Genetic analysis of iron homeostasis in C. elegans

描述(由申请人提供)：铁在许多生物过程中起着重要作用，包括DMA合成/呼吸作用、三羧酸循环、氧运输、血红素合成和光合作用。但铁也可能是有毒的，因为它能够产生自由基，破坏细胞中的大分子。因此，生物体进化出了感知、获取和储存铁的精确机制。我们的目标是更多地了解真核细胞如何感知铁，产生信号，并将信号转化为调节铁的动态平衡。具体地说，我们感兴趣的是了解转录因子如何感知铁，并调控动物细胞中的基因表达。虽然铁介导的转录因子已经在真菌和植物中被发现，但对动物细胞中铁介导的转录调控的机制和蛋白质知之甚少。我们发现秀丽线虫表达铁蛋白(CeFTN-1和CeFTN-2)，铁蛋白是铁储存的关键蛋白。我们的数据表明，Ceftn-1和Ceftn-2在转录上受铁的调控，而它们5‘上游区域存在的顺式调控序列是这种调控所必需的。我们建议鉴定顺式调控序列和与这些元件结合的铁敏感转录因子，并确定该转录因子如何感知铁。我们还将确定与铁信号通路有关的其他线虫基因。线虫为其他生物体的铁代谢研究提供了一种补充方法，因为它们可以进行遗传分析，它们有器官/组织系统，它们有编码哺乳动物铁稳态的蛋白质的同源基因。其具体目的是：1)鉴定Ceftn-1和Ceftn-2启动子中顺式作用的铁调节元件；2)纯化调控Ceftn-1和Ceftn-2基因的线虫转录因子，并确定该因子如何控制铁蛋白的表达；3)利用基于绿色荧光蛋白(GFP)的筛选方法鉴定线虫铁信号通路中涉及的基因。

DESCRIPTION (provided by applicant): Iron has an essential role in many biological processes, including DMA synthesis/respiration, the tricarboxylic acid cycle, oxygen transport, heme synthesis and photosynthesis. But iron can also be toxic due to its ability to generate free radicals that damage macromolecules in cells. Consequently, organisms have evolved precise mechanisms to sense, acquire and store iron. Our goal is to learn more about how eukaryotic cells sense iron, generate a signal and transduce the signal into regulating iron homeostasis. Specifically, we are interested in understanding how transcription factors sense iron, and regulate gene expression in animal cells. While iron-mediated transcription factors have been identified in fungi and plants, little is known about the mechanisms and the proteins regulating iron-mediated transcriptional regulation in animal cells. We have discovered that Caenorhabditis elegans express ferritin (CeFTN-1 and CeFTN-2), which are key iron storage proteins. Our data shows that Ceftn-1 and Ceftn-2 are transcriptionally regulated by iron, and that cis-regulatory sequences present in their 5' upstream regions are required for this regulation. We propose to identify the cis-regulatory sequences and the iron-sensing transcription factor that binds these elements, and to determine how this transcription factor senses iron. We will also identify other C. elegans genes involved in iron signaling pathways. C. elegans offers a complementary approach to iron metabolic studies in other organisms, in that they are amenable to genetic analysis, they have organ/tissue systems, and they have homologs of genes encoding proteins involved in mammalian iron homeostasis. The specific aims are 1) to characterize cis-acting iron-regulatory elements in Ceftn-1 and Ceftn-2 promoters, 2) to purify the C. elegans transcription factor that regulates the Ceftn-1 and Ceftn-2 genes, and to determine how this factor controls ferritin expression, and 3) to identify genes involved in iron signaling pathways in C. elegans using a green fluorescence protein (GFP)-based screen.