ENHANCER 10-KB-5 TO HUMAN EPSILON GLOBIN

人红系特异性转录增强子IOkb上游 已被鉴定。 DNA序列 包括这种增强剂已经显示出赋予高水平的 人β珠蛋白基因的位置非依赖性转录 在转基因小鼠中， 地中海贫血 这个增强子的位置表明 保持整个β样基因结构域的开放性， 转录因子 因此这个序列和蛋白质 激活它是β样珠蛋白基因转录所必需的。 本研究的目的是鉴定和纯化红细胞 特异性增强子激活蛋白，并确定最小的 位置非依赖性红细胞特异性所需序列 转录增强 为此目的， 将进行：l-研究体内和体外DMS 干扰和DNA酶保护研究，以确定普遍存在的 和红系特异性DNA序列基序和同源增强子 结合蛋白 2-电泳凝胶迁移率变动分析将 与色谱分离结合使用， 红细胞和非红细胞核提取物，以鉴定和纯化 增强子结合蛋白。 3-稳定的细胞系，其含有 具有/不具有额外的启动子上游DNA的增强子， 报告基因将产生，以确定最小的 位置非依赖性红细胞所需的增强子序列 特异性高水平基因转录。 4-的亚克隆 将进行限制性片段和Bal 31缺失以鉴定 红细胞特异性表达所需的最小序列。 红系特异性增强子结合蛋白的鉴定 并保持其开放的染色质构象是 理解反式作用蛋白及其在改变中的作用 染色质结构和红细胞分化的承诺。 位置所需的最小序列的鉴定 独立红细胞特异性转录增强， 构建有效的遗传载体所必需的 β地中海贫血和镰状细胞贫血的治疗。

A human erythroid specific transcriptional enhancer lOkb upstream of the epsilon globin gene has been identified. DNA sequences including this enhancer have been shown to confer high level position independent transcription of the human Beta globin gene in transgenic mice and are deleted in the human gamma-delta-beta thalassemias. The location of this enhancer element suggests it maintains openness of the entire Beta-like gene domain to transcriptional factors. Thus this sequence and the proteins which activate it are required for Beta-like globin gene transcription. The goal of our studies is to identify and purify erythroid specific enhancer activating proteins and identify the minimal sequence required for position independent erythroid specific transcriptional enhancement. To this end the following studies will be undertaken: l-Investigation of in vivo and in vitro DMS interference and DNase protection studies to identify ubiquitous and erythroid specific DNA sequence motifs and cognate enhancer binding proteins. 2-Electrophoretic gel mobility shift assays will be used in conjunction with chromatographic fractionation of erythroid and non-erythroid nuclear extracts to identify and purify enhancer binding proteins. 3-Stable cell lines containing the enhancer with/without additional epsilon upstream DNA driving a reporter gene will be produced in order to identify the minimal enhancer sequence required for position independent erythroid specific high level gene transcription. 4-Subcloning of restriction fragments and Bal 31 deletions will be made to identify the minimal sequence required for erythroid specific expression. Identification of erythroid specific proteins binding the enhancer and maintaining its open chromatin conformation is required for understanding of transacting proteins and their role in alteration of chromatin structure and commitment to erythroid differentiation. Identification of the minimal sequence required for position independent erythroid specific transcriptional enhancement is necessary for construction of effective vectors for the genetic therapy of Beta thalassemia and sickle cell anemia.