AbstractThe centromere is the chromosomal locus at which the kinetochore is assembled to direct chromosome segregation. The histone H3 variant, centromere protein A (CENP-A), is known to epigenetically mark active centromeres, but the mechanism by which CENP-A propagates at the centromere, replacing histone H3, remains poorly understood. Using fission yeast, here we show that the Ino80 adenosine triphosphate (ATP)-dependent chromatin-remodeling complex, which removes histone H3-containing nucleosomes from associated chromatin, promotes CENP-A^Cnp1 chromatin assembly at the centromere in a redundant manner with another chromatin-remodeling factor Chd1^Hrp1. CENP-A^Cnp1 chromatin actively recruits the Ino80 complex to centromeres to elicit eviction of histone H3-containing nucleosomes. Artificial targeting of Ino80 subunits to a non-centromeric DNA sequence placed in a native centromere enhances the spreading of CENP-A^Cnp1 chromatin into the non-centromeric DNA. Based on these results, we propose that CENP-A^Cnp1 chromatin employs the Ino80 complex to mediate the replacement of histone H3 with CENP-A^Cnp1, and thereby reinforces itself.