Karnani Neerja, Gaur Naseem Akhtar, Jha Sudhakar, Puri Neeti, Krishnamurthy Shankarling, Goswami Shyamal K, Mukhopadhyay Gauranga, Prasad Rajendra
School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
Yeast. 2004 Feb;21(3):219-39. doi: 10.1002/yea.1067.
CDR1 gene encoding an ATP-driven drug extrusion pump has been implicated in the development of azole-resistance in Candida albicans. Although the upregulation of CDR1 expression by various environmental factors has been documented, the molecular mechanism underlying such process is poorly understood. We have demonstrated earlier that the CDR1 promoter encompasses a large number of cis-regulatory elements, presumably mediating its response to various drugs. In this study we have identified a novel steroid responsive region (SRR) conferring beta-oestradiol and progesterone inducibility on the CDR1 promoter. The SRR is located -696 to -521 bp upstream of the transcription start site; it is modular in nature and can confer steroid responsiveness to a heterologous promoter (ADH1) linked to a GFP reporter gene. In vitro DNase I protection analyses of SRR revealed two progesterone responsive sequences (-628 to -594 and -683 to -648) and one beta-oestradiol responsive sequence (-628 to -577), which was further corroborated by the gel mobility shift assay. Deletion analyses within the SRR further delimited these steroid responsive sequences into two distinct elements, viz. SRE1 and SRE2. While SRE1 (-677 to -648) responds only to progesterone, SRE2 (-628 to -598) responded to both progesterone and beta-oestradiol. Both SRE1 and SRE2 were specific for steroids, as they did not respond to other drugs, such as cycloheximide, miconazole and terbinafine. In silico comparison of the SRE1/2 with the promoter sequences of other MDR (CDR2 and PDR5) and non-MDR (HSP90) steroid-responsive genes revealed a similarity with respect to conservation of three 5 bp stretches (AAGAA, CCGAA and ATTGG). Taken together, we have identified a novel steroid responsive cis-regulatory sequence in the CDR1 promoter, which presumably can be instrumental in understanding the steroid response cascade in Candida albicans.
编码一种由ATP驱动的药物外排泵的CDR1基因与白色念珠菌唑类耐药性的产生有关。尽管已有文献记载各种环境因素可上调CDR1的表达，但其潜在的分子机制仍知之甚少。我们之前已经证明，CDR1启动子包含大量顺式调控元件，推测这些元件介导其对各种药物的反应。在本研究中，我们鉴定出一个新的类固醇反应区域（SRR），该区域赋予CDR1启动子对β-雌二醇和孕酮的诱导性。SRR位于转录起始位点上游-696至-521 bp处；它本质上是模块化的，可赋予与绿色荧光蛋白报告基因相连的异源启动子（ADH1）类固醇反应性。对SRR进行的体外DNase I保护分析揭示了两个孕酮反应序列（-628至-594和-683至-648）和一个β-雌二醇反应序列（-628至-577），凝胶迁移率变动分析进一步证实了这一点。SRR内的缺失分析进一步将这些类固醇反应序列划分为两个不同的元件，即SRE1和SRE2。虽然SRE1（-677至-648）仅对孕酮有反应，但SRE2（-628至-598）对孕酮和β-雌二醇均有反应。SRE1和SRE2对类固醇具有特异性，因为它们对其他药物如环己酰亚胺、咪康唑和特比萘芬无反应。对SRE1/2与其他多药耐药（CDR2和PDR5）和非多药耐药（HSP90）类固醇反应基因的启动子序列进行的电子比较显示，在三个5 bp序列（AAGAA、CCGAA和ATTGG）的保守性方面存在相似性。综上所述，我们在CDR1启动子中鉴定出一个新的类固醇反应性顺式调控序列，该序列可能有助于理解白色念珠菌中的类固醇反应级联。
