Unknown,Transcriptomics,Genomics,Proteomics

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Function and regulation of Cph2 in Candida albicans

ABSTRACT: Candida albicans is associated with humans as both a harmless commensal organism and a pathogen. Cph2 is a transcription factor whose DNA binding domain is similar to mammalian sterol response element binding proteins (SREBPs). SREBPs are master regulators of cellular cholesterol levels, and are highly conserved from fungi to mammals. However, ergosterol biosynthesis is regulated by the zinc finger transcription factor Upc2 in C. albicans and several other yeasts. Cph2 is not necessary for ergosterol biosynthesis, but important for colonization in the murine gastrointestinal tract. Here we demonstrate that Cph2 is a membrane-associated transcription factor that is processed to release the N-terminal DNA binding domain like SREBPs; but its cleavage is not regulated by cellular levels of ergosterol or oxygen. ChIP-Seq shows that Cph2 binds to the promoters of HMS1 and other components of the regulatory circuit for GI tract colonization. In addition, 50% of Cph2 targets are also bound by Hms1 and other factors of the regulatory circuit. Several common targets function at the head of the glycolysis pathway. Thus, Cph2 is an integral part of the regulatory circuit for GI colonization that regulates glycolytic flux. RNA-seq shows a significant overlap in genes differentially regulated by Cph2 and hypoxia, and Cph2 is important for optimal expression of some hypoxia-responsive genes in glycolysis and the citric acid cycle. We suggest that Cph2 and Upc2 regulate hypoxia-responsive expression in different pathways, consistent with a synthetic lethal defect of the cph2 upc2 double mutant in hypoxia. Genome binding/occupancy profiling by high throughput sequencing. ChIP-seq of Cph2 was carried out in a wild-type strain carrying N-terminal myc-tagged Cph2 under the MAL2 promoter (MAL2-myc-Cph2N). IP and INPUT samples from 2 independent experiments, as well as a sample of untagged wild-type control, were sequenced.

ORGANISM(S): Candida albicans

SUBMITTER: Pietro Di Lena

PROVIDER: E-GEOD-71728 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In the pathogenic yeast Candida albicans, the zinc cluster transcription factor Upc2p has been shown to regulate expression of ERG11 and other genes involved in ergosterol biosynthesis upon exposure to azole antifungals. ERG11 encodes lanosterol demethylase, the target enzyme of this antifungal class. Over-expression of UPC2 reduces azole susceptibility, whereas its disruption results in hypersusceptibility to azoles and reduced accumulation of exogenous sterols. Constitutive up-regulation of ERG11 is a major cause of resistance to fluconazole in clinical isolates of C. albicans, yet the mechanism for this has yet to be determined. Using genome-wide gene expression profiling, we found UPC2 and other genes involved in ergosterol biosynthesis to be coordinately up-regulated with ERG11 in a fluconazole resistant clinical isolate as compared with a matched susceptible isolate from the same patient. Sequence analysis of the UPC2 alleles of these isolates revealed that the resistant isolate contained a single nucleotide substitution in one UPC2 allele that resulted in a G648D exchange in the encoded protein. Introduction of the mutated allele into a drug susceptible strain resulted in constitutive up-regulation of ERG11 and increased resistance to fluconazole. By comparing the gene expression profiles of the fluconazole resistant isolate and of strains carrying wild-type and mutated UPC2 alleles, we identified target genes that are controlled by Upc2p. Here we show for the first time that a gain-of-function mutation in UPC2 leads to increased expression of ERG11 and imparts resistance to fluconazole in clinical isolates of C. albicans. Keywords: genome-wide expression profiling Clinical isolates S1 (susceptible) and S2 (resistant), genome strain SC5314, UPC2 disruption strains (UPC2M4A and UPC2M4B), UPC2 re-integrant strains of non-mutated UPC2 allele (UPC2M2K21A and UPC2M2K21B), and UPC2 re-integrant strains of mutated UPC2 allele (UPC2M2K31A and UPC2M2K31B) were grown for 3 hours until log phase. RNA was extracted for each isolate/strain. Experiments were performed in duplicate.

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Function and regulation of Cph2 in Candida albicans

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