Pan Yu-Jiao, Cho Chao-Cheng, Kao Yu-Yun, Sun Chin-Hung
Department of Parasitology, College of Medicine, National Taiwan University, Taipei 100, Taiwan.
J Biol Chem. 2009 Jul 3;284(27):17975-88. doi: 10.1074/jbc.M109.012047. Epub 2009 May 7.
Synthesis of a protective cyst wall is required for survival outside of the host and for infection of Giardia lamblia. Little is known of gene regulation of the cyst wall proteins (CWPs) during differentiation into dormant cysts. WRKY homologues constitute a large family of DNA-binding proteins in plants that are involved in several key cellular functions, including disease resistance, stress response, dormancy, and development. A putative wrky gene has been identified in the G. lamblia genome. We found that wrky expression levels increased significantly during encystation. The epitope-tagged WRKY was translocated into the nuclei during encystation. Recombinant WRKY specifically bound to its own promoter and the encystation-induced cwp1 and cwp2 promoters. WRKY contains several key residues for DNA binding, and mutation analysis revealed that its binding sequences are similar to those of the known plant WRKY proteins and that two of them are positive cis-acting elements of the wrky and cwp2 promoters. Overexpression of WRKY increased the cwp1-2 and myb2 mRNA levels, and these gene promoters were bound by WRKY in vivo. Interestingly, the wrky and cwp1-2 genes were up-regulated by ERK1 (extracellular signal-related kinase 1) overexpression, suggesting that WRKY may be a downstream component of the ERK1 pathway. In addition, a WRKY mutant that cannot enter nuclei and an ERK1 mutant lacking the predicted kinase domain showed decreased cwp1-2 gene expression. Our results suggest that the WRKY family has been conserved during evolution and that WRKY is an important transactivator of the cwp1-2 genes during G. lamblia differentiation into dormant cysts.
合成保护性包囊壁是蓝氏贾第鞭毛虫在宿主体外存活和感染所必需的。关于在分化为休眠包囊过程中包囊壁蛋白（CWPs）的基因调控知之甚少。WRKY同源物在植物中构成一个大型的DNA结合蛋白家族，参与多种关键细胞功能，包括抗病性、应激反应、休眠和发育。在蓝氏贾第鞭毛虫基因组中已鉴定出一个假定的wrky基因。我们发现wrky表达水平在成囊过程中显著增加。带有表位标签的WRKY在成囊过程中易位至细胞核。重组WRKY特异性结合其自身启动子以及成囊诱导的cwp1和cwp2启动子。WRKY含有几个用于DNA结合的关键残基，突变分析表明其结合序列与已知植物WRKY蛋白的相似，且其中两个是wrky和cwp2启动子的正向顺式作用元件。WRKY的过表达增加了cwp1 - 2和myb2 mRNA水平，并且这些基因启动子在体内被WRKY结合。有趣的是，wrky和cwp1 - 2基因被ERK1（细胞外信号相关激酶1）过表达上调，表明WRKY可能是ERK1途径的下游组分。此外，不能进入细胞核的WRKY突变体和缺乏预测激酶结构域的ERK1突变体显示cwp1 - 2基因表达降低。我们的结果表明WRKY家族在进化过程中得以保留，并且WRKY是蓝氏贾第鞭毛虫分化为休眠包囊过程中cwp1 - 2基因的重要反式激活因子。
