Establishment of centromere identity is dependent on nuclear spatial organization.

The establishment of centromere-specific CENP-A chromatin is influenced by epigenetic and genetic processes. Central domain sequences from fission yeast centromeres are preferred substrates for CENP-A incorporation, but their use is context dependent, requiring adjacent heterochromatin. CENP-A overexpression bypasses heterochromatin dependency, suggesting that heterochromatin ensures exposure to conditions or locations permissive for CENP-A assembly. Centromeres cluster around spindle-pole bodies (SPBs). We show that heterochromatin-bearing minichromosomes localize close to SPBs, consistent with this location promoting CENP-A incorporation. We demonstrate that heterochromatin-independent de novo CENP-A chromatin assembly occurs when central domain DNA is placed near, but not far from, endogenous centromeres or neocentromeres. Moreover, direct tethering of central domain DNA at SPBs permits CENP-A assembly, suggesting that the nuclear compartment surrounding SPBs is permissive for CENP-A incorporation because target sequences are exposed to high levels of CENP-A and associated assembly factors. Thus, nuclear spatial organization is a key epigenetic factor that influences centromere identity.