NF-E2 BINDING SITES AND GLOBIN GENE REGULATION

本研究的目的是了解红细胞中高水平珠蛋白基因表达所需的分子事件。镰状细胞病和地中海贫血的治疗，其重点是增加胎儿珠蛋白表达，将进一步阐明珠蛋白基因激活的机制。先前的实验表明，珠蛋白调控序列中核因子-红细胞2 （NF-E2）的结合位点对珠蛋白基因表达很重要（11,45,60,78）。最近，研究表明珠蛋白基因的表达并不严格依赖于NF-E2（72）。我们提出的证据表明，NF-E2缺乏导致红细胞成熟和珠蛋白基因表达的部分阻断。我们还提供证据表明，除了NF-E2之外，其他蛋白质通过NF-E2位点激活珠蛋白表达。我们的假设是，珠蛋白基因的表达需要NF-E2的结合位点，在缺乏NF-E2的情况下，其他蛋白能够结合NF-E2位点，激活珠蛋白基因的表达。因此，本课题的具体目的是：1)确定NF-E2结合位点在珠蛋白基因表达中的作用；2)表征通过NF-E2位点激活珠蛋白基因表达的蛋白；3)深入了解NF-E2活化珠蛋白基因的机制。为了实现这些目标，在转基因小鼠和细胞系中提出了一系列的实验。为了研究NF-E2位点在珠蛋白基因表达中的作用，研究人员提出了在小鼠α -珠蛋白阳性调节元件（alphaPRE）中消融串联NF-E2结合位点的方法。实验被提议表征由于NF-E2的损失而导致的红细胞生成的变化。我们拟对原代红细胞提取物中的蛋白质进行生化表征。研究提出，在转基因小鼠中，以确定激活珠蛋白基因表达的蛋白质代替NF-E2。最近提供了与NF-E2有关的结构域的结构信息（69）。我们建议对这个结构域以及NF-E2 n端的第二个结构域进行功能映射。为了鉴定与这些基序相互作用的分子，我们提出酵母单/双杂交筛选。这些实验的结果将对珠蛋白基因表达的调控产生重要的见解。

The goal of this research to understand the molecular events that are required for high-level globin gene expression in erythroid cells. Therapy of sickle cell disease and the thalassemias, which has focused on increasing fetal globin expression, would be furthered by elucidating the mechanism of globin gene activation. Previous experiments have suggested that binding sites for Nuclear factor-erythroid 2 (NF-E2), in globin regulatory sequences, are important for globin gene expression (11,45,60,78). More recently, it has been shown that globin gene expression is not strictly dependent on NF-E2 (72). We present evidence that NF-E2 deficiency causes a partial block to erythroid maturation and globin gene expression. We also present evidence that proteins, other than NF-E2, activate globin expression through NF-E2 sites. It is our hypothesis that NF-E2 binding sites are required for globin gene expression, and that in the absence of NF-E2, other proteins are able to bind NF-E2 sites and activate globin gene expression. Therefore, the specific aims contained in this grant proposal are: 1) To determine the role of NF-E2 binding sites in globin gene expression; 2) To characterize the proteins that activate globin gene expression through NF-E2 sites; and 3) To gain insight into the mechanism of globin gene activation by NF-E2. To achieve each of these aims, a series of experiments is proposed in genetically modified mice and cell lines. To examine the role of NF-E2 sites in globin gene expression, ablation of tandem NF-E2 binding sites in the mouse alpha-globin positive regulatory element (alphaPRE) is proposed. Experiments are proposed to characterize changes in erythropoiesis due to loss of NF-E2. We propose to biochemically characterize proteins in extracts from primary erythroid cells. Studies are proposed, in transgenic mice, to identify proteins that activate globin gene expression in place of NF-E2. Structural information on a domain related to NF-E2, has recently been made available (69). We propose to functionally map this domain, as well as a second domain at the N-terminus of NF-E2. To identify molecules that interact with these motifs, we propose yeast one/two-hybrid screens. Results from these experiments should yield important insights into the regulation of globin gene expression.