RbAp46/48LIN-53and HAT-1 are required for initial CENP-AHCP-3deposition and de novo centromere formation inCaenorhabditis elegansembryos

ABSTRACT Foreign DNA microinjected into the Caenorhabditis elegans germline forms episomal extra-chromosomal arrays, or artificial chromosomes (ACs), in embryos. Injected linear, short DNA fragments concatemerize into high molecular weight (HMW)-DNA arrays that are visible as punctate DAPI-stained foci in oocytes, which undergo chromatinization and centromerization in embryos. The inner centromere, inner and outer kinetochore components, including AIR-2, CENP-A HCP-3 , Mis18BP1 KNL-2 and BUB-1, assemble onto the nascent ACs during the first mitosis. Yet, due to incomplete DNA replication of the nascent ACs, centromeric proteins are not oriented at the poleward faces of the nascent ACs in mitosis, resulting in lagging ACs. The DNA replication efficiency of ACs improves over several cell cycles. We found that a condensin subunit, SMC-4, but not the replicative helicase component, MCM-2, facilitates de novo CENP-A HCP-3 deposition on nascent ACs. Furthermore, H3K9ac, H4K5ac, and H4K12ac are highly enriched on newly chromatinized ACs. HAT-1 and RbAp46/48 LIN-53 , which are essential for de novo centromere formation and segregation competency of nascent ACs, also hyperacetylate histone H3 and H4. Different from centromere maintenance on endogenous chromosomes, where Mis18BP1 KNL-2 functions upstream of RbAp46/48 LIN-53 , RbAp46/48 LIN-53 depletion causes the loss of both CENP-A HCP-3 and Mis18BP1 KNL-2 initial deposition at de novo centromeres on ACs.