Aspergillus fumigatus is the major human filamentous fungal pathogen. While its importance as a pathogen is well-appreciated, our understanding of its biology is still at an early stage. One valuable area of study has been the analysis of gene expression in A. fumigatus. Dissection of the transcriptional circuits that control drug resistance and virulence in this fungus has been facilitated by the use of chromatin immunoprecipitation (ChIP), an approach pioneered in the mid-1980s. The use of ChIP coupled with next-generation sequencing (ChIP-seq) allows the determination of the genomic binding sites of a transcription factor of interest. Here, we describe the application of ChIP in A. fumigatus and detail different approaches to appropriately shear chromatin from this organism. This sheared chromatin can be used for analysis of factor association with single genes by qPCR or for downstream ChIP-seq applications.

The capacity of Aspergillus fumigatus to cause invasive pulmonary aspergillosis depends on its ability to adapt to dynamic and stressful microenvironments within the host. Epigenetic regulation, including histone deacetylation, plays a critical role in fungal adaptation to stress. Here, we investigated the role of the class I histone deacetylase HosA in A. fumigatus stress resistance, host cell interactions, and virulence. A ΔhosA mutant had increased susceptibility to intracellular oxidant stress induced by menadione. It also had impaired capacity to invade and damage two pulmonary epithelial cell lines in vitro. In a corticosteroid-immunosuppressed mouse model of invasive aspergillosis, mice infected with the ΔhosA mutant survived significantly longer than those infected with the wild-type strain, despite having similar pulmonary fungal burden. The ΔhosA mutant also induced a weaker inflammatory response than the wild-type strain. Transcriptomic analysis revealed that HosA regulates genes involved in secondary metabolite biosynthesis and energy metabolism, functioning as both an activator and repressor of distinct gene sets. Collectively, these results indicate that HosA is a key epigenetic regulator that governs A. fumigatus interactions with host cells and virulence during invasive pulmonary aspergillosis.IMPORTANCEEpigenetic modifications in Aspergillus fumigatus can be induced by environmental changes and stresses such as those induced by interaction with host cells. HosA, a class I histone deacetylase, has been shown to play a key role in regulating secondary metabolism in several Aspergillus species, but its function in A. fumigatus was previously unknown. We found that deletion of hosA increased susceptibility to intracellular, but not extracellular, oxidative stress. The ΔhosA mutant also exhibited significantly reduced pulmonary epithelial cell invasion and host cell damage, as well as attenuated virulence in immunosuppressed mice. Together, these findings indicate that HosA functions as a key epigenetic regulator that governs stress resistance, secondary metabolism, and fungal-host interactions. Defining the functions of HosA could provide critical insight into the epigenetic mechanisms that control fungal pathogenicity and may reveal a potential therapeutic target for invasive aspergillosis.

Candida glabrata is the second most common cause of invasive candidiasis and is widely known to have reduced susceptibility to fluconazole relative to many other Candida spp. Upc2A is a transcription factor that regulates ergosterol biosynthesis gene expression under conditions of sterol stress such as azole drug treatment or hypoxia. Through an in vitro microevolution experiment, we found that loss-of-function mutants of the ATF/CREB transcription factor CST6 suppresses the fluconazole hyper-susceptibility of the upc2A triangle mutant. Here, we confirm that the cst6 triangle upc2A triangle mutants are resistant to fluconazole but not to hypoxia relative to the upc2A triangle mutant. Sterol analysis of these mutants indicates that this suppression phenotype is not due to restoration of ergosterol levels in the cst6 triangle upc2A triangle mutant. Furthermore, increased expression of CDR1, the efflux pump implicated in the vast majority of azole-resistant C. glabrata strains, does not account for the suppression phenotype. Instead, our data suggest that this effect is due in part to increased expression of the adhesin EPA3, which has been shown by others to reduce fluconazole susceptibility in C. glabrata. In addition, we find that loss of both UPC2A and CST6 reduces the expression of mitochondrial and respiratory genes and that this also contributes to the suppression phenotype as well as to the resistance of cst6 triangle to fluconazole. These latter data further emphasize the connection between mitochondrial function and azole susceptibility.

