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Heat shock factor (HSF)-type regulators are stress-responsive transcription factors widely distributed among eukaryotes, including fungi. They carry a four-stranded winged helix-turn-helix DNA-binding domain considered as the signature domain for HSFs. The genome of the opportunistic yeast Candida albicans encodes four HSF members, namely, Sfl1, Sfl2, Skn7, and the essential regulator, Hsf1. C. albicans HSFs do not only respond to heat shock and/or temperature variation but also to CO 2 levels, oxidative stress, and quorum sensing, acting this way as central decision makers. In this minireview, I follow on the heels of my mSphere of Influence commentary (2020) to provide an overview of the repertoire of HSF regulators in Saccharomyces cerevisiae and C. albicans and describe how their genetic perturbation in C. albicans , coupled with genome-wide expression and location analyses, allow to map their transcriptional circuitry. I highlight how they can regulate, in common, a crucial developmental program: filamentous growth., Competing Interests: The author declares no conflict of interest.

The alternate growth of Candida albicans between a unicellular yeast form and a multicellular hyphal form is crucial for its ability to cause disease. Interestingly, both morphological forms support distinct functions during proliferation in the human host. We previously identified ORF19.217 (C2_08890W_A), encoding a zinc-finger transcription factor of the C 2 H 2 family, in a systematic screen of genes whose overexpression contributes to C. albicans ' morphological changes. Conditional overexpression of ORF19.217 with the strong tetracycline-inducible promoter (P TET ) resulted in a hyperfilamentous phenotype. We examined growth of the orf19.217 knockout-mutant in different hypha-inducing conditions and found that the mutant still formed hyphae under standard hypha-inducing conditions. To further investigate the function of Orf19.217 in C. albicans , we combined genome-wide expression (RNA-Seq) and location (ChIP-Seq) analyses. We found that Orf19.217 is involved in regulatory processes comprising hyphal morphogenesis and iron acquisition. Comparative analysis with existing C. albicans hyphal transcriptomes indicates that Orf19.217-mediated filamentation is distinct from a true hyphal program. Further, the orf19.217 knockout-mutant did not show increased sensitivity to iron deprivation, but ORF19.217 overexpression was able to rescue the growth of a hap5 -mutant, defective in a subunit of the CCAAT-complex, which is essential for iron acquisition. This suggested that Orf19.217 is involved in regulation of iron acquisition genes during iron deprivation and acts in a parallel pathway to the established CCAAT-complex. Interestingly, the orf19.217 -mutant turned out to be defective in its ability to form filaments under iron-deficiency. Taken together our findings propose that the transcription factor Orf19.217 stimulates expression of the hyphal regulators EFG1 and BRG1 to promote filamentous growth under iron deprivation conditions, allowing the fungus to escape these iron-depleted conditions. The transcription factor therefore appears to be particularly important for adaptation of C. albicans to diverse environmental conditions in the human host. In regard to the newly identified functions, we have given the regulator the name Irf1, Iron-dependent Regulator of Filamentation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wijlick, Znaidi, Hernández-Cervantes, Basso, Bachellier-Bassi and d’Enfert.)

Transcription factor Rme1 is conserved among ascomycetes and regulates meiosis and pseudohyphal growth in Saccharomyces cerevisiae. The genome of the meiosis-defective pathogen Candida albicans encodes an Rme1 homolog that is part of a transcriptional circuitry controlling hyphal growth. Here, we use chromatin immunoprecipitation and genome-wide expression analyses to study a possible role of Rme1 in C. albicans morphogenesis. We find that Rme1 binds upstream and activates the expression of genes that are upregulated during chlamydosporulation, an asexual process leading to formation of large, spherical, thick-walled cells during nutrient starvation. RME1 deletion abolishes chlamydosporulation in three Candida species, whereas its overexpression bypasses the requirement for chlamydosporulation cues and regulators. RME1 expression levels correlate with chlamydosporulation efficiency across clinical isolates. Interestingly, RME1 displays a biphasic pattern of expression, with a first phase independent of Rme1 function and dependent on chlamydospore-inducing cues, and a second phase dependent on Rme1 function and independent of chlamydospore-inducing cues. Our results indicate that Rme1 plays a central role in chlamydospore development in Candida species.

Sadri Znaidi works in the field of molecular mycology with a focus on functional genomics in Candida albicans In this mSphere of Influence article, he reflects on how the paper "An iron homeostasis regulatory circuit with reciprocal roles in Candida albicans commensalism and pathogenesis" by Chen et al. (C. Chen, K. Pande, S. D. French, B. B. Tuch, and S. M. Noble, Cell Host Microbe 10:118-135, 2011, https://doi.org/10.1016/j.chom.2011.07.005) made an impact on his research on how transcriptional regulatory networks function to control C. albicans ' ability to efficiently interact with the host environment., (Copyright © 2020 Znaidi.)

