Metal-Regulatory Factor 1 (MTF-1) Role in Development and Stress Response

金属反应元件结合转录因子-1(MTF-1)是一种普遍表达的转录因子，在金属稳态、胚胎发育、肿瘤进展、氧化应激或缺氧信号转导等多种基因的调控中发挥重要作用。本研究的总体目标是研究MTF-1与NRF2的重叠作用，NRF2是调节氧化应激反应的主要转录因子，在应激反应基因的发育和转录共同调节中发挥作用。本项目的具体目的是(1)确定MTF-1在斑马鱼胚胎发育中的作用，(2)确定已知应激反应基因表达的表型变化，并通过MTF-1和NRF2的‘敲除’识别新的靶基因，以及(3)确定MTF-1在协同调节NRF2靶基因转录激活中的相互作用。将采用各种技术和方法来实现这些目标。这些技术将包括开发转基因斑马鱼品系、吗啉敲除基因表达、利用寡核苷酸微阵列进行基因表达谱分析，以及用于微阵列分析和计算启动子分析的先进生物信息学技术。一种新型的转基因斑马鱼品系，它表达一种结构性核、显性阴性的MTF-1亚型，以及三个在肠道、肝脏、胰腺和神经元中表达绿色或黄色荧光蛋白的转基因斑马鱼品系，将被用来观察与MTF-1‘敲除’相关的潜在发育表型。其次，将使用寡核苷酸微阵列来确定MTF-1或NRF2“敲除”后基因表达的变化，以发现在发育过程中受这两种转录因子共同调节的新靶基因，或者对镉或TBHQ(氧化应激)的响应。第三，MTF-1和NRF2在金属或氧化应激转录反应中的相互作用将通过选择目标基因进行计算启动子分析来进一步表征，以确认MTF-1和NRF2识别位点的存在，并将选择一个基因通过体内启动子分析对镉或氧化应激做出额外表征。

The metal response element-binding transcription factor-1 (MTF-1) is a ubiquitously expressed transcription factor that plays a primary role in the regulation of a variety of genes involved in metal homeostasis, embryonic development, tumor progression, and oxidative stress or hypoxia signaling. The overall objective ofthis research is to characterize the overlapping role of MTF-1 with NRF2, the primary transcription factor for regulating the oxidative stress response, in development and transcriptional co-regulation of stress responsive genes. The specific aims ofthis project are to (1) determine role of MTF-1 in zebrafish embryonic development, (2) determine phenotypic changes in expression of known stress response genes and identify novel target genes via MTF-1 and NRF2 'knockdowns', and (3) determine the interactive role of MTF-1 in coregulating transcriptional activation of NRF2 target genes. A variety of techniques and approaches will be employed to achieve these aims. These techniques will include development of transgenic zebrafish lines, morpholino knockdown of gene expression, gene expression profiling with oligonucleotide microarrays, and advanced bioinformatic techniques for microarray analysis and computational promoter analysis. A novel transgenic zebrafish strain that expresses a constitutively nuclear, dominant-negative MTF-1 isoform and three transgenic zebrafish lines that express green or yellow fluorescent protein in the gut, liver, pancreas and neurons will be used to visualize potential developmental phenotypes associated with the MTF-1 'knockdowns'. Second, oligonucleotide microarrays will be used to determine changes in gene expression after MTF-1 or NRF2 'knockdown' to discover novel target genes co-regulated by both transcription factors during development or in response to cadmium or tBHQ (oxidative stress). Third, the interactive role of MTF-1 with NRF2 in the transcriptional response to metals or oxidative stress will be further characterized by selecting target genes for computational promoter analysis to confirm the presence of MTF-1 and NRF2 recognition sites, and one gene will be selected for additional characterization by in vivo promoter analysis in response to cadmium or oxidative stress.