Candida albicans is one of the most common causes of human fungal disease. An intensively studied C. albicans virulence traits is its ability to adopt both yeast and filamentous morphologies. Accordingly, the regulation of the yeast-to-filament transition has been an area of intense study in medical mycology. A long-standing mechanistic paradigm in this field is that the cAMP-protein kinase A pathway phosphorylates a master transcriptional regulator of C. albicans filamentation, Efg1, to drive filamentation. This model has been generalized over the years despite the fact that the initial work showed that it applied to only specific in vitro filamentation conditions. Here, we reinvestigated the protein kinase A-Efg1 paradigm by generating new strains containing alleles of EFG1 with the putative protein kinase A phosphorylation site (T208) mutated to either Ala or Glu which blocks or mimics phosphorylation, respectively. We integrated these alleles into efg1∆∆ mutants so that they are expressed from the endogenous promoter. We assayed the effect of these Efg1 mutations on (i) in vitro filamentation under a wide range of inducing conditions, (ii) biofilm formation, (iii) in vivo filamentation, (iv) virulence in a model of disseminated candidiasis, and (v) gene expression during in vitro filamentation. Using updated genetic approaches, we found no evidence that blocking or mimicking protein kinase phosphorylation of Efg1 affected its function during filamentation, biofilm formation, or infection. Therefore, additional studies will be required to identify the mechanisms by which Efg1 is regulated during hyphal morphogenesis.IMPORTANCECandida albicans is a common human fungal pathogen causing both superficial mucosal and life-threatening invasive disease. The virulence of C. albicans is associated with its ability to form filamentous hyphae and pseudohyphae. The regulation of this process is widely attributed to the phosphorylation of a transcription factor Efg1 by the protein kinase A signaling pathway. Since its initial description 25 years ago, this model has informed the design and interpretation of many studies of C. albicans. Through the use of updated genetic methods, we have found that protein kinase A-mediated phosphorylation of Efg1 is dispensable for filamentation, biofilm formation, and virulence in an experimental model of candidiasis. Although these data are negative in nature, the centrality of the protein kinase A-Efg1 paradigm to C. albicans pathogenesis studies increases their impact and should catalyze new studies to understand how this master regulator of C. albicans biology is itself regulated.

Resistance to endocrine therapy (ET) remains a major clinical challenge in the treatment of estrogen receptor-positive (ER⁺) breast cancer, underscoring the need for novel therapeutic targets. To identify genetic drivers of ET resistance, we conducted an in vivo genome-wide CRISPR-Cas9 screen in MCF7 cells implanted into ovariectomized nude mice under estrogen-deprived conditions. NFKB1 emerged as a top candidate whose loss promoted estrogen-independent tumor growth and recurrence. Functional studies confirmed that NFKB1 deficiency enhanced tumorigenicity and conferred resistance to tamoxifen and fulvestrant both in vitro and in vivo. Mechanistically, transcriptomic and biochemical analyses revealed that NFKB1 loss activated canonical NF-κB signaling, leading to inflammatory gene induction and hyperactivation of ER signaling. Importantly, pharmacologic inhibition of NF-κB signaling restored ET sensitivity in NFKB1-deficient cells. Clinically, NFKB1 downregulation was enriched in ER⁺ breast tumors and associated with poor patient outcomes. Collectively, these findings establish NFKB1 as a key suppressor of ET resistance, uncover a mechanistic link between inflammation and ER reactivation, and highlight NF-κB signaling as a therapeutic vulnerability in NFKB1-deficient ER⁺ breast cancer.

