REGULATION OF HUMAN DELTA GLOBIN GENE EXPRESSION

我们研究了成人红系细胞低水平表达β-珠蛋白基因的分子机制，为治疗操作血红蛋白(HbA2)的合成提供了便利。以前，我们通过计算机比对确定了CCAAC存在一个突变的CP1结合基序(CCAAT Box)，位于-70bp处，而缺乏红系特异性因子EKLF在-85bp处的结合序列，这些变化在灵长类动物和人类三角洲-珠蛋白基因中是保守的。在Delta-启动子连接的荧光素酶报告系统中，在-70bp处恢复CCAAT序列和/或在-85bp处插入一致的EKLF结合位点，可显著提高K562和MEL细胞以及人原代成年红系细胞(HAEC)的活性。因此，我们的体外实验结果表明，突变的CCAAT盒(CCAAC)和启动子中EKLF结合位点的缺失至少在转录水平上对成人红系细胞中三角洲-珠蛋白基因的低水平表达负有部分责任。我们还获得了在Delta启动子序列中插入EKLF结合位点的转基因小鼠，这些转基因小鼠的构建中包含了座位控制区和人β和Delta-珠蛋白基因。这些转基因研究表明，人类Delta珠蛋白的表达显著增加。目前正在进行研究，以评估这些转基因小鼠的三角洲珠蛋白链合成。此外，我们还构建了几个框内嵌合转录分子。我们正在鉴定针对有缺陷的β-珠蛋白启动子的嵌合蛋白。这种新的转录因子将在细胞系、原代细胞和转基因小鼠中进行靶向Delta珠蛋白基因的测试。

We investigated the molecular mechanisms governing the low levels of expressed delta-globin gene in adult erythroid cells, which could facilitate the therapeutic manipulation of hemoglobin (HbA2) synthesis. Previously, we determined, by computer alignment, the presence of a mutated CP1 binding motif (CCAAT box) to CCAAC located at -70bp and the lack of a binding sequence for the erythroid specific factor EKLF at - 85bp, changes that have been conserved among primates and human delta- globin genes. Restoration of the CCAAT sequence at -70bp and/or insertion of a consensus EKLF binding site at -85bp position in a delta-promoter linked luciferase reporter system resulted in significantly increased activity in both K562 and MEL cells, as well as human primary adult erythroid cells (hAEC). Our in vitro results therefore implicated the mutated CCAAT box (CCAAC) and the lack of an EKLF binding site in the promoter at least partially responsible for the low level of delta-globin gene expression in adult erythroid cells at the transcriptional level. We also generated transgenic mice with EKLF binding site insertion in delta promoter sequence within the constructs containing locus control region and human beta- and delta- globin genes. These transgenics demonstrate a significant increased of human delta globin expression. Studies are underway to evaluate the delta globin chain synthesis in these transgenic mice. In addition, we haveconstructed several in-frame chimeric transcription molecules. We are in the process of identifying the chimeric protein which specifically targets the defective delta-globin promoter. The novel transcription factor will be tested for targeting delta globin gene in cell lines, primary cells and transgenic mice.