Regulation Of Human Delta Globin Gene Expression

我们研究了控制低水平表达的δ-珠蛋白基因的分子机制，这可能有助于血红蛋白A2（HbA 2）合成的治疗操作。在此之前，我们证明，恢复的CCAAT序列在？70 bp和/或在δ启动子连接的荧光素酶报告系统中在-85bp位置插入共有EKLF结合位点导致K562和MEL细胞以及人原代成体红系细胞（hAEC）中的活性显著增加。因此，我们的体外研究结果暗示突变的CCAAT盒（CCAAC）和启动子中EKLF结合位点的缺乏至少部分地导致了成人红系细胞中d-珠蛋白基因在转录水平上的低水平表达。我们还产生了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EKLF结合位点的转基因小鼠。我们正在开发一种新的实时PCR定量δ-珠蛋白表达水平的具体分析。正在进行研究以评估这些转基因小鼠中δ珠蛋白链的合成。此外，我们构建了五个框内嵌合转录分子，其含有来自EKLF的反式激活结构域与来自EKLF的两个锌指和来自加塔-1的一个锌指连接。其中一个嵌合构建体与δ-荧光素酶报告基因构建体共转染到K562细胞中，使报告基因活性增加3-4倍。我们将检测具有不同接头序列组合的嵌合蛋白的结合亲和力，并鉴定可以靶向缺陷型d-珠蛋白启动子的特异性融合蛋白。将在细胞系和原代细胞以及转基因小鼠中测试这种新的转录因子是否靶向δ珠蛋白基因。

We investigated the molecular mechanisms governing the low level expressed delta-globin gene, which could facilitate the therapeutic manipulation of hemoglobin A2 (HbA2) synthesis. Previously, we demonstrated that 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 implicate the mutated CCAAT box (CCAAC) and the lack of an EKLF binding site in the promoter at least partially responsible for the low level d-globin gene expression in adult erythroid cells at 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. We are developing a new assay with real-time PCR to quantitate the delta-globin expression level specifically. Studies are underway to evaluate the delta globin chain synthesis in these transgenic mice. In addition, we constructed five in-frame chimeric transcription molecules containing a transactivation domain from EKLF linked with two zinc-fingers from EKLF and one zinc-finger from GATA-1. One of the chimeric constructs, cotransfected with delta-luciferase reporter construct into K562 cells, increased the reporter activity by 3-4 fold. We will assay the binding affinity of the chimeric proteins with different combination of linker sequences and identify the specific fusion protein that can target the defective d-globin promoter. This novel transcription factor will be tested for targeting delta globin gene in cell lines and primary cells as well as transgenic mice.