Cryptococcus is the leading cause of fungal meningitis in humans and a major source of mortality in people living with HIV. Infection starts in the lungs and results in meningoencephalitis after dissemination via the bloodstream. Alveolar macrophages are the first immune cells to encounter the infection and are usually capable of clearing it. In immunocompromised hosts, Cryptococcus survives and grows inside macrophages, establishing pulmonary and ultimately central nervous system infection. The initial interaction with macrophages is a key event in pathogenesis, and we have demonstrated that macrophage exposure induces an enhanced virulence phenotype in Cryptococcus. This process can be replicated in vitro: yeast cells exposed to macrophages in culture demonstrate enhanced virulence in both zebrafish and mouse models. To understand this induction process, we investigated its effect on the cryptococcal transcriptome to determine what cryptococcal factors are required. Ada2 is a subunit of the transcription modulating Spt-Ada-Gcn5-acetyltransferase complex and is required for cryptococcal virulence. We find that a cryptococcal strain lacking Ada2 is not capable of enhanced virulence after macrophage exposure. We further show that Pdr802, a transcription factor with a significant but poorly understood role in cryptococcal pathogenesis, acts downstream of Ada2 during macrophage exposure, and loss of Pdr802 function is sufficient to block macrophage-enhanced virulence. These findings shed light on the earliest stages of cryptococcal pathogenesis and offer new clues to the functions of Pdr802.IMPORTANCECryptococcus is a fungal pathogen affecting mostly immunocompromised people, especially those with HIV. Infection starts in the lungs but ultimately disseminates to cause meningitis, leading to over 50% mortality in patients with AIDS. Cryptococcal meningitis is difficult to treat, and understanding the early stages of infection will be the first step toward preventing it. Lung macrophages are the first immune cells to encounter Cryptococcus, and while they clear the infection in some cases, in immunocompromised patients, they instead serve as a niche for cryptococcal growth. How can macrophage interaction promote infection? We have shown that in a susceptible host, cryptococcal virulence is increased after initial contact with macrophages. This effect can be replicated in vitro, as cryptococcal cells pre-exposed to cultured macrophages are more virulent in animal models. We have identified two cryptococcal genes that regulate this change in virulence, setting the stage for a better understanding of cryptococcal pathogenesis.

Treatment of fungal infections associated with the filamentous fungus Aspergillus fumigatus is becoming more problematic as this organism is developing resistance to the main chemotherapeutic drug at an increasing rate. Azole drugs represent the current standard-of-care in treatment of aspergillosis with this drug class acting by inhibiting a key step in biosynthesis of the fungal sterol ergosterol. Azole compounds block the activity of the lanosterol α-14 demethylase, encoded by the cyp51A gene. A common route of azole resistance involves an increase in transcription of cyp51A. This transcriptional increase requires the function of a Zn2Cys6 DNA-binding domain-containing transcription activator protein called AtrR. AtrR was identified through its action as a positive regulator of expression of an ATP-binding cassette transporter (abcC/cdr1B here called abcG1). Using both deletion and alanine scanning mutagenesis, we demonstrate that a conserved C-terminal domain in A. fumigatus is required for expression of abcG1 but dispensable for cyp51A transcription. This domain is also found in several other fungal pathogen AtrR homologues consistent with a conserved gene-selective function of this protein segment being conserved. Using RNA-seq, we find that this gene-specific transcriptional defect extends to several other membrane transporter-encoding genes including a second ABC transporter locus. Our data reveal that AtrR uses at least two distinct mechanisms to induce gene expression and that normal susceptibility to azole drugs cannot be provided by maintenance of wild-type expression of the ergosterol biosynthetic pathway when ABC transporter expression is reduced.

