The transcription factor Ahr1 links cell size control to amino acid metabolism in the opportunistic yeast Candida albicans.

In most eukaryotes, size homeostasis is exerted in late G1 phase as cells commit to division, called Start in yeast and the Restriction Point in metazoans. At the cellular level, size is dictated by the balance between cellular growth and division such that each cell division is accompanied by a doubling in cell mass. Our systematic screen for size mutants revealed that hundreds of genes markedly altered cell size in the opportunistic yeast Candida albicans, but only few of these overlapped with size control genes in the model yeast Saccharomyces cerevisiae. Here, we characterized one of the potent size regulators in C. albicans, the zinc-finger transcription factor Ahr1 that is unique to Candida yeasts of the CTG-clade. We found that Ahr1 acts as both a repressor of Start and a transcriptional regulator of amino acid metabolic genes. Consistently, Ahr1 was required for amino acid and nitrogen-source modulation of cell size. Genetic interactions with deletions of different known Start regulators in C. albicans revealed functional relationship of Ahr1 with the AGC family protein kinase Sch9. Collectively, this work uncovered a novel network of the nutrient-dependent size control in C. albicans and emphasizes the impact of nitrogen and amino acid metabolisms in size homeostasis in this pathogenic fungus.

Hof1 plays a checkpoint-related role in MMS-induced DNA damage response in Candida albicans.

Cells depend on robust DNA damage recognition and repair systems to maintain genomic integrity for survival in a mutagenic environment. In the pathogenic yeast Candida albicans, a subset of genes involved in the response to DNA damage-induced genome instability and morphological changes has been found to regulate virulence. To better understand the virulence-linked DNA repair network, we screened for methyl methane sulfonate (MMS) sensitivity within the GRACE conditional expression collection and identified 56 hits. One of these potential DNA damage repair-associated genes, a HOF1 conditional mutant, unexpectedly had a previously characterized function in cytokinesis. Deletion of HOF1 resulted in MMS sensitivity and genome instability, suggesting Hof1 acts in the DNA damage response. By probing genetic interactions with distinct DNA repair pathways, we found that Hof1 is genetically linked to the Rad53 pathway. Furthermore, Hof1 is down-regulated in a Rad53-dependent manner and its importance in the MMS response is reduced when Rad53 is overexpressed or when RAD4 or RAD23 is deleted. Together, this work expands our understanding of the C. albicans DNA repair network and uncovers interplay between the cytokinesis regulator Hof1 and the Rad53-mediated checkpoint.

The p38/HOG stress-activated protein kinase network couples growth to division in Candida albicans.

Cell size is a complex trait that responds to developmental and environmental cues. Quantitative size analysis of mutant strain collections disrupted for protein kinases and transcriptional regulators in the pathogenic yeast Candida albicans uncovered 66 genes that altered cell size, few of which overlapped with known size genes in the budding yeast Saccharomyces cerevisiae. A potent size regulator specific to C. albicans was the conserved p38/HOG MAPK module that mediates the osmostress response. Basal HOG activity inhibited the SBF G1/S transcription factor complex in a stress-independent fashion to delay the G1/S transition. The HOG network also governed ribosome biogenesis through the master transcriptional regulator Sfp1. Hog1 bound to the promoters and cognate transcription factors for ribosome biogenesis regulons and interacted genetically with the SBF G1/S machinery, and thereby directly linked cell growth and division. These results illuminate the evolutionary plasticity of size control and identify the HOG module as a nexus of cell cycle and growth regulation.

MAP Kinase Regulation of the Candida albicans Pheromone Pathway.

