Jiangye Chen

Bistable expression of WOR1, a master regulator of white–opaque switching in Candida albicans

Candida albicans, a commensal organism and a pathogen of humans, can switch stochastically between a white phase and an opaque phase without an intermediate phase. The white and opaque phases have distinct cell shapes and gene expression programs. Once switched, each phase is stable for many cell divisions. White–opaque switching is under a1–α2 repression and therefore only happens in a or α cells. Mechanisms that control the switching are unknown. Here, we identify Wor1 (white–opaque regulator 1) as a master regulator of white–opaque switching. The deletion of WOR1 blocks opaque cell formation. The ectopic expression of WOR1 converts all cells to stable opaque cells in a or α cells. In addition, the ectopic expression of WOR1 in a/α cells is sufficient to induce opaque cell formation. Importantly, WOR1 expression displays an all-or-none pattern. It is undetectable in white cells, and it is highly expressed in opaque cells. The ectopic expression of Wor1 induces the transcription of WOR1 from the WOR1 locus, which correlates with the switch to opaque phase. We present genetic evidence for feedback regulation of WOR1 transcription. The feedback regulation explains the bistable and stochastic nature of white–opaque switching.

Crk1, a Novel Cdc2-Related Protein Kinase, Is Required for Hyphal Development and Virulence in Candida albicans

ABSTRACT Both mitogen-activated protein kinases and cyclin-dependent kinases play a role in hyphal development in Candida albicans. Using an oligonucleotide probe-based screen, we have isolated a new member of the Cdc2 kinase subfamily, designated Crk1 (Cdc2-related kinase). The protein sequence of Crk1 is most similar to those ofSaccharomyces cerevisiae Sgv1 and human Pkl1/Cdk9. InS. cerevisiae, CRK1 suppresses some, but not all, of the defects associated with an sgv1 mutant. Deleting both copies of CRK1 in C. albicansslows growth slightly but leads to a profound defect in hyphal development under all conditions examined. crk1/crk1mutants are impaired in the induction of hypha-specific genes and are avirulent in mice. Consistent with this, ectopic expression of the Crk1 kinase domain (CRK1N) promotes filamentous or invasive growth in S. cerevisiae and hyphal development in C. albicans. The activity of Crk1 inS. cerevisiae requires Flo8 but is independent of Ste12 and Phd1. Similarly, Crk1 promotes filamentation through a route independent of Cph1 and Efg1 in C. albicans. RAS1V13 can also activate filamentation in acph1/cph1 efg1/efg1 double mutant. Interestingly,CRK1N produces florid hyphae in ras1/ras1strains, while RAS1V13 generates feeble hyphae in crk1/crk1 strains.

Cyclin Y, a novel membrane-associated cyclin, interacts with PFTK1

MINT‐7147695, MINT‐7147713: pftk1 (uniprotkb:O94921) and CCNY (uniprotkb:Q8ND76) colocalize (MI:0403) by fluorescence microscopy (MI:0416)

Efg1-mediated Recruitment of NuA4 to Promoters Is Required for Hypha-specific Swi/Snf Binding and Activation in Candida albicans

Efg1 is essential for hyphal development and virulence in the human pathogenic fungus Candida albicans. How Efg1 regulates gene expression is unknown. Here, we show that Efg1 interacts with components of the nucleosome acetyltransferase of H4 (NuA4) histone acetyltransferase (HAT) complex in both yeast and hyphal cells. Deleting YNG2, a subunit of the NuA4 HAT module, results in a significant decrease in the acetylation level of nucleosomal H4 and a profound defect in hyphal development, as well as a defect in the expression of hypha-specific genes. Using chromatin immunoprecipitation, Efg1 and the NuA4 complex are found at the UAS regions of hypha-specific genes in both yeast and hyphal cells, and Efg1 is required for the recruitment of NuA4. Nucleosomal H4 acetylation at the promoters peaks during initial hyphal induction in an Efg1-dependent manner. We also find that Efg1 bound to the promoters of hypha-specific genes is critical for recruitment of the Swi/Snf chromatin remodeling complex during hyphal induction. Our data show that the recruitment of the NuA4 complex by Efg1 to the promoters of hypha-specific genes is required for nucleosomal H4 acetylation at the promoters during hyphal induction and for subsequent binding of Swi/Snf and transcriptional activation.

Roles of Candida albicans Sfl1 in Hyphal Development

ABSTRACT The ability to switch between different morphological forms is an important feature of Candida albicans and is relevant to its pathogenesis. Many conserved positive and negative transcription factors are involved in morphogenetic regulation of the two dimorphic fungi Candida albicans and Saccharomyces cerevisiae. In S. cerevisiae, the transcriptional repressor Sfl1 and the activator Flo8 function antagonistically in invasive and filamentous growth. We have previously reported that Candida albicans Flo8 is a transcription factor essential for hyphal development and virulence in C. albicans. To determine whether a similar negative factor exists in C. albicans, we identified Candida albicans Sfl1 as a functional homolog of the S. cerevisiae sfl1 mutant. Sfl1 is a negative regulator of hyphal development in C. albicans. Deletion of C. albicans SFL1 enhanced filamentous growth and hypha-specific gene expression in several media and at several growth temperatures. Overexpression of the SFL1 led to a significant reduction of filament formation. Both deletion and overexpression of the SFL1 attenuated virulence of C. albicans in a mouse model. Deleting FLO8 in an sfl1/sfl1 mutant completely blocked hyphal development in various growth conditions examined, suggesting that C. albicans Sfl1 may act as a negative regulator of filamentous growth by antagonizing Flo8 functions. We suggest that, similar to the case for S. cerevisiae, a combination of dual control by activation and repression of Flo8 and Sfl1 may contribute to the fine regulatory network in C. albicans morphogenesis responding to different environmental cues.

