DNA. Our results reveal a pre- viously undemonstrated role for chromatin barriers in chromosome segregation and in the prevention of genome instability. KEYWORDS centromere genome instability chromatin CENP-A barrier Centromeres are unique loci that direct chromosome segregation dur- ing mitosis and meiosis. Mammalian centromeres encompass hun- dreds to thousands of kilobases of repetitive arrays that assemble into structurally and functionally distinct chromatin domains. For example, centromeric core chromatin is enriched in atypical nucleosomes in which histone H3 is replaced by the evolutionarily conserved centro- mere-specific histone H3 variant CENP-A. Core chromatin is the structural foundation of the three-dimensional kinetochore, a multi- protein complex that links the chromosome to the mitotic spindle during cell division. Epigenetic mechanisms are involved in determin- ing the genomic location of CENP-A deposition because centromere- associated DNA sequences vary among organisms, among chromo- somes of a single organism, and between individuals of the same organism (Henikoff and Furuyama 2010). A second centromeric chromatin domain, pericentric heterochromatin, is characterized by the presence of the conserved heterochromatin protein 1 and nucle- osomes di- or trimethylated at lysines 9 and/or 27 of histone H3 (Verdaasdonk and Bloom 2011). Pericentric heterochromatin may provide tension and/or rigidity at centromeres during cell division (Black et al. 2004) and is functionally required for chromosome co- hesion in some organisms (Bernard et al. 2001). In addition to being structurally and functionally distinct, core and pericentric heterochro- matin domains are also spatially distinct. Linear chromatin fibers of centromeric loci appear as alternating, nonoverlapping bocks of core and pericentric heterochromatin domains (Blower et al. 2002; Sullivan and Karpen 2004; Dunleavy et al. 2011). The specification and pres- ervation of discrete chromatin subdomains is closely linked with cen- tromere activity and genome stability. For example, some malignant cells are characterized by an increase in the size of the core chromatin domain and a reduction in pericentric heterochromatin marks (Sullivan et al. 2011). Similarly, many tumors that display aberrant cell division and genome instability are associated with aberrant histone modifications (Nakano et al. 2008; Bergmann et al. 2011; Slee et al. 2012). Defects in meiotic chromosome segregation also are correlated with changes in subdomain size and integrity (Scott et al. 2006). Copyright © 2014 Gaither et al. doi: 10.1534/g3.114.010397 Manuscript received November 26, 2013; accepted for publication February 7, 2014; published Early Online February 13, 2014. This is an open-access article distributed under the terms of the Creative Commons Attribution Unported License (http://creativecommons.org/licenses/ by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Supporting information is available online at http://www.g3journal.org/lookup/ suppl/doi:10.1534/g3.114.010397/-/DC1 1 Present address: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany. 2 Corresponding author: Institute for Genome Sciences and Policy, Duke University, 101 Science Drive Box 3382, Durham, NC 27708. E-mail: kristin.scott@duke.edu Volume 4 | April 2014 | 633