Genomewide Location Analysis of Candida albicans Upc2p, a Regulator of Sterol Metabolism and Azole Drug Resistance

Abstract

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 upc2⌬/upc2⌬ 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.Candida albicans is an important human fungal pathogen, in terms of both its clinical significance and its use as an experimental model for scientific investigation. This opportunistic pathogen is a natural component of the human flora, colonizing skin and the gastrointestinal and genitourinary tracts (4). Although many infections involve the colonization of surface mucosal membranes (oral thrush and vaginal candidiasis, for example), immunosuppressed patients can be subject to potentially lethal systemic infections (45).Many antifungal drugs used to treat C. albicans infections function by targeting ergosterol, the analogue of cholesterol in mammalian cells and the major sterol of fungal cell membranes. Polyenes, such as amphotericin B, bind directly to ergosterol and perturb general membrane functions, resulting in low selectivity and high toxicity (2, 35). Azoles, including fluconazole and voriconazole, are more widely used and target the enzyme lanosterol demethylase (Erg11p) in the ergosterol biosynthesis pathway, with the consequence that ergosterol is depleted and replaced by unusual sterols, altering the fluidity of the membrane and the activities of membrane-bound proteins (e.g., enzymes...

Regulation of hypoxia adaptation: an overlooked virulence attribute of pathogenic fungi?

“…Znaidi et al (84) observed that upregulation of ERG11 during hypoxia is strictly Upc2p dependent. Upc2p also binds the promoters of four genes encoding transcription factors (INO2, ACE2, SUT1, and UPC2 itself).…”

Role of the Yap Family in the Transcriptional Response to Oxidative Stress in Yeasts

“…Recently, it was proposed that drug resistance and the response to oxidative stress are interconnected processes in C. albicans (Znaidi et al, 2009). This connection exists through the activity of several TFs like Cap1p, Tac1p, Mrr1p or Upc2p for which (i) gain-of-function mutations were observed as being related to clinical azole resistance and (ii) genome-wide studies identified several of their target genes as being involved in the general response to oxidative stress Morschhauser et al, 2007;Znaidi et al, 2009;Znaidi et al, 2008). Also, it is interesting to remind that, in S. cerevisiae, YAP1 was initially described as a gene involved in pleiotropic drug resistance (Wu et al, 1993).…”

Nuclear receptor-like transcription factors in fungi

“…This suggests that Upc2p/Ecm22p orthologs in pathogenic fungi might play important roles in azole resistance. Indeed, C. albicans harbors a single Upc2p/Ecm22p ortholog (CaUpc2p) that has been implicated in azole resistance (Oliver et al 2007;Dunkel et al 2008;Hoot et al 2008;Znaidi et al 2008). If CaUpc2p by analogy with other zinc cluster transcription factors also interacts with cognate ligands, it might be possible to identify high-affinity small-molecule antagonists that lock CaUpc2p in an inactive conformation, and which could serve as cotherapeutics to enhance the efficacy of standard azole therapy.…”