Nonidentical function of Upc2A binding sites in the Candida glabrata CDR1 promoter

Increased expression of the Candida glabrata CDR1 gene, encoding an ATP-binding cassette membrane transporter, is routinely observed in fluconazole-resistant isolates of this pathogenic yeast. CDR1 transcription has been well-documented to be due to activity of the Zn2Cys6 zinc cluster-containing transcription factor Pdr1. Gain-of-function mutations in the gene encoding this factor are the most commonly observed cause of fluconazole hyper-resistance in clinical isolates. We have recently found that the sterol-responsive transcription factor Upc2A also acts to control CDR1 transcription, providing a direct link between ergosterol biosynthesis and expression of Pdr1 target genes. While this earlier work implicated Upc2A as an activator of CDR1 transcription, our further analyses revealed the presence of a second Upc2A binding site that negatively regulated CDR1 expression. This Upc2A binding site designated a sterol-responsive element (SRE) was found to have significant lower affinity for Upc2A DNA-binding than the previously described SRE. This new SRE was designated SRE2 while the original, positively acting site was named SRE1. A mutant version of SRE2 prevented in vitro DNA-binding by recombinant Upc2A and, when introduced into the CDR1 promoter, caused decreased fluconazole susceptibility and increased CDR1 expression. This negative effect caused by loss of SRE2 was shown to be Pdr1 independent, consistent with the presence of at least one additional activator of CDR1 transcription. The ability of Upc2A to exert either positive or negative effects on gene expression resembles behavior of mammalian nuclear receptor proteins and reveals an unexpectedly complex nature for SRE effects on gene regulation.