ENHANCER 10-KB-5 TO HUMAN EPSILON GLOBIN

人红系特异性转录增强子1OkB上游 表珠蛋白基因的一部分已经被鉴定出来。DNA序列 包括这种增强剂，已经被证明可以提供高水平的 人β珠蛋白基因的位置非依赖性转录 在转基因小鼠中，并在人的伽马-β-β基因中缺失 地中海贫血。这个增强子元件的位置表明了它 保持整个类Beta基因域的开放性 转录因子。因此，这个序列和它的蛋白质 激活它是贝塔样珠蛋白基因转录所必需的。 我们研究的目标是鉴定和提纯红系。 特异的增强子激活蛋白和鉴定最小的 位置非依赖性红系特异性所需的序列 转录增强。为此，开展了以下研究 将承担：L-体内和体外DMS的研究 干扰和DNA酶保护研究以识别无处不在的 和红系特异性DNA序列基序和同源增强子 结合蛋白。2-电泳凝胶迁移率改变分析将 与层析分级法联合使用 红系和非红系核抽提物的鉴定和纯化 增强子结合蛋白。3-稳定的细胞系，含有 带有/不带有额外epsilon上游DNA的增强子驱动 会产生报告基因，以鉴定最小的 位置非依赖性红系所需的增强子序列 特异的高水平基因转录。4-亚克隆 将进行限制片段和Bal 31缺失以识别 红系特异性表达所需的最小序列。 与增强子结合的红系特异性蛋白的鉴定 而维持其开放的染色质构象是 对交易蛋白及其在变化中的作用的理解 染色质结构和对红系分化的承诺。 确定位置所需的最小序列 独立的红系特异性转录增强是 为构建有效的遗传载体所必需的 贝塔地中海贫血和镰状细胞性贫血的治疗。

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.