We investigated the relationships of the Cek1 and Cek2 mitogen-activated protein (MAP) kinases and the putative MAP kinase phosphatase Cpp1 in the mating process of Candida albicans Mutants of the CPP1 gene are hyperresponsive to pheromone, generating large halos, high levels of projections, and an increase in pheromone-responsive gene expression. Mating-type-homozygous opaque cells that lack both kinases are sterile, consistent with previous observations, although several lines of evidence show that the two kinases do not simply provide redundant functions in the mating process. Loss of CEK1 reduces mating significantly, to about 0.3% of wild-type strains, and also reduces projection formation and pheromone-mediated gene expression. In contrast, loss of CEK2 has less of an effect, reducing mating to approximately one-third that of the wild-type strain and moderately reducing projection formation but having little influence on the induction of gene expression. However, loss of Cek2 function reduces adaptation to pheromone-mediated arrest. The mutation enhances pheromone response halos to a level similar to that of cpp1 mutants, although the cpp1 mutants are considerably more mating defective than the cek2 mutant. The double cek2 cpp1 mutant shows enhanced responsiveness relative to either single mutant in terms of gene expression and halo formation, suggesting the kinase and phosphatase roles in the adaptation process are independent. Analysis of protein phosphorylation shows that Cek1 undergoes pheromone-mediated phosphorylation of the activation loop, and this phosphorylation is enhanced in cells lacking either the Cpp1 phosphatase or the Cek2 kinase. In addition, Cek1-GFP shows enhanced nuclear localization in response to pheromone treatment. In contrast, Cek2 shows no evidence for pheromone-mediated phosphorylation or pheromone-mediated nuclear localization. Intriguingly, however, deletion of CPP1 enhances both the phosphorylation state and the nuclear localization of Cek2-GFP. Overall, these results identify a complex interaction among the MAP kinases and MAP kinase phosphatase that function in the C. albicans mating pathway.IMPORTANCE MAP kinases and their regulators are critical components of eukaryotic signaling pathways implicated in normal cell behavior as well as abnormal behaviors linked to diseases such as cancer. The mating pathway of the yeast Saccharomycescerevisiae was central in establishing the MAP kinase paradigm. Here we investigate the mating pathway in a different ascomycete, the fungal pathogen C. albicans In this dimorphic fungus MAP kinases are also implicated in the mating response, with two MAP kinases apparently playing redundant roles in the mating process. This work establishes that while some level of mating can occur in the presence of a single kinase, the Cek1 kinase is most important for mating, while the Cek2 kinase is involved in adaptation to signaling. While both kinases appear to be themselves regulated by dephosphorylation through the action of the Cpp1 phosphatase, this process appears important for mating only in the case of Cek1.

Mms21: A Putative SUMO E3 Ligase in Candida albicans That Negatively Regulates Invasiveness and Filamentation, and Is Required for the Genotoxic and Cellular Stress Response.

In the life cycle of the fungal pathogen Candida albicans, the formation of filamentous cells is a differentiation process that is critically involved in host tissue invasion, and in adaptation to host cell and environmental stresses. Here, we have used the Gene Replacement And Conditional Expression library to identify genes controlling invasiveness and filamentation; conditional repression of the library revealed 69 mutants that triggered these processes. Intriguingly, the genes encoding the small ubiquitin-like modifier (SUMO) E3 ligase Mms21, and all other tested members of the sumoylation pathway, were both nonessential and capable of triggering filamentation upon repression, suggesting an important role for sumoylation in controlling filamentation in C. albicans We have investigated Mms21 in detail. Both Mms21 nulls (mms21Δ/Δ) and SP [Siz/Pias (protein inhibitor of activated signal transducer and activator of transcription)] domain (SUMO E3 ligase domain)-deleted mutants displayed invasiveness, filamentation, and abnormal nuclear segregation; filament formation occurred even in the absence of the hyphal transcription factor Efg1. Transcriptional analysis of mms21Δ/Δ showed an increase in expression from two- to eightfold above that of the wild-type for hyphal-specific genes, including ECE1, PGA13, PGA26, HWP1, ALS1, ALS3, SOD4, SOD5, UME6, and HGC1 The Mms21-deleted mutants were unable to recover from DNA-damaging agents like methyl methane sulfonate, hydroxyurea, hydrogen peroxide, and UV radiation, suggesting that the protein is important for genotoxic stress responses. In addition, the mms21Δ/Δ mutant displayed sensitivity to cell wall and thermal stresses, and to different antifungal drugs. All these findings suggest that Mms21 plays important roles in cellular differentiation, DNA damage and cellular stress responses, and in response to antifungal drugs.

