Orc4 spatiotemporally stabilizes centromeric chromatin

Area of Science:

• Cell Biology
• Genetics
• Epigenetics

Background:

• Centromeric chromatin establishment and propagation rely on epigenetic mechanisms and the histone CENPA.
• Replication origins, binding proteins, and timing are key to centromere function.
• Candida albicans features unique, epigenetically regulated regional centromeres that replicate early and cluster.

Purpose of the Study:

• To investigate the genome-wide occupancy of the replication initiation protein Orc4 in Candida albicans.
• To understand the relationship between Orc4, DNA replication timing, and centromere organization.
• To elucidate the roles of Orc4, Mcm2, and Scm3 in maintaining centromeric chromatin and CENPA stability.

Main Methods:

• Genome-wide occupancy analysis of Orc4.
• Hi-C and replication timing analyses.
• Polymer modeling of Candida albicans chromosomes.

Main Results:

• Orc4 is abundant at all centromeres and binds to motifs including tRNA genes that replicate early.
• Early replicating Orc4-bound regions show stronger self-interaction and preferential localization around kinetochores.
• Orc4 and Mcm2 are essential for cell viability and CENPA stability; Orc4 localizes constitutively to centromeres.

Conclusions:

• Spatiotemporal localization of Orc4, Mcm2, and Scm3 maintains centromeric chromatin stability in Candida albicans.
• Orc4's association with early replicating DNA and kinetochores is vital for centromere function.
• New CENPA molecules are recruited during late anaphase/telophase, coinciding with Scm3 localization.