JCB_201605001 101..114

The centromere is a critical genomic region where the kinetochore is assembled and mediates the interaction between chromosome and spindle microtubules in the process of faithful chromosome segregation. The centromere position must be specified at a single locus on each chromosome to prevent chromosome instability in most organisms, and the specification of the centromere position is an important step during chromosome segregation. Centromeres with repetitive sequences are found in many organisms (Fukagawa and Earnshaw, 2014a). For example, most human and mouse chromosomes contain α satellite and minor satellite sequences, respectively. Although DNA sequence may contain information significant for the centromere function, a recent consensus theory suggests that the DNA sequence itself is not crucial for the centromere specification, but that the centromere is specified at a particular position by sequence-independent epigenetic mechanisms (Allshire and Karpen, 2008; Perpelescu and Fukagawa, 2011; Fukagawa and Earnshaw, 2014a). This theory is based on the discovery and characterization of human neocentromeres, which do not possess α satellite sequences, but contain most of the kinetochore components and can contribute to faithful chromosome segregation (Marshall et al., 2008; Fukagawa and Earnshaw, 2014b). A centromere-specific histone H3 variant, CENP-A, was identified at most centromeres described to date, including neocentromeres. Additionally, because CENP-A represents an upstream factor required for kinetochore assembly (McKinley and Cheeseman, 2016), it has recently been suggested that CENP-A carries an epigenetic mark for the centromere specification (Black and Cleveland, 2011; Westhorpe and Straight, 2013). The formation of human neocentromeres is observed in some diseases (Voullaire et al., 1993; du Sart et al., 1997; Marshall et al., 2008; Fukagawa and Earnshaw, 2014b), and it is possible that the functional and structural aspects of neocentromeres are somewhat different from the naturally occurring centromeres. However, chromatin immunoprecipitation (ChIP) combined with massive parallel sequencing (ChIP-seq), using anti–CENP-A antibodies revealed the existence of native nonrepetitive centromeres at horse (Wade et al., 2009), chicken (Shang et al., 2010, 2013), and orangutan (Lomiento et al., 2013) chromosomes. Because these nonrepetitive centromeres are functional, this suggests that they are functionally equivalent to the centromeres with repetitive sequences. In general, the characterization of centromeric chromatin is difficult because of the existence of highly repetitive sequences. The mapping of DNAs obtained by ChIP experiments Centromeres are specified by sequence-independent epigenetic mechanisms, and the centromere position may drift at each cell cycle, but once this position is specified, it may not be frequently moved. Currently, it is unclear whether the centromere position is stable. To address this question, we systematically analyzed the position of nonrepetitive centromeres in 21 independent clones isolated from a laboratory stock of chicken DT40 cells using chromatin immunoprecipitation combined with massive parallel sequencing analysis with anti–CENP-A antibody. We demonstrated that the centromere position varies among the clones, suggesting that centromere drift occurs during cell proliferation. However, when we analyzed this position in the subclones obtained from one isolated clone, the position was found to be relatively stable. Interestingly, the centromere drift was shown to occur frequently in CENP-U– and CENP-S–deficient cells. Based on these results, we suggest that the centromere position can change after many cell divisions, but this drift is suppressed in short-term cultures, and the complete centromere structure contributes to the suppression of the centromere drift. Constitutive centromere-associated network controls centromere drift in vertebrate cells