ABSTRACT Treatment of fungal infections associated with the filamentous fungus Aspergillus fumigatus is becoming more problematic as this organism is developing resistance to the main chemotherapeutic drug at an increasing rate. Azole drugs represent the current standard-of-care in the treatment of aspergillosis with this drug class acting by inhibiting a key step in the biosynthesis of the fungal sterol ergosterol. Azole compounds block the activity of the lanosterol α-14 demethylase, encoded by the cyp51A gene. A common route of azole resistance involves an increase in transcription of cyp51A . This transcriptional increase requires the function of a Zn2Cys6 DNA-binding domain-containing transcription activator protein called AtrR. AtrR was identified through its action as a positive regulator of expression of an ATP-binding cassette transporter ( abcC / cdr1B here called abcG1 ). Using both deletion and alanine scanning mutagenesis, we demonstrate that a conserved C-terminal domain in A. fumigatus is required for the expression of abcG1 but dispensable for cyp51A transcription. This domain is also found in several other fungal pathogen AtrR homologs consistent with a conserved gene-selective function of this protein segment being conserved. Using RNA sequencing (RNA-seq), we find that this gene-specific transcriptional defect extends to several other membrane transporter-encoding genes including a second ABC transporter locus. Our data reveal that AtrR uses at least two distinct mechanisms to induce gene expression and that normal susceptibility to azole drugs cannot be provided by maintenance of wild-type expression of the ergosterol biosynthetic pathway when ABC transporter expression is reduced. IMPORTANCE Aspergillus fumigatu s is the primary human filamentous fungal pathogen. The principal chemotherapeutic drug used to control infections associated with A. fumigatus is the azole compound. These drugs are well-tolerated and effective, but resistance is emerging at an alarming rate. Most resistance is associated with mutations that lead to overexpression of the azole target enzyme, lanosterol α-14 demethylase, encoded by the cyp51A gene. A key regulator of cyp51A gene expression is the transcription factor AtrR. Very little is known of the molecular mechanisms underlying the effect of AtrR on gene expression. Here, we use deletion and clustered amino acid substitution mutagenesis to map a region of AtrR that confers gene-specific activation on target genes of this transcription factor. This region is highly conserved across AtrR homologs from other pathogenic species arguing that its importance in transcriptional regulation is maintained across evolution.

Background: In all eukaryotic cell types, the unfolded protein response (UPR) upregulates factors that promote protein folding and misfolded protein clearance to help alleviate endoplasmic reticulum (ER) stress. Yet, ER stress in the liver is uniquely accompanied by the suppression of metabolic genes, the coordination and purpose of which are largely unknown. Methods: Here, we combined in silico machine learning, in vivo liver-specific deletion of the master regulator of hepatocyte differentiation HNF4α, and in vitro manipulation of hepatocyte differentiation state to determine how the UPR regulates hepatocyte identity and toward what end. Results: Machine learning identified a cluster of correlated genes that were profoundly suppressed by persistent ER stress in the liver. These genes, which encode diverse functions including metabolism, coagulation, drug detoxification, and bile synthesis, are likely targets of the master regulator of hepatocyte differentiation HNF4α. The response of these genes to ER stress was phenocopied by liver-specific deletion of HNF4α. Strikingly, while deletion of HNF4α exacerbated liver injury in response to an ER stress challenge, it also diminished UPR activation and partially preserved ER ultrastructure, suggesting attenuated ER stress. Conversely, pharmacological maintenance of hepatocyte identity in vitro enhanced sensitivity to stress. Conclusions: Together, our findings suggest that the UPR regulates hepatocyte identity through HNF4α to protect ER homeostasis even at the expense of liver function.

In all eukaryotic cell types, the unfolded protein response (UPR) upregulates factors that promote protein folding and misfolded protein clearance to help alleviate endoplasmic reticulum (ER) stress. Yet ER stress in the liver is uniquely accompanied by the suppression of metabolic genes, the coordination and purpose of which is largely unknown. Here, we used unsupervised machine learning to identify a cluster of correlated genes that were profoundly suppressed by persistent ER stress in the liver. These genes, which encode diverse functions including metabolism, coagulation, drug detoxification, and bile synthesis, are likely targets of the master regulator of hepatocyte differentiation HNF4α. The response of these genes to ER stress was phenocopied by liver-specific deletion of HNF4 α. Strikingly, while deletion of HNF4α exacerbated liver injury in response to an ER stress challenge, it also diminished UPR activation and partially preserved ER ultrastructure, suggesting attenuated ER stress. Conversely, pharmacological maintenance of hepatocyte identity in vitro enhanced sensitivity to stress. Several pathways potentially link HNF4α to ER stress sensitivity, including control of expression of the tunicamycin transporter MFSD2A; modulation of IRE1/XBP1 signaling; and regulation of Pyruvate Dehydrogenase. Together, these findings suggest that HNF4α activity is linked to hepatic ER homeostasis through multiple mechanisms.

