TOS9 regulates white-opaque switching in Candida albicans

In Candida albicans, the a1-alpha2 complex represses white-opaque switching, as well as mating. Based upon the assumption that the a1-alpha2 corepressor complex binds to the gene that regulates white-opaque switching, a chromatinimmunoprecipitation-microarray analysis strategy was used to identify 52 genes that bound to the complex. One of these genes, TOS9, exhibited an expression pattern consistent with a "master switch gene." TOS9 was only expressed in opaque cells, and its gene product, Tos9p, localized to the nucleus. Deletion of the gene blocked cells in the white phase, misexpression in the white phase caused stable mass conversion of cells to the opaque state, and misexpression blocked temperature-induced mass conversion from the opaque state to the white state. A model was developed for the regulation of spontaneous switching between the opaque state and the white state that includes stochastic changes of Tos9p levels above and below a threshold that induce changes in the chromatin state of an as-yet-unidentified switching locus. TOS9 has also been referred to as EAP2 and WOR1.     

A histone deacetylation inhibitor and mutant promote colony-type switching of the human pathogen Candida albicans

Most strains of Candida albicans undergo high frequency phenotypic switching. Strain WO-1 undergoes the white-opaque transition, which involves changes in colony and cellular morphology, gene expression, and virulence. We have hypothesized that the switch event involves heritable changes in chromatin structure. To test this hypothesis, we transiently exposed cells to the histone deacetylase inhibitor trichostatin-A (TSA). Treatment promoted a dramatic increase in the frequency of switching from white to opaque, but not opaque to white. Targeted deletion of HDA1, which encodes a deacetylase sensitive to TSA, had the same selective effect. These results support the model that the acetylation of histones plays a selective role in regulating the switching process.     

Differential phagocytosis of white versus opaque Candida albicans by Drosophila and mouse phagocytes

The human fungal pathogen Candida albicans resides asymptomatically in the gut of most healthy people but causes serious invasive diseases in immunocompromised patients. Many C. albicans strains have the ability to stochastically switch between distinct white and opaque cell types, but it is not known with certainty what role this switching plays in the physiology of the organism. Here, we report a previously undescribed difference between white and opaque cells, namely their interaction with host phagocytic cells. We show that both Drosophila hemocyte-derived S2 cells and mouse macrophage-derived RAW264.7 cells preferentially phagocytose white cells over opaque cells. This difference is seen both in the overall percentage of cultured cells that phagocytose white versus opaque C. albicans and in the average number of C. albicans taken up by each phagocytic cell. We conclude that susceptibility to phagocytosis by cells of the innate immune system is an important distinction between white and opaque C. albicans, and propose that one role of switching from the prevalent white form into the rarer opaque form may be to allow C. albicans to escape phagocytosis.     

Candida albicans Zcf37, a zinc finger protein, is required for stabilization of the white state

Candida albicans, the most prevalent human fungal pathogen, can switch stochastically between white and opaque phases. In this study, we identified Zcf37, a zinc finger protein, as a new regulator of white-opaque switching. Deletion of ZCF37 increased white-to-opaque switching frequency and stabilized the opaque state. Overexpression of ZCF37 promoted conversion of opaque cells to white phase, but needed existence of Efg1, a key regulator required for maintenance of the white state. Deletion of EFG1 abolished the effect of ectopically expressed Zcf37 on opaque-to-white switching, whereas ectopic expression of EFG1 promoted white cell formation without presence of Zcf37. Our results suggest that Zcf37 acts as an activator of white cell formation and a repressor of opaque state and functions upstream of Efg1.     

Why does Candida albicans switch?

White–opaque switching in Candida albicans was first discovered in 1987. Fifteen years later, and three years after the discovery of the mating system, it was demonstrated that the switch from white to opaque was an essential step in the mating process. But this latter discovery did not reveal why C. albicans had this requirement, when Saccharomyces cerevisiae and other hemiascomycetes did not. The discovery that mating-competent opaque cells signaled mating-incompetent white cells, through the release of pheromones, to become adhesive and form biofilms provided a clue to this fundamental question. Opaque cells appeared to signal white cells to form biofilms that facilitated mating by protecting the fragile gradients of the pheromone that directed chemotropism, a process necessary for fusion. Here, we explore the discoveries and observations that have led to this hypothesis, and the ancillary questions that have risen that are related to the regulation of the unique pheromone response, the evolution of this response and the relationship between pheromone-enhanced white cell biofilms and 'asexual' biofilms formed by a /α cells. This discussion, therefore, focuses on a unique and complex component of the basic biology of C. albicans that relates switching, mating and pathogenesis.     

