DNA methylation regulates phenotype-dependent transcriptional activity in Candida albicans.

DNA methylation is a common epigenetic signaling mechanism associated with silencing of repeated DNA and transcriptional regulation in eukaryotes. Here we report that DNA methylation in the human fungal pathogen Candida albicans is primarily localized within structural genes and modulates transcriptional activity. Major repeat sequences and multigene families are largely free of DNA methylation. Among the genes subject to DNA methylation are those associated with dimorphic transition between yeast and hyphal forms, switching between white and opaque cells, and iron metabolism. Transcriptionally repressed methylated loci showed increased frequency of C-to-T transitions during asexual growth, an evolutionarily stable pattern of repression associated mutation that could bring about genetic alterations under changing environmental or host conditions. Dynamic differential DNA methylation of structural genes may be one factor contributing to morphological plasticity that is cued by nutrition and host interaction.

Centromere size and position in Candida albicans are evolutionarily conserved independent of DNA sequence heterogeneity.

The centromere regions (CEN) of all eight chromosomes in Candida albicans have been characterized in terms of nucleotide sequence and size. The boundaries of each of the eight CEN DNA regions were mapped by chromatin immunoprecipitation-PCR using polyclonal rabbit antibodies generated against C. albicans centromere-specific protein CaCse4p (CENP-A homolog). A single 3-4.5 kb unique DNA sequence on each chromosome was found to be bound to CaCse4p. Sequence analysis revealed that the eight CEN regions in C. albicans lack any conserved DNA sequence motifs common to the group; all are quite different in overall DNA sequence. In contrast to centromeres in many organisms, the C. albicans centromeres are generally free of repeated DNA elements and transposons. However, a few small inverted repeats and long terminal repeats do occur in the centromeric and pericentric regions on a few chromosomes. We also characterized the CEN DNAs in four groups of phylogenetically divergent C. albicans strains, estimated to be separated from each other by 1-3 million years. The same eight different and unique 3-4.5 kb DNA sequences are utilized as centromeres in all of these strains. The chromosomal locations and the sizes of CEN DNAs have remained conserved, in agreement with the idea that CEN function in C. albicans is templated by heritable epigenetic mechanisms.

Formation of functional centromeric chromatin is specified epigenetically in Candida albicans.

In the pathogenic yeast Candida albicans, the 3-kb centromeric DNA regions (CEN) of each of the eight chromosomes have different and unique DNA sequences. The centromeric histone CaCse4p (CENP-A homolog) occurs only within these 3-kb CEN regions to form specialized centromeric chromatin. Centromere activity was maintained on small chromosome fragments derived in vivo by homologous recombination of a native chromosome with linear DNA fragments containing a telomere and a selectable marker. An in vivo derived 85-kb truncated chromosome containing the 3-kb CEN7 locus on 69 kb of chromosome 7 DNA was stably and autonomously maintained in mitosis, indicating that preexisting active CEN chromatin remains functional through many generations. This same 85-kb chromosome fragment, isolated as naked DNA (devoid of chromatin proteins) from C. albicans and reintroduced back into C. albicans cells by standard DNA transformation techniques, was unable to reform functional CEN chromatin and was mitotically unstable. Comparison of active and inactive CEN chromatin digested with micrococcal nuclease revealed that periodic nucleosome arrays are disrupted at active centromeres. Chromatin immunoprecipitation with antibodies against CaCse4p confirmed that CEN7 introduced into C. albicans cells as naked DNA did not recruit CaCse4p or induce its spread to a duplicate region only 7 kb away from active CEN7 chromatin. These results indicate that CaCse4p recruitment and centromere activation are epigenetically specified and maintained in C. albicans.

Inner kinetochore of the pathogenic yeast Candida glabrata.