Transcriptional regulation of azole resistance in the filamentous fungus Aspergillus fumigatus is a key step in development of this problematic clinical phenotype. We and others have previously described a C2H2-containing transcription factor called FfmA that is required for normal levels of voriconazole susceptibility. Null alleles of ffmA exhibit a strongly compromised growth rate even in the absence of any external stress. Here, we employ an acutely repressible doxycycline-off form of ffmA to rapidly deplete FfmA protein from the cell. Using this approach, we carried out RNA-seq analyses to probe the transcriptome cells acutely deprived of FfmA. A total of 2,000 genes were differentially expressed upon acute depletion of FfmA, illustrating the broad transcriptomic effect of this factor. Interestingly, the transcriptome changes observed upon this acute depletion of FfmA expression only shared limited overlap with those found in an ffmAΔ null strain analyzed by others. Chromatin immunoprecipitation coupled with high throughput DNA sequencing analysis (ChIP-seq) identified 530 genes that were bound by FfmA. More than 300 of these genes were also bound by AtrR, a transcription factor important in azole drug resistance, demonstrating striking regulatory overlap with FfmA. However, while AtrR is an upstream activation protein with known specificity, our data suggest that FfmA is a chromatin-associated factor that binds DNA in a manner dependent on other factors. We provide evidence that AtrR and FfmA interact in the cell and show reciprocal expression modulation. Interaction of AtrR and FfmA is required for normal gene expression in A. fumigatus.

Candida albicans is a diploid human fungal pathogen that displays significant genomic and phenotypic heterogeneity over a range of virulence traits and in the context of a variety of environmental niches. Here, we show that the effects of Rob1 on biofilm and filamentation virulence traits is dependent on both the specific environmental condition and the clinical strain of C. albicans . The C. albicans reference strain SC5314 is a ROB1 heterozygote with two alleles that differ by a single nucleotide polymorphism at position 946 resulting in a serine or proline containing isoform. An analysis of 224 sequenced C. albicans genomes indicates that SC5314 is the only ROB1 heterozygote documented to date and that the dominant allele contains a proline at position 946. Remarkably, the ROB1 alleles are functionally distinct and the rare ROB1 946S allele supports increased filamentation in vitro and increased biofilm formation in vitro and in vivo, suggesting it is a phenotypic gain-of-function allele. SC5314 is amongst the most highly filamentous and invasive strains characterized to date. Introduction of the ROB1 946S allele into a poorly filamenting clinical isolate increases filamentation and conversion of an SC5314 laboratory strain to a ROB1 946S homozygote increases in vitro filamentation and biofilm formation. In a mouse model of oropharyngeal infection, the predominant ROB1 946P allele establishes a commensal state while the ROB1 946S phenocopies the parent strain and invades into the mucosae. These observations provide an explanation for the distinct phenotypes of SC5314 and highlight the role of heterozygosity as a driver of C. albicans phenotypic heterogeneity. Importance:Candida albicans is a commensal fungus that colonizes human oral cavity and gastrointestinal tracts but also causes mucosal as well as invasive disease. The expression of virulence traits in C. albicans clinical isolates is heterogenous and the genetic basis of this heterogeneity is of high interest. The C. albicans reference strain SC5314 is highly invasive and expresses robust filamentation and biofilm formation relative to many other clinical isolates. Here, we show that SC5314 derivatives are heterozygous for the transcription factor Rob1 and contain an allele with a rare gain-of-function SNP that drives filamentation, biofilm formation, and virulence in a model of oropharyngeal candidiasis. These finding explain, in part, the outlier phenotype of the reference strain and highlight the role of heterozygosity plays in the strain-to-strain variation of diploid fungal pathogens.

Cryptococcus spp. are environmental fungi that first must adapt to the host environment before they can cause life-threatening meningitis in immunocompromised patients. Host CO 2 concentrations are 100-fold higher than the external environment and strains unable to grow at host CO 2 concentrations are not pathogenic. Using a genetic screening and transcriptional profiling approach, we report that the TOR pathway is critical for C. neoformans adaptation to host CO 2 partly through Ypk1-dependent remodeling of phosphatidylserine asymmetry at the plasma membrane. We also describe a C. neoformans ABC/PDR transporter ( PDR9 ) that is highly expressed in CO 2 -sensitive environmental strains, suppresses CO 2 -induced phosphatidylserine/phospholipid remodeling, and increases susceptibility to host concentrations of CO 2 . Interestingly, regulation of plasma membrane lipid asymmetry by the TOR-Ypk1 axis is distinct in C. neoformans compared to S. cerevisiae . Finally, host CO 2 concentrations suppress the C. neoformans pathways that respond to host temperature (Mpk1) and pH (Rim101), indicating that host adaptation requires a stringent balance among distinct stress responses.

