NF-E2 BINDING SITES AND GLOBIN GENE REGULATION

这项研究的目的是了解红系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主要集中在增加胎儿珠蛋白的表达，通过阐明珠蛋白基因激活的机制将进一步促进治疗。以前的实验表明，珠蛋白调控序列中的核因子-红系2(NF-E2)结合部位对珠蛋白基因的表达很重要(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核因子-E2(72)。我们提出的证据表明，核因子-E2缺乏导致红系成熟和珠蛋白基因表达的部分阻断。我们还提供了证据表明，除了核因子-E2，蛋白质还通过核因子-E2位点激活珠蛋白的表达。我们的假设是，珠蛋白基因的表达需要NF-E2结合位点，并且在没有NF-E2的情况下，其他蛋白质能够与NF-E2结合并激活珠蛋白基因的表达。因此，这项拨款提案中包含的具体目标是：1)确定核因子-E2结合位点在珠蛋白基因表达中的作用；2)表征通过核因子-E2结合位点激活珠蛋白基因表达的蛋白质；以及3)深入了解核因子-E2激活珠蛋白基因的机制。为了实现这些目标中的每一个，建议在转基因小鼠和细胞系中进行一系列实验。为了研究核因子-E2位点在珠蛋白基因表达中的作用，提出了消融小鼠α-珠蛋白正调控元件(AlphaPRE)中串联的核因子-E2结合位点。有人建议用实验来描述由于失去核因子-E2而导致的红细胞生成的变化。我们建议对原代红系细胞提取液中的蛋白质进行生化表征。有人建议在转基因小鼠中进行研究，以确定能够激活珠蛋白基因表达的蛋白质，而不是核因子-E2。关于与核因子-E2相关的结构域的结构信息，最近已被提供(69)。我们建议在功能上定位这个结构域，以及在NF-E2的N末端的第二个结构域。为了确定与这些基序相互作用的分子，我们提出了酵母单/双杂交筛选。这些实验的结果应该会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.