Functional Analysis of cis- and trans-Acting Elements of the Candida albicans CDR2 Promoter with a Novel Promoter Reporter System
Functional Analysis of cis- and trans-Acting Elements of the Candida albicans CDR2 Promoter with a Novel Promoter Reporter System
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DOI：
10.1128/ec.00069-09
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发表时间：
2009-08-01
期刊：
影响因子：
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通讯作者：
Sanglard, Dominique
中科院分区：
文献类型：
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作者：
Coste, Alix T.;Crittin, Jerome;Sanglard, Dominique
唑类耐药的白色念珠菌可以介导的ATP结合盒转运基因CDR 1和CDR 2的上调。这两个基因都受一个称为药物反应元件（DRE）的顺式作用元件和转录因子Tac 1 p的调控，DRE具有共有序列5 '-CGGAWATCGGATATTTTTTT-3'。为了详细分析调控CDR 1和CDR 2所必需的DRE序列以及TAC 1等位基因的性质，设计了一个单杂交系统。该系统基于P-（CDR 2）-HIS 3报告系统，其中组氨酸营养缺陷型的互补可以通过由CDR 2诱导药物（例如雌二醇）激活报告系统来监测。我们的研究结果表明，大多数DRE内的修饰，但特别是在CGG三联体的水平，强烈降低CDR 2的表达。CDR 2 DRE被从共调节基因（CDR 1、RTA 3和IFU 5）的启动子推导出的推定DRE取代。令人惊讶的是，即使Tac 1 p能够结合这些推定的DRE，如染色质免疫沉淀所示，来自RTA 3和IFU 5的DRE在功能上不能取代CDR 2 DRE。单杂交系统也被用于鉴定来自临床C.白色念珠菌分离物或通过随机诱变插入到TAC 1野生型等位基因中。总共在13个不同的位置鉴定了17个不同的GOF突变。其中5个（G980 E、N972 D、A736 V、T225 A和N977 D）已经在临床分离株中描述，另外4个（G980 W、A736 T、N972 S和N972 I）发生在已经描述的位置，因此表明GOF突变可以发生在Tac 1 p中有限数量的位置。总之，本研究建立的单杂交系统快速、有效，可用于C.白色念珠菌。
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.