Transcriptional regulation of azole resistance in the filamentous fungus Aspergillus fumigatus is a key step in development of this problematic clinical phenotype. We and others have previously described a C2H2-containing transcription factor called FfmA that is required for normal levels of voriconazole susceptibility and expression of an ATP-binding cassette transporter gene called abcG1 . Null alleles of ffmA exhibit a strongly compromised growth rate even in the absence of any external stress. Here we employ an acutely repressible doxycycline-off form of ffmA to rapidly deplete FfmA protein from the cell. Using this approach, we carried out RNA-seq analyses to probe the transcriptome of A. fumigatus cells that have been deprived of normal FfmA levels. We found that 2000 genes were differentially expressed upon depletion of FfmA, consistent with the wide-ranging effect of this factor on gene regulation. Chromatin immunoprecipitation coupled with high throughput DNA sequencing analysis (ChIP-seq) identified 530 genes that were bound by FfmA using two different antibodies for immunoprecipitation. More than 300 of these genes were also bound by AtrR demonstrating the striking regulatory overlap with FfmA. However, while AtrR is clearly an upstream activation protein with clear sequence specificity, our data suggest that FfmA is a chromatin-associated factor that may bind to DNA in a manner dependent on other factors. We provide evidence that AtrR and FfmA interact in the cell and can influence one another’s expression. This interaction of AtrR and FfmA is required for normal azole resistance in A. fumigatus .

The most commonly used antifungal drugs are the azole compounds that interfere with biosynthesis of the fungal-specific sterol: ergosterol. The pathogenic yeast Candida glabrata commonly acquires resistance to azole drugs like fluconazole via mutations in a gene encoding a transcription factor called PDR1. These PDR1 mutations lead to overproduction of drug transporter proteins like the ATP-binding cassette transporter Cdr1. In other Candida species, mutant forms of a transcription factor called Upc2 are associated with azole resistance, owing to the important role of this protein in control of expression of genes encoding enzymes involved in the ergosterol biosynthetic pathway. Recently, the C. glabrata Upc2A factor was demonstrated to be required for normal azole resistance, even in the presence of a hyperactive mutant form of PDR1. Using genome-scale approaches, we define the network of genes bound and regulated by Upc2A. By analogy to a previously described hyperactive UPC2 mutation found in Saccharomyces cerevisiae, we generated a similar form of Upc2A in C. glabrata called G898D Upc2A. Chromatin immunoprecipitation coupled with Next Generation Sequencing (ChIP-seq) demonstrated that wild-type Upc2A binding to target genes was strongly induced by fluconazole while G898D Upc2A bound similarly, irrespective of drug treatment. We also carried out RNA-seq analysis to determine the genes that were direct or indirect targets of Upc2A transcriptional control. In addition to the well-described ERG genes as Upc2A transcriptional targets, we found a large group of genes encoding components of the translational apparatus along with membrane proteins. These Upc2A-regulated membrane protein-encoding genes are often targets of the Pdr1 transcription factor, demonstrating the high degree of overlap between these two regulatory networks. Finally, we provide evidence that Upc2A impacts the Pdr1-Cdr1 system during the anaerobic response and also modulates resistance to caspofungin. These studies provide a new perspective of Upc2A as a master regulator of lipid and membrane protein biosynthesis. Author summary In the pathogenic yeast Candida glabrata, expression of the genes encoding enzymes in the ergosterol biosynthetic pathway is controlled by the transcription factor Upc2A. C. glabrata has a low intrinsic susceptibility to azole therapy and acquires fluconazole resistance at high frequency. These azole resistant mutants typically contain substitution mutations in a gene encoding the transcription factor Pdr1. Pdr1 does not appear to regulate ergosterol genes and instead induces expression of genes encoding drug transport proteins like CDR1. Here we establish that extensive overlap exists between the regulatory networks defined by Upc2A and Pdr1. Genomic approaches are used to describe the hundreds of genes regulated by Upc2A that far exceed the well-described impact of this factor on genes involved in ergosterol biosynthesis. The overlap between Upc2A and Pdr1 is primarily described by co-regulation of genes encoding membrane transporters like CDR1. We provide evidence that Upc2A impacts the transcriptional control of the FKS1 gene, producing a target of a second major class of antifungal drugs, the echinocandins. Our data are consistent with Upc2A playing a role as a master regulator coordinating the synthesis of membrane structural components, both at the level of lipids and proteins, to produce properly functional biological membranes.

