Centromeric islands constrain CENP-A domain size and position

Centromere identity is specified epigenetically by nucleosomes containing the histone H3 variant CENP-A, yet centromeres are almost invariably embedded within repetitive DNA, suggesting that underlying DNA sequences contribute to the organization of centromeric chromatin. In Drosophila melanogaster, CENP-A primarily occupies retroelement-rich islands flanked by simple satellite DNA, but their functional contribution remains unclear. Here, we delete the ~50-kb island Giglio from centromere 3 while preserving the flanking satellites and residual CENP-A chromatin. Despite removal of the DNA sequence associated with most centromeric CENP-A, centromere identity and overall CENP-A levels are preserved at the native locus, indicating that the island itself is not required for centromere identity. Instead, Giglio deletion increases variability in CENP-A chromatin organization, producing highly heterogeneous domain sizes and preferential redistribution of the CENP-A domain into the adjacent dodeca satellite. Together, these findings demonstrate that Giglio is dispensable for centromere identity but constrains the size and position of the CENP-A domain, establishing centromeric islands as key determinants of CENP-A domain architecture.