Integration of Growth and Cell Size via the TOR Pathway and the Dot6 Transcription Factor in Candida albicans.

In most species, size homeostasis appears to be exerted in late G1 phase as cells commit to division, called Start in yeast and the Restriction Point in metazoans. This size threshold couples cell growth to division, and, thereby, establishes long-term size homeostasis. Our former investigations have shown that hundreds of genes markedly altered cell size under homeostatic growth conditions in the opportunistic yeast Candida albicans, but surprisingly only few of these overlapped with size control genes in the budding yeast Saccharomyces cerevisiae Here, we investigated one of the divergent potent size regulators in C. albicans, the Myb-like HTH transcription factor Dot6. Our data demonstrated that Dot6 is a negative regulator of Start, and also acts as a transcriptional activator of ribosome biogenesis (Ribi) genes. Genetic epistasis uncovered that Dot6 interacted with the master transcriptional regulator of the G1 machinery, SBF complex, but not with the Ribi and cell size regulators Sch9, Sfp1, and p38/Hog1. Dot6 was required for carbon-source modulation of cell size, and it is regulated at the level of nuclear localization by the TOR pathway. Our findings support a model where Dot6 acts as a hub that integrates growth cues directly via the TOR pathway to control the commitment to mitotic division at G1.

Chemogenomic Profiling of the Fungal Pathogen Candida albicans.

There is currently a small number of classes of antifungal drugs, and these drugs are known to target a very limited set of cellular functions. We derived a set of approximately 900 nonessential, transactivator-defective disruption strains from the tetracycline-regulated GRACE collection of strains of the fungal pathogen Candida albicans This strain set was screened against classic antifungal drugs to identify gene inactivations that conferred either enhanced sensitivity or increased resistance to the compounds. We examined two azoles, fluconazole and posaconazole; two echinocandins, caspofungin and anidulafungin; and a polyene, amphotericin B. Overall, the chemogenomic profiles within drug classes were highly similar, but there was little overlap between classes, suggesting that the different drug classes interacted with discrete networks of genes in C. albicans We also tested two pyridine amides, designated GPI-LY7 and GPI-C107; these drugs gave very similar profiles that were distinct from those of the echinocandins, azoles, or polyenes, supporting the idea that they target a distinct cellular function. Intriguingly, in cases where these gene sets can be compared to genetic disruptions conferring drug sensitivity in other fungi, we find very little correspondence in genes. Thus, even though the drug targets are the same in the different species, the specific genetic profiles that can lead to drug sensitivity are distinct. This implies that chemogenomic screens of one organism may be poorly predictive of the profiles found in other organisms and that drug sensitivity and resistance profiles can differ significantly among organisms even when the apparent target of the drug is the same.

SRYTH: A New Yeast Two-Hybrid Method.

Many biological processes are regulated by protein-protein interactions, and the analysis of these interactions has been a productive endeavor contributing to our understanding of cellular organization and function. The yeast two-hybrid technique is a widely used, powerful method of analyzing protein-protein interactions. The currently used formats, however, have inherent limitations, providing an opportunity to develop new alternatives that extend our ability to detect protein-protein interactions of biological relevance. Here we present a two-hybrid system named SRYTH (Ste11p/Ste50p related yeast two-hybrid) based on the Ste11p/Ste50p interaction that uses the activation of the HOG pathway of Saccharomyces cerevisiae as a reporter for interactions. The system is suitable for detecting cytoplasmic protein interactions in their natural subcellular environment, and has been successfully used to investigate protein-protein interactions, including transcription factor associations, in Candida albicans.