The ability of cells to sort, transport, and secrete proteins depends largely on the function of organelles that comprise the secretory pathway. The Golgi complex is an organelle that plays a central role in processing cargo proteins as they transit the secretory pathway. In particular, the precise addition and removal of sugars from glycoproteins occur through a series of Golgi-associated enzymatic reactions. This organelle also serves to store calcium and as a site to initiate microtubule assembly. The molecular mechanisms underlying the ability of the Golgi complex to carry out these manifold tasks have fascinated cell biologists for many years.

Two of the major classes of antifungal drugs in clinical use target ergosterol biosynthesis. Despite its importance, our understanding of the transcriptional regulation of ergosterol biosynthesis genes in pathogenic fungi is essentially limited to the role of hypoxia and sterol-stress-induced transcription factors such as Upc2 and Upc2A as well as homologs of sterol response element binding (SREB) factors. To identify additional regulators of ergosterol biosynthesis in Candida glabrata, an important human fungal pathogen with reduced susceptibility to ergosterol biosynthesis inhibitors relative to other Candida spp., we used a serial passaging strategy to isolate suppressors of the fluconazole hypersusceptibility of a upc2AΔ deletion mutant. This led to the identification of loss-of-function mutations in two genes: ROX1, the homolog of a hypoxia gene transcriptional suppressor in Saccharomyces cerevisiae, and CST6, a transcription factor that is involved in the regulation of carbon dioxide response in C. glabrata. Here, we describe a detailed analysis of the genetic interaction of ROX1 and UPC2A. In the presence of fluconazole, loss of Rox1 function restores ERG11 expression to the upc2AΔ mutant and inhibits the expression of ERG3 and ERG6, leading to increased levels of ergosterol and decreased levels of the toxic sterol 14α methyl-ergosta-8,24(28)-dien-3β, 6α-diol, relative to the upc2AΔ mutant. Our observations establish that Rox1 is a negative regulator of ERG gene biosynthesis and indicate that a least one additional positive transcriptional regulator of ERG gene biosynthesis must be present in C. glabrata. IMPORTANCE Candida glabrata is one of the most important human fungal pathogens and has reduced susceptibility to azole-class inhibitors of ergosterol biosynthesis. Although ergosterol is the target of two of the three classes of antifungal drugs, relatively little is known about the regulation of this critical cellular pathway. Sterols are both essential components of the eukaryotic plasma membrane and potential toxins; therefore, sterol homeostasis is critical for cell function. Here, we identified two new negative regulators in C. glabrata of ergosterol (ERG) biosynthesis gene expression. Our results also indicate that in addition to Upc2A, the only known activator of ERG genes, additional positive regulators of this pathway must exist.

The most commonly used antifungal drugs are the azole compounds, which interfere with biosynthesis of the fungal-specific sterol: ergosterol. The pathogenic yeast Candida glabrata commonly acquires resistance to azole drugs like fluconazole via mutations in a gene encoding a transcription factor called PDR1. These PDR1 mutations lead to overproduction of drug transporter proteins like the ATP-binding cassette transporter Cdr1. In other Candida species, mutant forms of a transcription factor called Upc2 are associated with azole resistance, owing to the important role of this protein in control of expression of genes encoding enzymes involved in the ergosterol biosynthetic pathway. Recently, the C. glabrata Upc2A factor was demonstrated to be required for normal azole resistance, even in the presence of a hyperactive mutant form of PDR1. Using genome-scale approaches, we define the network of genes bound and regulated by Upc2A. By analogy to a previously described hyperactive UPC2 mutation found in Saccharomyces cerevisiae, we generated a similar form of Upc2A in C. glabrata called G898D Upc2A. Analysis of Upc2A genomic binding sites demonstrated that wild-type Upc2A binding to target genes was strongly induced by fluconazole while G898D Upc2A bound similarly, irrespective of drug treatment. Transcriptomic analyses revealed that, in addition to the well-described ERG genes, a large group of genes encoding components of the translational apparatus along with membrane proteins were responsive to Upc2A. These Upc2A-regulated membrane protein-encoding genes are often targets of the Pdr1 transcription factor, demonstrating the high degree of overlap between these two regulatory networks. Finally, we provide evidence that Upc2A impacts the Pdr1-Cdr1 system and also modulates resistance to caspofungin. These studies provide a new perspective of Upc2A as a master regulator of lipid and membrane protein biosynthesis.