Self-induction of a/a or alpha/alpha biofilms in Candida albicans is a pheromone-based paracrine system requiring switching

Like MTL-heterozygous (a/α) cells, white MTL-homozygous (a/a or α/α) cells of Candida albicans, to which a minority of opaque cells of opposite mating type have been added, form thick, robust biofilms. The latter biofilms are uniquely stimulated by the pheromone released by opaque cells and are regulated by the mitogen-activated protein kinase signal transduction pathway. However, white MTL-homozygous cells, to which opaque cells of opposite mating type have not been added, form thinner biofilms. Mutant analyses reveal that these latter biofilms are self-induced. Self-induction of a/a biofilms requires expression of the α-receptor gene STE2 and the α-pheromone gene MFα, and self-induction of α/α biofilms requires expression of the a-receptor gene STE3 and the a-pheromone gene MFa. In both cases, deletion of WOR1, the master switch gene, blocks cells in the white phenotype and biofilm formation, indicating that self-induction depends upon low frequency switching from the white to opaque phenotype. These results suggest a self-induction scenario in which minority opaque a/a cells formed by switching secrete, in a mating-type-nonspecific fashion, α-pheromone, which stimulates biofilm formation through activation of the α-pheromone receptor of majority white a/a cells. A similar scenario is suggested for a white α/α cell population, in which minority opaque α/α cells secrete a-pheromone. This represents a paracrine system in which one cell type (opaque) signals a second highly related cell type (white) to undergo a complex response, in this case the formation of a unisexual white cell biofilm.     

Genetic analysis implicates the Set3/Hos2 histone deacetylase in the deposition and remodeling of nucleosomes containing H2A.Z

Histone variants and histone modification complexes act to regulate the functions of chromatin. In Saccharomyces cerevisiae the histone variant H2A.Z is encoded by HTZ1. Htz1 is dispensable for viability in budding yeast, but htz1Δ is synthetic sick or lethal with the null alleles of about 200 nonessential genes. One of the strongest of these interactions is with the deletion of SET3, which encodes a subunit of the Set3/Hos2 histone deacetylase complex. Little is known about the functions of Set3, and interpreting these genetic interactions remains a highly challenging task. Here we report the results of a forward genetic screen to identify bypass suppressors of the synthetic slow-growth phenotype of htz1Δ set3Δ. Among the identified loss-of-function suppressors are genes encoding subunits of the HDA1 deacetylase complex, the SWR1 complex, the H2B deubiquitination module of SAGA, the proteasome, Set1, and Sir3. This constellation of suppressor genes is uncommon among the global set of htz1Δ synthetic interactions. BDF1, AHC1, RMR1, and CYC8 were identified as high-copy suppressors. We also identified interactions with SLX5 and SLX8, encoding the sumoylation-targeted ubiquitin ligase complex. In the context of htz1Δ set3Δ, suppressors in the SWR1 and the H2B deubiquitination complexes show strong functional similarity, as do suppressors in the silencing genes and the proteasome. Surprisingly, while both htz1Δ set3Δ and swr1Δ set3Δ have severe slow-growth phenotypes, the htz1Δ swr1Δ set3Δ triple mutant grows relatively well. We propose that Set3 has previously unrecognized functions in the dynamic deposition and remodeling of nucleosomes containing H2A.Z.     

