HUMAN GLOBIN AND ERYTHROPOIETIN RECEPTOR GENE REGULATION

描述：(改编自调查人员摘要)。这项建议有如下几点 它的长期目标是阐明 人类两个重要的专门化基因系统的表达调控 红系细胞：在发育过程中高水平表达的珠蛋白基因。 红系分化后期与促红细胞生成素 受体(EPO-R)基因在体内表达水平相对较低 红系分化的早期阶段。顺式和反式 调控这两种基因表达的因素在行为上有所不同 将对红系特异性基因系统进行比较和对比。这个 将研究的珠蛋白基因调控的特定方面是 从胎儿血红蛋白(HBF)转换为成人血红蛋白(HBA)，使用 转基因小鼠作为血红蛋白转换的模型系统。大DNA 同时含有胎儿(伽马)和成人(贝塔)人类珠蛋白基因的片段 已经被我们自己和其他人证明是以一种受监管的 在转基因小鼠中(即，它们在发育过程中发生变化)。鉴于 其他人则强调了在这个激活轨迹的过程中的作用 位于珠蛋白基因上游的区域(LAR)，我们的研究将集中在 主要是关于40kb KpnI DNA中DNA序列的作用 包含伽马、德尔塔和贝塔珠蛋白基因的片段。研究将会 使用正常的DNA片段和相应的DNA进行 从持续表达Hb的成人个体中分离到的片段 F因非缺失型或缺失型遗传性不同 胎儿血红蛋白或β-地中海贫血的持续性。对中国传统文化的研究 人类EPO-R基因将由基因组特征组成 克隆的染色体基因的结构，特别强调 基因启动子区域的结构和功能。这位将军 可采用的方法包括：克隆和DNA序列分析 EPO-R基因；顺式作用元件的基因操纵研究 然后是基因延迟分析、DNase I足迹和甲基化 干扰技术；研究DNase I超敏部位 邻近和远程侧翼DNA用于识别可能的LAR 序列；潜在LAR序列的功能活性测试 组织培养细胞和转基因小鼠。这些研究应该导致一个 更透彻地理解调控的分子机制 珠蛋白基因的表达发生在发育过程中，可能表明 刺激个体产生Hb-F的未来途径 患有血红蛋白疾病的患者可能会从这一过程中受益。 更一般地说，分子调控机制的比较 EPO-R基因表达调控珠蛋白基因表达应 为组织特异性基因表达过程提供新的见解 在红系细胞分化过程中。

DESCRIPTION: (Adapted from investigator's abstract). This proposal has as its long-term objective the elucidation of the molecular mechanisms that regulate the expression of two important specialized gene systems in human erythroid cells: the globin genes that are expressed at high levels during the later stages of erythroid differentiation and the erythropoietin receptor (EPO-R) gene that is expressed at relatively low levels during the early stages of erythroid differentiation. The cis- and trans-acting factors that regulate the expression of these two behaviorly different yet erythroid specific gene systems will be compared and contrasted. The particular aspect of globin gene regulation that will be investigated is the switch from fetal hemoglobin (HbF) to adult hemoglobin (HbA), using the transgenic mouse as a model system for hemoglobin switching. Large DNA fragments containing both fetal (gamma) and adult (beta) human globin genes have been shown by ourselves and others to be expressed in a regulated fashion (i.e., they switch during development) in transgenic mice. Whereas others have emphasized the role in this process of the locus activating region (LAR) located upstream of the globin gene, our studies will focus primarily on the role of the DNA sequences within the 40 kb KpnI DNA fragment encompassing the gamma, delta and beta globin genes. Studies will be carried out using both normal DNA fragments and corresponding DNA fragments isolated from individuals with persistent adult expression of Hb F due to different forms of nondeletion or deletion type hereditary persistence of fetal hemoglobin or delta-beta thalassemia. Studies of the human EPO-R gene will consist of the characterization of the genomic structure of the cloned chromosomal gene with particular emphasis on the structure and function of the promoter region of the gene. The general methodology to be utilized include: cloning and DNA sequence analysis of the EPO-R gene; studies of cis-acting elements by gene manipulation followed by gene retardation assays, DNase I footprinting and methylation interference techniques; studies DNase I hypersensitive sites in neighboring and remote flanking DNA for identification of possible LAR sequences; testing of functional activity of potential LAR sequences in tissue culture cells and transgenic mice. These studies should lead to a more thorough understanding of the molecular mechanisms by which regulation of globin gene expression occurs during development and perhaps indicate future approaches for the stimulation of Hb F production in individuals with hemoglobinopathies who might benefit clinically from such a process. In more general terms, the comparison of molecular mechanisms regulating EPO-R gene expression to those regulating globin gene expression should provide new insights on the process of tissue-specific gene expression during erythroid cell differentiation.