DNA replication initiator proteins facilitate CENPA loading on early replicating compact chromatin
2018
The process of centromere formation enables the cell to conserve established genetic and epigenetic information from the previous cell cycle and reuse it for future episodes of chromosome segregation. CENPA asserts the role of an epigenetic requirement in maintaining active centromeres. Active centromeres are subject to position effects which can cause its site of assembly to drift occasionally. Determinants of neocentromere formation, when a native centromere is inactivated, remain elusive. To dissect factors for centromere/neocentromere formation, here, we employed the budding yeast Candida albicans, whose centromeres have unique and different DNA sequences, and exhibit classical epigenetic regulation. We used CENPA-mediated reversible silencing of a marker gene, URA3, as an assay to select cells with ectopic centromeres. We defined pericentric boundaries for C. albicans centromeres by Hi-C analysis and these were located in early replicating domains. The pericentric boundaries primed with CENPA served as sites of neocentromere formation in isolates with ectopic centromeres, indicating that the number of non-centromeric CENPA molecules determines neocentromere location. To understand the importance of early replication timing of centromeres, we identified genome-wide binding sites of the Origin Recognition Complex subunit, Orc4. A fraction of these Orc4 enriched regions located within tDNA, cluster towards early replicating regions, and frequently interact among themselves than the late replicating regions, demonstrating the spatiotemporal distribution of these regions. Strikingly, Orc4 is highly enriched at centromeres of C. albicans and along with the helicase component Mcm2, stabilizes the kinetochore, suggesting a role of pre-replication complex proteins as epigenetic determinants of centromere identity.
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