The Swi/Snf chromatin remodeling complex is essential for hyphal development in Candida albicans.

The ability of dimorphic transition between yeast and hyphal forms in Candida albicans is one of the vital determinants for its pathogenicity and virulence. We isolated C. albicans SWI1 as a suppressor of the invasive growth defect in a Saccharomyces cerevisiae mutant. Expression of C. albicans SWI1 in S. cerevisiae partially complemented the growth defect of a swi1 mutant in the utilization of glycerol. Swi1 is in a complex with Snf2 in C. albicans, and both proteins are localized in the nucleus independent of the growth form. Deleting SWI1 or SNF2 in C. albicans prevented true hyphal formation and resulted in constitutive pseudohypha‐like growth in all media examined. Furthermore, swi1/swi1 mutant was defective in hypha‐specific gene expression and avirulent in a mouse model of systemic infection. These data strongly suggest the conserved Swi/Snf complex in C. albicans is required for hyphal development and pathogenicity.

Function and subcellular localization of Gcn5, a histone acetyltransferase in Candida albicans

Candida albicans is an opportunistic fungal pathogen commonly found in humans. It has the ability to switch reversibly between three growth forms: budding yeast, pseudohypha, and hypha. The transition between yeast and hyphal growth forms is critical for the pathogenesis of C. albicans. During the yeast-to-hypha morphologic transition, gene expression is regulated by transcriptional regulators including histone modifying complexes and chromatin remodeling complexes. We previously reported that Esa1, a catalytic subunit in the histone acetyltransferase complex NuA4, is essential for the hyphal development of C. albicans. In this study, we analyzed the functional roles of Gcn5, a catalytic subunit in the histone acetyltransferase complex SAGA, in C. albicans. Gcn5 is required for the invasive and filamentous growth of C. albicans. Deletion of GCN5 impaired hyphal elongation in sensing serum and attenuated the virulence of C. albicans in a mouse systemic infection model. The C. albicans gcn5/gcn5 mutant cells also exhibited sensitivity to cell wall stress. Functional analysis showed that the HAT domain and Bromodomain in Gcn5 play distinct roles in morphogenesis and cell wall stress response of C. albicans. Our results show that the conserved residue Glu188 is crucial for the Gcn5 HAT activity and for Gcn5 function during filamentous growth. In addition, the subcellular distribution of ectopically expressed GFP-Gcn5 correlates with the different growth states of C. albicans. In stationary phase, Gcn5 accumulated in the nucleus, while during vegetative growth it localized in the cytoplasm in a morpha-independent manner. Our results suggest that the nuclear localization of Gcn5 depends on the existence of its N-terminal NLS and HAT domains.

Molecular mechanism for Rabex-5 GEF activation by Rabaptin-5

Rabex-5 and Rabaptin-5 function together to activate Rab5 and further promote early endosomal fusion in endocytosis. The Rabex-5 GEF activity is autoinhibited by the Rabex-5 CC domain (Rabex-5CC) and activated by the Rabaptin-5 C2-1 domain (Rabaptin-5C21) with yet unknown mechanism. We report here the crystal structures of Rabex-5 in complex with the dimeric Rabaptin-5C21 (Rabaptin-5C212) and in complex with Rabaptin-5C212 and Rab5, along with biophysical and biochemical analyses. We show that Rabex-5CC assumes an amphipathic α-helix which binds weakly to the substrate-binding site of the GEF domain, leading to weak autoinhibition of the GEF activity. Binding of Rabaptin-5C21 to Rabex-5 displaces Rabex-5CC to yield a largely exposed substrate-binding site, leading to release of the GEF activity. In the ternary complex the substrate-binding site of Rabex-5 is completely exposed to bind and activate Rab5. Our results reveal the molecular mechanism for the regulation of the Rabex-5 GEF activity. DOI: http://dx.doi.org/10.7554/eLife.02687.001

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.

Distinct and Redundant Roles of the Two MYST Histone Acetyltransferases Esa1 and Sas2 in Cell Growth and Morphogenesis of Candida albicans

ABSTRACT Candida albicans is associated with humans, as both a harmless commensal organism and a pathogen. Adaption to human body temperature is extremely important for its growth and morphogenesis. Saccharomyces cerevisiae Esa1, a member of the MYST family HATs (histone acetyltransferases) and the catalytic subunit of the NuA4 complex, and its homologues in other eukaryotes have been shown to be essential for cell growth. To investigate the functional roles of two MYST family HATs, Esa1 and Sas2 in C. albicans, we deleted ESA1 and SAS2 in the C. albicans genome and performed cell growth analyses. Our results demonstrated that C. albicans Esa1 is not essential for general growth but is essential for filamentous growth. The esa1/esa1 mutant cells exhibited sensitivity to thermal, genotoxic, and oxidative stresses but tolerance to cold, osmotic, and cell wall stresses. In contrast, the sas2/sas2 mutant adapted to growth at higher temperatures and promoted filament formation at lower temperatures, resembling the phenotype of a C. albicans strain overexpressing ESA1. Cells with deletions of both ESA1 and SAS2 were inviable, reflecting the functional redundancy in cell growth. C. albicans Esa1 and Sas2 have distinct and synergistic effects on histone acetylation at H4K5, H4K12, and H4K16. Esa1 contributes mainly to acetylation of H4K5 and H4K12, whereas Sas2 contributes to acetylation of H4K16. Our findings suggest that C. albicans Esa1 and Sas2 play opposite roles in cell growth and morphogenesis and contribute coordinately to histone acetylation and gene regulation.