REGULATION OF HUMAN DELTA GLOBIN GENE EXPRESSION

点击翻译按钮获取中文摘要

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.