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Eukaryotic Cell, August 2009, p. 1250-1267, Vol. 8, No. 8
1535-9778/09/$08.00+0     doi:10.1128/EC.00069-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Functional Analysis of cis- and trans-Acting Elements of the Candida albicans CDR2 Promoter with a Novel Promoter Reporter System {triangledown} ,{dagger}

Alix T. Coste,1 Jérôme Crittin,1 Christopher Bauser,2 Bettina Rohde,2 and Dominique Sanglard1*

Institute of Microbiology, University of Lausanne and University Hospital Center, CH-1011 Lausanne, Switzerland,1 GATC Biotech, Konstanz, Germany2

Received 1 March 2009/ Accepted 17 June 2009

Azole resistance in Candida albicans can be mediated by the upregulation of the ATP binding cassette transporter genes CDR1 and CDR2. Both genes are regulated by a cis-acting element called the drug-responsive element (DRE), with the consensus sequence 5'-CGGAWATCGGATATTTTTTT-3', and the transcription factor Tac1p. In order to analyze in detail the DRE sequence necessary for the regulation of CDR1 and CDR2 and properties of TAC1 alleles, a one-hybrid system was designed. This system is based on a P(CDR2)-HIS3 reporter system in which complementation of histidine auxotrophy can be monitored by activation of the reporter system by CDR2-inducing drugs such as estradiol. Our results show that most of the modifications within the DRE, but especially at the level of CGG triplets, strongly reduce CDR2 expression. The CDR2 DRE was replaced by putative DREs deduced from promoters of coregulated genes (CDR1, RTA3, and IFU5). Surprisingly, even if Tac1p was able to bind these putative DREs, as shown by chromatin immunoprecipitation, those from RTA3 and IFU5 did not functionally replace the CDR2 DRE. The one-hybrid system was also used for the identification of gain-of-function (GOF) mutations either in TAC1 alleles from clinical C. albicans isolates or inserted in TAC1 wild-type alleles by random mutagenesis. In all, 17 different GOF mutations were identified at 13 distinct positions. Five of them (G980E, N972D, A736V, T225A, and N977D) have already been described in clinical isolates, and four others (G980W, A736T, N972S, and N972I) occurred at already-described positions, thus suggesting that GOF mutations can occur in a limited number of positions in Tac1p. In conclusion, the one-hybrid system developed here is rapid and powerful and can be used for characterization of cis- and trans-acting elements in C. albicans.

* Corresponding author. Mailing address: Institute of Microbiology, University Hospital Lausanne and University Hospital Center (CHUV), rue du Bugnon 48, CH-1011 Lausanne, Switzerland. Phone: 00 41 21 314 40 83. Fax: 00 41 21 314 40 60. E-mail: Dominique.Sanglard{at}chuv.ch

{triangledown} Published ahead of print on 26 June 2009.

{dagger} Supplemental material for this article may be found at http://ec.asm.org/.

Eukaryotic Cell, August 2009, p. 1250-1267, Vol. 8, No. 8
1535-9778/09/$08.00+0     doi:10.1128/EC.00069-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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