The R egulation of A ce2 and M orphogenesis (RAM) pathway is an important regulatory network in the human fungal pathogen Candida albicans . The RAM pathway’s two most well-studied components, the NDR/Lats kinase Cbk1 and its putative substrate, the transcription factor Ace2, have a wide range of phenotypes and functions. It is not clear, however, which of these functions are specifically due to the phosphorylation of Ace2 by Cbk1. To address this question, we first compared the transcriptional profiles of CBK1 and ACE2 deletion mutants. This analysis indicates that, of the large number of genes whose expression is affected by deletion of CBK1 and ACE2 , only 5.5% of those genes are concordantly regulated. Our data also suggest that Ace2 directly or indirectly represses a large set of genes during hyphal morphogenesis. Second, we generated strains containing ACE2 alleles with alanine mutations at the Cbk1 phosphorylation sites. Phenotypic and transcriptional analysis of these ace2 mutants indicates that, as in Saccharomyces cerevisiae , Cbk1 regulation is important for daughter cell localization of Ace2 and cell separation during yeast phase growth. In contrast, Cbk1 phosphorylation of Ace2 plays a minor role in C. albicans yeast-to-hyphae transition. We have, however, discovered a new function for the Cbk1-Ace2 axis. Specifically, Cbk1 phosphorylation of Ace2 prevents the hyphae-to-yeast transition. To our knowledge, this is one of the first regulators of the C. albicans hyphae-to-yeast transition to be described. Finally, we present an integrated model for the role of Cbk1 in the regulation of hyphal morphogenesis in C. albicans . Importance R egulation of A ce2 and M orphogenesis (RAM) pathway is a key regulatory network that plays a role in many aspects of C. albicans pathobiology. In addition to characterizing the transcriptional effects of this pathway, we discovered that Cbk1 and Ace2, a key RAM pathway regulator-effector pair, mediate a specific set of the overall functions of the RAM pathway. We have also discovered a new function for the Cbk1-Ace2 axis; suppression of the hyphae-to-yeast transition. Very few regulators of this transition have been described and our data indicate that maintenance of hyphal morphogenesis requires suppression of yeast phase growth by Cbk1-regulated Ace2.

SOX2 and OCT4 are key regulators of embryonic stem cell pluripotency. They are overexpressed in prostate cancers and have been associated with cancer stem cell (CSC) properties. However, reliable tools for detecting and targeting SOX2/OCT4-overexpressing cells are lacking, limiting our understanding of their roles in prostate cancer initiation, progression, and therapeutic resistance. Here, we show that a fluorescent reporter called SORE6 can identify SOX2/OCT4-overexpressing prostate cancer cells. Among tumor cells, the SORE6 reporter identified a small fraction with CSC hallmarks: rapid self-renewal, the capability to form tumors and metastasize, and resistance to chemotherapies. Transcriptome and biochemical analyses identified PI3K/AKT signaling as critical for maintaining the SORE6+ population. Moreover, a SORE6-driven herpes simplex virus thymidine kinase (TK) expression construct could selectively ablate SORE6+ cells in tumors, blocking tumor initiation and progression, and sensitizing tumors to chemotherapy. This study demonstrates a key role of SOX2/OCT4-associated prostate cancer stem cells in tumor development and therapeutic resistance, and identifies the SORE6 reporter system as a useful tool for characterizing CSCs functions in a native tumor microenvironment.