Sfl1p and Sfl2p are two homologous heat shock factor-type transcriptional regulators that antagonistically control morphogenesis in Candida albicans, while being required for full pathogenesis and virulence. To understand how Sfl1p and Sfl2p exert their function, we combined genome-wide location and expression analyses to reveal their transcriptional targets in vivo together with the associated changes of the C. albicans transcriptome. We show that Sfl1p and Sfl2p bind to the promoter of at least 113 common targets through divergent binding motifs and modulate directly the expression of key transcriptional regulators of C. albicans morphogenesis and/or virulence. Surprisingly, we found that Sfl2p additionally binds to the promoter of 75 specific targets, including a high proportion of hyphal-specific genes (HSGs; HWP1, HYR1, ECE1, others), revealing a direct link between Sfl2p and hyphal development. Data mining pointed to a regulatory network in which Sfl1p and Sfl2p act as both transcriptional activators and repressors. Sfl1p directly represses the expression of positive regulators of hyphal growth (BRG1, UME6, TEC1, SFL2), while upregulating both yeast form-associated genes (RME1, RHD1, YWP1) and repressors of morphogenesis (SSN6, NRG1). On the other hand, Sfl2p directly upregulates HSGs and activators of hyphal growth (UME6, TEC1), while downregulating yeast form-associated genes and repressors of morphogenesis (NRG1, RFG1, SFL1). Using genetic interaction analyses, we provide further evidences that Sfl1p and Sfl2p antagonistically control C. albicans morphogenesis through direct modulation of the expression of important regulators of hyphal growth. Bioinformatic analyses suggest that binding of Sfl1p and Sfl2p to their targets occurs with the co-binding of Efg1p and/or Ndt80p. We show, indeed, that Sfl1p and Sfl2p targets are bound by Efg1p and that both Sfl1p and Sfl2p associate in vivo with Efg1p. Taken together, our data suggest that Sfl1p and Sfl2p act as central "switch on/off" proteins to coordinate the regulation of C. albicans morphogenesis.

Candida albicans is the most frequently encountered human fungal pathogen, causing both superficial infections and life-threatening systemic diseases. Functional genomic studies performed in this organism have mainly used knock-out mutants and extensive collections of overexpression mutants are still lacking. Here, we report the development of a first generation C. albicans ORFeome, the improvement of overexpression systems and the construction of two new libraries of C. albicans strains overexpressing genes for components of signaling networks, in particular protein kinases, protein phosphatases and transcription factors. As a proof of concept, we screened these collections for genes whose overexpression impacts morphogenesis or growth rates in C. albicans. Our screens identified genes previously described for their role in these biological processes, demonstrating the functionality of our strategy, as well as genes that have not been previously associated to these processes. This article emphasizes the potential of systematic overexpression strategies to improve our knowledge of regulatory networks in C. albicans. The C. albicans plasmid and strain collections described here are available at the Fungal Genetics Stock Center. Their extension to a genome-wide scale will represent important resources for the C. albicans community.

Upc2p, a transcription factor of the zinc cluster family, is an important regulator of sterol biosynthesis and azole drug resistance in Candida albicans. To better understand Upc2p function in C. albicans, we used genomewide location profiling to identify the transcriptional targets of Upc2p in vivo. A triple hemagglutinin epitope, introduced at the C terminus of Upc2p, conferred a gain-of-function effect on the fusion protein. Location profiling identified 202 bound promoters (P < 0.05). Overrepresented functional groups of genes whose promoters were bound by Upc2p included 12 genes involved in ergosterol biosynthesis (NCP1, ERG11, ERG2, and others), 18 genes encoding ribosomal subunits (RPS30, RPL32, RPL12, and others), 3 genes encoding drug transporters (CDR1, MDR1, and YOR1), 4 genes encoding transcription factors (INO2, ACE2, SUT1, and UPC2), and 6 genes involved in sulfur amino acid metabolism (MET6, SAM2, SAH1, and others). Bioinformatic analyses suggested that Upc2p binds to the DNA motif 5'-VNCGBDTR that includes the previously characterized Upc2p binding site 5'-TCGTATA. Northern blot analysis showed that increased binding correlates with increased expression for the analyzed Upc2p targets (ERG11, MDR1, CDR1, YOR1, SUT1, SMF12, and CBP1). The analysis of ERG11, MDR1, and CDR1 transcripts in wild-type and upc2Delta/upc2Delta strains grown under Upc2p-activating conditions (lovastatin treatment and hypoxia) showed that Upc2p regulates its targets in a complex manner, acting as an activator or as a repressor depending upon the target and the activating condition. Taken together, our results indicate that Upc2p is a key regulator of ergosterol metabolism. They also suggest that Upc2p may contribute to azole resistance by regulating the expression of drug efflux pump-encoding genes in addition to ergosterol biosynthesis genes.

