REGULATION OF ZETA GLOBIN GENE EXPRESSION

该项目的广泛的长期目标是确定 调节珠蛋白基因表达的分子基础， 这些研究涉及造血通路的解剖， 发展过程中的承诺。 该项目的具体目标是： 1. 验证zeta珠蛋白的发育调控 表达是由于胚胎和非胚胎之间的差异 红系细胞中的蛋白质-DNA相互作用发生在近端 ζ-珠蛋白启动子， 2. 检测上述蛋白质-DNA的功能意义 在近端zeta-珠蛋白启动子内的相互作用， 将改变的启动子导入红系细胞系和转基因小鼠， 3. 确定一个新的上游正调控区的特征 负责高水平zeta-珠蛋白表达的基因， 胚胎红系细胞系K562， 4. 表征对zeta调节重要的反式作用因子， 通过使用表达克隆分离cDNA克隆的珠蛋白表达 策略和/或将因子纯化为同质性。 该项目与健康的关系有三个方面。 第一，研究 zeta珠蛋白基因调控的研究将有助于全面了解 正常个体和地中海贫血患者中珠蛋白表达的调节 患者 其次，由于调节ζ-珠蛋白表达的机制 与其他球蛋白基因所使用的不同， 将鉴定出可用于体细胞基因治疗的元件 表达构建体。 第三，由于ζ-珠蛋白是最早的 在发育过程中表达的造血基因，了解其 监管将有助于更好地了解正常情况 造血和异常造血病症如白血病， 不适当的胚胎基因表达发生。 研究设计和方法包括蛋白质-DNA的表征 通过电泳迁移率分析zeta-珠蛋白启动子内的相互作用 迁移分析、DNA酶I足迹和蛋白质-DNA交联 研究，通过测试调节元件的功能分析， 定点突变对Zeta珠蛋白启动子功能的影响 白血病细胞系、转基因小鼠和胚状体，以及分子 克隆和/或纯化负责 zeta-珠蛋白表达的发育调节。

The broad, long-term objectives of this project are to determine the molecular basis for regulation of globin gene expression, and to extend these studies to the dissection of pathways involved in hematopoietic commitment during development. The specific aims of this project are to: 1. Test the hypothesis that development regulation of zeta-globin expression is due to differences between embryonic and non-embryonic erythroid cells in protein-DNA-interactions occurring within the proximal zeta-globin promoter, 2. Test the functional significance of the above protein-DNA interactions within the proximal zeta-globin promoter by introducing altered promoters into both erythroid cell lines and transgenic mice, 3. Identify the features of a novel upstream positive regulatory region that are responsible for high-level zeta-globin expression in the embryonic erythroid cell line K562, 4. Characterize a trans-acting factor important for regulation of zeta- globin expression by isolating a cDNA clone using expression cloning strategies and/or purifying the factor to homogeneity. The health-relatedness of this project is threefold. First, the study of zeta-globin gene regulation will contribute to overall understanding of regulation of globin expression in normal individuals and thalassemic patients. Second, since mechanisms that regulate zeta-globin expression are distinct from those used by other globulin genes, novel regulatory elements will be identified that can be adapted for somatic gene therapy expression constructs. Third, since zeta-globin is one of the first hematopoietic genes expressed during development, understanding its regulation will contribute to better understanding of normal hematopoiesis and abnormal hematopoietic conditions such as leukemia, in which inappropriate embryonic gene expression occurs. The research design and methods include characterization of protein-DNA interactions within the zeta-globin promoter by electrophoretic mobility shift assays, DNAaseI footprinting, and protein-DNA cross-linking studies, functional analysis of regulatory elements by testing the effects of site-directed mutations on zeta-globin promoter function in leukemic cell lines, transgenic mice, and embryoid bodies, and molecular cloning and/or purification of DNA-binding activities responsible for developmental regulation of zeta-globin expression.