Hap2-Ino80-facilitated transcription promotes de novo establishment of CENP-A chromatin.

Centromeres are maintained epigenetically by the presence of CENP-A, an evolutionarily conserved histone H3 variant, which directs kinetochore assembly and hence centromere function. To identify factors that promote assembly of CENP-A chromatin, we affinity-selected solubilized fission yeast CENP-ACnp1 chromatin. All subunits of the Ino80 complex were enriched, including the auxiliary subunit Hap2. Chromatin association of Hap2 is Ies4-dependent. In addition to a role in maintenance of CENP-ACnp1 chromatin integrity at endogenous centromeres, Hap2 is required for de novo assembly of CENP-ACnp1 chromatin on naïve centromere DNA and promotes H3 turnover on centromere regions and other loci prone to CENP-ACnp1 deposition. Prior to CENP-ACnp1 chromatin assembly, Hap2 facilitates transcription from centromere DNA. These analyses suggest that Hap2-Ino80 destabilizes H3 nucleosomes on centromere DNA through transcription-coupled histone H3 turnover, driving the replacement of resident H3 nucleosomes with CENP-ACnp1 nucleosomes. These inherent properties define centromere DNA by directing a program that mediates CENP-ACnp1 assembly on appropriate sequences.