Structure-function analysis of centromeres in the cryptococcus species complex

Abstract

In this work, we identified and characterized centromeres and studied their structural organization in a group of Cryptococcus species of the fungal phylum Basidiomycota. Further, we examined the spatio-temporal dynamics of the centromere-kinetochore complex in one of these species, Cryptococcus neoformans, an opportunistic pathogen that infects immunocompromised as well as immunocompetent people worldwide. Since centromeres have been shown to be evolving rapidly, we were interested in studying centromere evolution in closely related Cryptococcus species. Thus, we sought to identify centromeres in four species of the Cryptococcus species complex (Table 1.2). Chromatin immunoprecipitation (ChIP), with conserved kinetochore proteins CENP-A and CENP-C, followed by next generation sequencing (ChlP-seq) or qPCR was used to identify and/or validate the centromeres. This analysis led to the identification of large regional centromeres in all four Cryptococcus species. In all cases, each centromere resides in a long ORF-free and transcriptionally silent region on the respective chromosome. Sequence analysis of centromere DNA sequences revealed that they are enriched with retrotransposons. Retrotransposons present in the pathogenic species of the Cryptococcus species complex differ from those present in the non-pathogenic species hinting that centromeres are evolving rapidly in the Cryptococcus species complex. Among the pathogenic species in Cryptococcus species complex, one of the species was known to be RNAi-deficient (R265) whereas the other two are RNAi-proficient (H99 and JEC21). We discovered a correlation between the presence of the RNAi machinery in the genome and the length of the centromere of the respective species - the centromere length in the RNAi-deficient species is significantly reduced as compared to the RNAi proficient strains. Comparison of retrotransposons present in centromeres of the three pathogenic species revealed that RNAi proficient species harbour a set of full-length retroelements; the RNAi-deficient species, on the other hand, completely lacks them. Using an experimental evolution based approach, we showed that in the RNAi mutants of H99, that usually possesses long retrotransposon-rich centromeres, undergo structural alterations across the centromere regions. Using in silico analysis based on a specific set of properties of centromeres, we also identified centromeres in three Ustilago species - another basidiomycetous species complex which are major plant pathogens. Like Cryptococcus, RNAi is lost in one of the three species in Ustilago species complex. Comparison of putative centromere length in the three Ustilago species revealed that the RNAi-deficient species ((/. maydis) has shorter centromeres than those of the RNAi-proficient species (U. hordei and U. bromivora). Taken together, we could identify centromeres in six pathogenic basidiomycetes belonging to two different species complexes. Both species complexes consist of one RNAideficient species with shorter centromeres compared to those of the RNAi-proficient species. Based on these observations, we conclude that RNAi components may be shaping up the structure of the centromere in basidiomycetous yeast species.