The evolutionary rewiring of the ribosomal protein transcription pathway modifies the interaction of transcription factor heteromer Ifh1-Fhl1 (interacts with forkhead 1-forkhead-like 1) with the DNA-binding specificity element.

The genes encoding the ribosomal proteins of fungi form a regulon whose expression is enhanced under good growth conditions and down-regulated under starvation conditions. The fungal pathogen Candida albicans contains an evolutionarily ancient control circuit for this regulon where a heteromer made up of the transcription regulators Ifh1 (interacts with Forkhead 1) and Fhl1 (Forkhead-like 1) is targeted to the ribosomal protein genes by the DNA binding factor Tbf1. In the more recently evolved circuit in the model yeast Saccharomyces cerevisiae (Sc), the generalist repressor-activator protein Rap1 now directs the Ifh1-Fhl1 module to the ribosomal protein genes. Even though overall sequence similarity is low for the respective Fhl1 and Ifh1 subunits, in both species, the Ifh1 protein links to the Forkhead-associated domain of Fhl1 through its FHB domain. Intriguingly, correlated with the transition to the Rap1-regulated circuit, the Sc-Ifh1 contains a Rap1 binding domain that is not present in the C. albicans protein. Because no extensive common sequences are found in Tbf1 and Rap1, it appears that these targeting proteins must connect to the Ifh1-Fhl1 module in distinct ways. Two-hybrid and co-immunoprecipitation analysis has been used to show that in C. albicans Tbf1 is linked to the heterodimer through direct association with Fhl1. By contrast, in S. cerevisiae, the linkage of the heteromer to Rap1 occurs through Ifh1. Thus, in the ascomycetes, the Ifh1-Fhl1 heterodimer has reconfigured its protein associations to remain connected to the ribosomal protein regulon despite rewiring of the targeting transcription factor from Tbf1 to Rap1.

The zinc cluster transcription factor Ahr1p directs Mcm1p regulation of Candida albicans adhesion.

Biofilm development by Candida albicans requires cell adhesion for the initial establishment of the biofilm and the continued stability after hyphal development occurs; however, the regulation of the process has not been fully established. Using chromatin immunoprecipitation coupled to microarray analysis (ChIP-chip) we have characterized a regulon containing the Mcm1p factor that is required for the initial surface adhesion during biofilm formation. In the yeast Saccharomyces cerevisiae several Mcm1p regulons have been characterized in which regulatory specificity is achieved through cofactors binding a sequence adjacent to the Mcm1p binding site. This new Mcm1p regulon in C. albicans also requires a cofactor, which we identify as the transcription factor Ahr1p. However, in contrast to the other yeast regulons, Ahr1p alone binds the target promoters, which include several key adhesion genes, and recruits Mcm1p to these sites. Through transcription profiling and qPCR analysis, we demonstrate that this Ahr1p-Mcm1p complex directly activates these adhesion genes. When the regulatory circuit was disrupted by deleting AHR1, the strain displayed reduced adherence to a polystyrene surface. We also demonstrate a role for the regulon in hyphal growth and in virulence. Our work thus establishes a new mechanism of Mcm1p-directed regulation distinct from those observed for other Mcm1p co-regulators.

Evolutionary tinkering with conserved components of a transcriptional regulatory network.

Gene expression variation between species is a major contributor to phenotypic diversity, yet the underlying flexibility of transcriptional regulatory networks remains largely unexplored. Transcription of the ribosomal regulon is a critical task for all cells; in S. cerevisiae the transcription factors Rap1, Fhl1, Ifh1, and Hmo1 form a multi-subunit complex that controls ribosomal gene expression, while in C. albicans this regulation is under the control of Tbf1 and Cbf1. Here, we analyzed, using full-genome transcription factor mapping, the roles, in both S. cerevisiae and C. albicans, of each orthologous component of this complete set of regulators. We observe dramatic changes in the binding profiles of the generalist regulators Cbf1, Hmo1, Rap1, and Tbf1, while the Fhl1-Ifh1 dimer is the only component involved in ribosomal regulation in both fungi: it activates ribosomal protein genes and rDNA expression in a Tbf1-dependent manner in C. albicans and a Rap1-dependent manner in S. cerevisiae. We show that the transcriptional regulatory network governing the ribosomal expression program of two related yeast species has been massively reshaped in cis and trans. Changes occurred in transcription factor wiring with cellular functions, movements in transcription factor hierarchies, DNA-binding specificity, and regulatory complexes assembly to promote global changes in the architecture of the fungal transcriptional regulatory network.