A major mechanism of azole resistance in Candida albicans is overexpression of the genes encoding the ATP binding cassette transporters Cdr1p and Cdr2p due to gain-of-function mutations in Tac1p, a transcription factor of the zinc cluster family. To identify the Tac1p regulon, we analyzed four matched sets of clinical isolates representing the development of CDR1- and CDR2-mediated azole resistance by using gene expression profiling. We identified 31 genes that were consistently up-regulated with CDR1 and CDR2, including TAC1 itself, and 12 consistently down-regulated genes. When a resistant strain deleted for TAC1 was examined similarly, expression of almost all of these genes returned to levels similar to those in the matched azole-susceptible isolate. Using genome-wide location (ChIP-chip) analysis (a procedure combining chromatin immunoprecipitation with hybridization to DNA intergenic microarrays), we found 37 genes whose promoters were bound by Tac1p in vivo, including CDR1 and CDR2. Sequence analysis identified nine new genes whose promoters contain the previously reported Tac1p drug-responsive element (CGGN(4)CGG), including TAC1. In total, there were eight genes whose expression was modulated in the four azole-resistant clinical isolates in a TAC1-dependent manner and whose promoters were bound by Tac1p, qualifying them as direct Tac1p targets: CDR1, CDR2, GPX1 (putative glutathione peroxidase), LCB4 (putative sphingosine kinase), RTA3 (putative phospholipid flippase), and orf19.1887 (putative lipase), as well as IFU5 and orf19.4898 of unknown function. Our results show that Tac1p binds under nonactivating conditions to the promoters of its targets, including to its own promoter. They also suggest roles for Tac1p in regulating lipid metabolism (mobilization and trafficking) and oxidative stress response in C. albicans.

The Candida albicans PDR16 gene, encoding a putative phosphatidylinositol transfer protein, is co-induced with the multidrug transporter genes CDR1 and CDR2 in azole-resistant (A(R)) clinical isolates and upon fluphenazine exposure of azole-susceptible (A(S)) cells, suggesting that it is regulated by Tac1p, the transcriptional activator of CDR genes. Deleting TAC1 in an A(R) isolate (5674) overexpressing PDR16, CDR1 and CDR2 decreased the expression of the three genes and fluconazole resistance to levels similar to those detected in the matched A(S) isolate (5457), demonstrating that Tac1p is responsible for PDR16 upregulation in that strain. Deleting TAC1 in the A(S) strain SC5314 abolished CDR2 induction by fluphenazine and decreased that of PDR16 and CDR1, uncovering the participation of an additional factor in the regulation of PDR16 and CDR1 expression. Sequencing of the TAC1 alleles identified one homozygous mutation in strain 5674, an Asn to Asp substitution at position 972 in the C-terminus of Tac1p. Introduction of the Asp(972) allele in a tac1Delta/Delta mutant caused high levels of fluconazole resistance and TAC1, PDR16, CDR1 and CDR2 constitutive induction. These results demonstrate that: (i) Tac1p controls PDR16 expression; (ii) Asn(972) to Asp(972) is a gain-of-function mutation; and (iii) Tac1p is positively autoregulated, directly or indirectly.

The Schizosaccharomyces pombe fep1(+) gene encodes a GATA transcription factor that represses the expression of iron transport genes in response to elevated iron concentrations. This transcriptional response is altered only in strains harboring a combined deletion of both tup11(+) and tup12(+) genes. This suggests that Tup11 is capable of negatively regulating iron transport gene expression in the absence of Tup12 and vice versa. The tup11(+)- and tup12(+)-encoded proteins resemble the Saccharomyces cerevisiae Tup1 corepressor. Using yeast two-hybrid analysis we show that Tup11 and Fep1 physically interact with each other. The C-terminal region from amino acids 242 to 564 of Fep1 is required for interaction with Tup11. Within this region, a minimal domain encompassing amino acids 405-541 was sufficient for Tup11-Fep1 association. Deletion mapping analysis revealed that the WD40-repeat sequence motifs of Tup11 are necessary for its interaction with Fep1. Analysis of Tup11 mutants with single amino acid substitutions in the WD40 repeats suggested that the Fep1 transcription factor interacts with a putative flat upper surface on the predicted beta-propeller structure of this motif. Further analysis by in vivo coimmunoprecipitation showed that Tup11 and Fep1 are physically associated. In vitro pull-down experiments further verified a direct interaction between the Fep1 C terminus and the Tup11 C-terminal WD40 repeat domain. Taken together, these results describe the first example of a physical interaction between a corepressor and an iron-sensing factor controlling the expression of iron uptake genes.