REGULATION OF HUMAN DELTA GLOBIN GENE EXPRESSION

人类珠蛋白基因表达的调节

• 批准号: 6289738
• 负责人: GRIFFIN P. RODGERS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6289738
• 关键词: cell line; chimeric proteins; gene expression; genetic promoter element; genetic regulation; genetic regulatory element; genetic transcription; genetically modified animals; globin; laboratory mouse; nucleic acid sequence; reporter genes; transcription factor

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.

我们研究了控制成人红系细胞中低水平表达的δ珠蛋白基因的分子机制，这可以促进血红蛋白（HbA2）合成的治疗操作。之前，我们通过计算机比对确定了位于 -70bp 处的 CCAAC 的突变 CP1 结合基序（CCAAT 盒）的存在，以及在 -85bp 处缺乏红细胞特异性因子 EKLF 的结合序列，这些变化在灵长类动物和人类 δ-珠蛋白基因中是保守的。在δ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位置处插入共有EKLF结合位点，导致K562和MEL细胞以及人原代成体红细胞（hAEC）的活性显着增加。因此，我们的体外结果暗示突变的CCAAT盒（CCAAC）和启动子中缺乏EKLF结合位点至少部分地导致了成体红细胞中转录水平上的δ-珠蛋白基因表达水平低。我们还产生了转基因小鼠，其在含有基因座控制区和人β-和δ-珠蛋白基因的构建体内的δ启动子序列中插入了EKLF结合位点。这些转基因表明人类δ珠蛋白表达显着增加。目前正在进行研究评估这些转基因小鼠的 δ 珠蛋白链合成。此外，我们还构建了几种框内嵌合转录分子。我们正在鉴定专门针对有缺陷的δ珠蛋白启动子的嵌合蛋白。该新型转录因子将在细胞系、原代细胞和转基因小鼠中针对δ珠蛋白基因进行测试。