Potential interface between ribosomal protein production and pre-rRNA processing.

It has become clear that in Saccharomyces cerevisiae the transcription of ribosomal protein genes, which makes up a major proportion of the total transcription by RNA polymerase II, is controlled by the interaction of three transcription factors, Rap1, Fhl1, and Ifh1. Of these, only Rap1 binds directly to DNA and only Ifh1 is absent when transcription is repressed. We have examined further the nature of this interaction and find that Ifh1 is actually associated with at least two complexes. In addition to its association with Rap1 and Fhl1, Ifh1 forms a complex (CURI) with casein kinase 2 (CK2), Utp22, and Rrp7. Fhl1 is loosely associated with the CURI complex; its absence partially destabilizes the complex. The CK2 within the complex phosphorylates Ifh1 in vitro but no other members of the complex. Two major components of this complex, Utp22 and Rrp7, are essential participants in the processing of pre-rRNA. Depletion of either protein, but not of other proteins in the early processing steps, brings about a substantial increase in ribosomal protein mRNA. We propose a model in which the CURI complex is a key mediator between the two parallel pathways necessary for ribosome synthesis: the transcription and processing of pre-rRNA and the transcription of ribosomal protein genes.

HABP1/p32/gC1qR induces aberrant growth and morphology in Schizosaccharomyces pombe through its N-terminal alpha helix.

Hyaluronan binding protein (HABP1), located on human chromosome 17p13.3, was identified and characterized as being involved in cellular signaling from our laboratory. Here, we demonstrate that HABP1 expression in Schizosaccharomyces pombe induces growth inhibition, morphological abnormalities like elongation, multinucleation and aberrant cell septum formation in several strains of S. pombe, implicating its role in cell cycle progression and cytokinesis. This argument is further strengthened by an observed delay in the maximal expression of cell cycle regulatory proteins like CDC 2 and CDC 25 coupled to the direct interaction of HABP1 with CDC 25. In order to pinpoint the interacting domain of HABP1, its N- and C-terminal truncated variants (DeltaN.HABP1 and DeltaC.HABP1, respectively) were utilized which revealed that while expression of the former did not alter the phenotype, the latter generated morphological changes similar to those imparted upon HABP1 expression. It was also noted that along with HABP1, DeltaC.HABP1 too directly interacts with CDC 25 while DeltaN.HABP1 does not. Taken together, these data suggest that HABP1 induces morphological changes and modulates the cell cycle by interacting with proteins like CDC 25 through its N-terminal alpha-helix.

Presence of a human Hyaluronan binding protein 1 (HABP1) pseudogene-like sequence in Methanosarcina barkeri suggests its linkage in evolution.

The gene encoding Hyaluronan binding protein 1 (HABP1) and its homologs have been reported across eukaryotes, from yeast to human. We have reported the presence of processed pseudogenes in several human chromosomes, along with the location of the HABP1 gene on chromosome 17p12-p13. In this study, we report not only the presence of HABP1 pseudogene in other animal species, but also the presence of a homologous sequence in Methanosarcina barkeri, an ancient life form. This sequence has 44.8% homology to the human HABP1 cDNA and 45.3% homology with the HABP1 pseudogene in human chromosome 21. This sequence has a high G + C content (57%), characteristic of archaea, a family to which M. barkeri belongs. The presence of this HABP1 cDNA like fragment in M. barkeri might enable us to shed light on the evolution of the HABPl gene and whether it was present in a common ancestral organism before the lineages separated.