Analysis of phase-specific gene expression at the single-cell level in the white-opaque switching system of Candida albicans

The opportunistic fungal pathogen Candida albicans can switch spontaneously and reversibly between different cell forms, a capacity that may enhance adaptation to different host niches and evasion of host defense mechanisms. Phenotypic switching has been studied intensively for the white-opaque switching system of strain WO-1. To facilitate the molecular analysis of phenotypic switching, we have constructed homozygous ura3 mutants from strain WO-1 by targeted gene deletion. The two URA3 alleles were sequentially inactivated using the MPA(R)-flipping strategy, which is based on the selection of integrative transformants carrying a mycophenolic acid (MPA) resistance marker that is subsequently deleted again by site-specific, FLP-mediated recombination. To investigate a possible cell type-independent switching in the expression of individual phase-specific genes, two different reporter genes that allowed the analysis of gene expression at the single-cell level were integrated into the genome, using URA3 as a selection marker. Fluorescence microscopic analysis of cells in which a GFP reporter gene was placed under the control of phase-specific promoters demonstrated that the opaque-phase-specific SAP1 gene was detectably expressed only in opaque cells and that the white-phase-specific WH11 gene was detectably expressed only in white cells. When MPA(R) was used as a reporter gene, it conferred an MPA-resistant phenotype on opaque but not white cells in strains expressing it from the SAP1 promoter, which was monitored at the level of single cells by a significantly enlarged size of the corresponding colonies on MPA-containing indicator plates. Similarly, white but not opaque cells became MPA resistant when MPA(R) was placed under the control of the WH11 promoter. The analysis of these reporter strains showed that cell type-independent phase variation in the expression of the SAP1 and WH11 genes did not occur at a detectable frequency. The expression of these phase-specific genes of C. albicans in vitro, therefore, is tightly linked to the cell type.     

Overlapping Functions between SWR1 Deletion and H3K56 Acetylation in Candida albicans

Nucleosome destabilization by histone variants and modifications has been implicated in the epigenetic regulation of gene expression, with the histone variant H2A.Z and acetylation of H3K56 (H3K56ac) being two examples. Here we find that deletion of SWR1, the major subunit of the SWR1 complex depositing H2A.Z into chromatin in exchange for H2A, promotes epigenetic white-opaque switching in Candida albicans. We demonstrate through nucleosome mapping that SWR1 is required for proper nucleosome positioning on the promoter of WOR1, the master regulator of switching, and that its effects differ in white and opaque cells. Furthermore, we find that H2A.Z is enriched adjacent to nucleosome-free regions at the WOR1 promoter in white cells, suggesting a role in the stabilization of a repressive chromatin state. Deletion of YNG2, a subunit of the NuA4 H4 histone acetyltransferase (HAT) that targets SWR1 activity through histone acetylation, produces a switching phenotype similar to that of swr1, and both may act downstream of the GlcNAc signaling pathway. We further uncovered a genetic interaction between swr1 and elevated H3K56ac with the discovery that the swr1 deletion mutant is highly sensitive to nicotinamide. Our results suggest that the interaction of H2A.Z and H3K56ac regulates epigenetic switching at the nucleosome level, as well as having global effects.     

A conserved histone deacetylase with a role in the regulation of cytokinesis in Schizosaccharomyces pombe

ABSTRACT: BACKGROUND: In Schizosaccharomyces pombe the SET domain protein, Set3p - together with its interacting partners, Snt1p, and Sif2p - form a complex that aids in preventing cell division failure upon mild cytokinetic stress. Intriguingly, the human orthologs of these genes (MLL5, NCOR2, and TBL1X) are also important for the faithful completion of cytokinesis in tissue culture cells. Since MLL5, NCOR2, and TBL1X form a complex with the histone de-acetylase, HDAC3, we sought to determine if an orthologous counterpart played a regulatory role in fission yeast cytokinesis. RESULTS: In this report we identify the hos2 gene as the fission yeast HDAC3 ortholog. We show that Hos2p physically interacts with Set3p, Snt1p, and Sif2p, and that hos2Delta mutants are indeed compromised in their ability to reliably complete cell division in the presence of mild cytokinetic stresses. Furthermore, we demonstrate that over-expression of hos2 causes severe morphological and cytokinetic defects. Lastly, through recombinase mediated cassette exchange, we show that expression of human HDAC3 complements the cytokinetic defects exhibited by hos2Delta cells. CONCLUSIONS: These data support a model in which Hos2p functions as a critical functional component of the Set3p-Snt1p-Sif2p complex with respect to its role in cytokinesis. The ability of human HDAC3 to complement the cytokinesis defects associated with the deletion of the hos2 gene suggests that further analysis of this system could translate into a theoretical framework for understanding how the orthologous MLL5 complex functions to regulate cytokinesis in human cells.