Genome-Wide Microarrray Analysis Reveals Roles for the REF-1 Family Member HLH-29 in Ferritin Synthesis and Peroxide Stress Response

In Caenorhabditis elegans, the six proteins that make up the REF-1 family have been identified as functional homologs of the Hairy/Enhancer of Split (HES) proteins. These transcription factors act in both Notch dependent and Notch-independent pathways to regulate embryonic events during development; however, their post-embryonic functions are not well defined. As a first step toward understanding how the REF-1 family works together to coordinate post-embryonic events, we used gene expression microarray analysis to identify transcriptional targets of HLH-29 in L4/young adult stage animals. Here we show that HLH-29 targets are genes needed for the regulation of growth and lifespan, including genes required for oxidative stress response and fatty acid metabolism, and the ferritin genes, ftn-1 and ftn-2. We show that HLH-29 regulates ftn-1 expression via promoter sequences upstream of the iron-dependent element that is recognized by the hypoxia inducible factor, HIF-1. Additionally, hlh-29 mutants are more resistant to peroxide stress than wild-type animals and ftn-1(RNAi) animals, even in the presence of excess iron. Finally we show that HLH-29 acts parallel to DAF-16 but upstream of the microphthalmia transcription factor ortholog, HLH-30, to regulate ftn-1 expression under normal growth conditions.

在秀丽隐杆线虫（Caenorhabditis elegans）中，构成REF-1家族的六种蛋白质已被鉴定为Hairy/增强子分裂（Hairy/Enhancer of Split, HES）蛋白的功能同源物。此类转录因子可在Notch依赖与非依赖通路中发挥调控作用，参与发育过程中的胚胎事件调控，但其胚胎后功能尚未得到明确阐释。作为探究REF-1家族如何协同调控胚胎后事件的首要步骤，我们采用基因表达微阵列分析技术，鉴定了L4期/年轻成虫阶段秀丽隐杆线虫体内HLH-29的转录靶标。本研究证实，HLH-29的靶基因涵盖生长与寿命调控相关基因，包括氧化应激响应、脂肪酸代谢所需基因，以及铁蛋白基因ftn-1与ftn-2。我们发现，HLH-29可通过缺氧诱导因子（hypoxia inducible factor, HIF-1）识别的铁依赖元件上游的启动子序列，调控ftn-1的表达。此外，即便在过量铁存在的环境中，hlh-29突变体对过氧化物应激的耐受性仍高于野生型动物与ftn-1(RNAi)动物。最后，我们证实在正常生长条件下，HLH-29通过平行于DAF-16、且位于小眼症转录因子同源物HLH-30上游的通路，调控ftn-1的表达。