The human pathogenic yeast Candida glabrata is the second most common Candida pathogen after Candida albicans, causing both bloodstream and mucosal infections. The centromere (CEN) DNA of C. glabrata (CgCEN), although structurally very similar to that of Saccharomyces cerevisiae, is not functional in S. cerevisiae. To further examine the structure of the C. glabrata inner kinetochore, we isolated several C. glabrata homologs of S. cerevisiae inner kinetochore protein genes, namely, genes for components of the CBF3 complex (Ndc10p, Cep3p, and Ctf13p) and genes for the proteins Mif2p and Cse4p. The amino acid sequence identities of these proteins were 32 to 49% relative to S. cerevisiae. CgNDC10, CgCEP3, and CgCTF13 are required for growth in C. glabrata and are specifically found at CgCEN, as demonstrated by chromatin immunoprecipitation experiments. Cross-complementation experiments revealed that the isolated genes, with the exception of CgCSE4, are species specific and cannot functionally substitute for the corresponding genes in S. cerevisiae deletion strains. Likewise, the S. cerevisiae CBF3 genes NDC10, CEP3, and CTF13 cannot functionally replace their homologs in C. glabrata CBF3 deletion strains. Two-hybrid analysis revealed several interactions between these proteins, all of which were previously reported for the inner kinetochore proteins of S. cerevisiae. Our findings indicate that although many of the inner kinetochore components have evolved considerably between the two closely related species, the organization of the C. glabrata inner kinetochore is similar to that in S. cerevisiae.

The CENP-A homolog CaCse4p in the pathogenic yeast Candida albicans is a centromere protein essential for chromosome transmission.

The gene encoding CaCse4p, a homolog of the evolutionarily conserved histone H3-like kinetochore protein CENP-A, has been cloned from the human pathogenic diploid yeast Candida albicans. To study the phenotype of C. albicans diploid cells depleted of CaCse4p, we deleted one copy of CaCSE4 and brought the other copy under control of a regulated PCK1 promoter (repressed by glucose and induced by succinate). Inability of this strain to grow on glucose medium indicates that CaCse4p is essential for cell viability. Shutdown of CaCSE4 expression resulted in a sharp decline of CaCse4p levels with concomitant loss of cell viability. Examination of these CaCse4p-depleted cells revealed a mitosis-specific arrest phenotype with accumulation of large-budded cells containing single G(2) nuclei at or near the bud neck along with short mitotic spindles. Subcellular localization of CaCse4p by anti-CaCse4p antibodies in both budding and filamentous C. albicans cells revealed an intense dot-like signal always colocalized with 4',6-diamidino-2-phenylindole-stained nuclei. Unlike higher eukaryotes but similar to the budding yeast Saccharomyces cerevisiae, centromere separation in the budding yeast form of C. albicans occurs before anaphase, at a very early stage of the cell cycle. In the filamentous mode of cell division, however, centromere separation appears to occur in early anaphase. Coimmunostaining with anti-CaCse4p and antitubulin antibodies shows that CaCse4p localizes near spindle pole bodies, analogous to the localization pattern observed for kinetochore proteins in S. cerevisiae. CaCse4p promises to be a highly useful reagent for the study of centromere/kinetochore structure in C. albicans.

Centromeric DNA sequences in the pathogenic yeast Candida albicans are all different and unique.

In an approach to clone and characterize centromeric DNA sequences of Candida albicans by chromatin immunoprecipitation, we have used antibodies directed against an evolutionarily conserved histone H3-like protein, CaCse4p (CENP-A homolog). Sequence analysis of clones obtained by this procedure reveals that only eight relatively small regions (approximately 3 kb each) of the Can. albicans genome are selectively enriched. These CaCse4-bound sequences are located within 4- to 18-kb regions lacking ORFs and occur once in each of the eight chromosomes of Can. albicans. Binding of another evolutionarily conserved kinetochore protein, CaMif2p (CENP-C homolog), colocalizes with CaCse4p. Deletion of the CaCse4p-binding region of chromosome 7 results in a high rate of loss of the altered chromosome, confirming that CaCse4p, a centromeric histone in the CENP-A family, indeed identifies the functional centromeric DNA of Can. albicans. The CaCse4p-rich regions not only lack conserved DNA motifs of point (<400 bp) centromeres and repeated elements of regional (>40 kb) centromeres, but also each chromosome of Can. albicans contains a different and unique CaCse4p-rich centromeric DNA sequence, a centromeric property previously unobserved in other organisms.