Background: Centromeres are critically important for chromosome stability and integrity. Most eukaryotes have regional centromeres that include long tracts of repetitive DNA packaged into pericentric heterochromatin. Neocentromeres, new sites of functional kinetochore assembly, can form at ectopic loci because no DNA sequence is strictly required for assembly of a functional kinetochore. Candida albicans, the pathogen that causes thrush, has small regional centromeres lacking pericentric heterochromatin.
Objectives: We sought to understand the properties of centromeres in C. albicans and to understand mechanisms by which centromere dysfunction causes aneuploidy and associated drug resistance.
Methods: We replaced CEN5 DNA with the counter-selectable URA3 gene. Neocentromere regions that arose were identified by chromatin immunoprecipitation with an antibody to a centromere-specific histone, CENP-A/Cse4p, followed by PCR analysis or high-throughput sequencing.
Results: We functionally delimited CEN5 DNA to an inverted repeat flanking a core region. All cen5Æ::URA3 transformants stably retained both copies of Chr5, indicating that a functional neocentromere had assembled efficiently on a Chr5 homolog. Strains selected to maintain only the cen5::URA3 homolog and no wild-type Chr5 homolog grew well, indicating that neocentromere function is independent of the presence of any wild-type CEN5 DNA. Similar results were obtained with other selectable markers. Two classes of neocentromere (neoCEN) strains were distinguishable: 'distal neoCENs' and 'proximal neoCENs'. Neocentromeres in the distal neoCEN strains, formed at loci ~200-450 kb from cen5::URA3 on either chromosome arm. In proximal neoCEN strains, neocentromeres formed directly adjacent to cen5::URA3 and upon counter-selection, moved onto the URA3 DNA, resulting in silencing of its expression.
Conclusion: In C. albicans, deletion of centromere DNA results in efficient neocentromere formation at numerous different loci. Furthermore, once formed, neocentromere can move to adjacent loci.
Funding was supported by MinnCResT fellowship (NIHT32-DE007288) to C.K. and NIHR01AI062427 and R01AI075096 awards to J.B.