Abstract
Deletion of native centromeres in the human fungal pathogen Cryptococcus deuterogattii leads to neocentromere formation. Native centromeres span truncated transposable elements, while neocentromeres do not and instead span actively expressed genes. To explore the epigenetic organization of neocentromeres, we analyzed the distribution of the heterochromatic histone modification H3K9me2, 5mC DNA methylation and the euchromatin mark H3K4me2. Native centromeres are enriched for both H3K9me2 and 5mC DNA methylation marks and are devoid of H3K4me2, while neocentromeres do not exhibit any of these features. Neocentromeres in cen10Δ mutants are unstable and chromosome-chromosome fusions occur. After chromosome fusion, the neocentromere is inactivated and the native centromere of the chromosome fusion partner remains as the sole, active centromere. In the present study, the active centromere of a fused chromosome was deleted to investigate if epigenetic memory promoted the re-activation of the inactive neocentromere. Our results show that the inactive neocentromere is not re-activated and instead a novel neocentromere forms directly adjacent to the deleted centromere of the fused chromosome. To study the impact of transcription on centromere stability, the actively expressed URA5 gene was introduced into the CENP-A bound regions of a native centromere. The introduction of the URA5 gene led to a loss of CENP-A from the native centromere, and a neocentromere formed adjacent to the native centromere location. Remarkably, the inactive, native centromere remained enriched for heterochromatin, yet the integrated gene was expressed and devoid of H3K9me2. A cumulative analysis of multiple CENP-A distribution profiles revealed centromere drift in C. deuterogattii, a previously unreported phenomenon in fungi. The CENP-A-binding shifted within the ORF-free regions and showed a possible association with a truncated transposable element. Taken together, our findings reveal that neocentromeres in C. deuterogattii are highly unstable and are not marked with an epigenetic memory, distinguishing them from native centromeres.
Highlights
To undergo proper cell division, chromosomes require a functional centromere that mediates binding of the kinetochore and microtubules to the chromosome [1]
We report that the native centromeres of the human fungal pathogen Cryptococcus deuterogattii are enriched with heterochromatin marks
We found that neocentromere formation can be triggered by deletion of the native centromere and by disrupting its function via insertion of a gene
Summary
To undergo proper cell division, chromosomes require a functional centromere that mediates binding of the kinetochore and microtubules to the chromosome [1]. The kinetochore assembles on a specific centromeric histone H3 variant (known as CENP-A, CENH3, or Cse depending on the species), which replaces the canonical histone H3 within the centromere [2]. Fungal regional centromeres are well studied; in particular, the centromeres of Candida albicans and closely related species have been the subject of extensive investigation [9,10,11]. Native centromeres of Schizosaccharomyces pombe, another ascomycetous fungus, have a higher-order structure with a CENP-A-enriched central core, flanked by heterochromatic inner and outer repeats that serve as pericentric regions [17]. The pericentric regions are enriched for H3K9me2/3, and this modification is RNAi-dependent and essential for CENP-A localization to the central core [18,19]
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