We have been investigating the molecular mechanisms which govern the low level expressed delta-globin gene, the minor adult beta-like globin gene. It is our hope that this research will facilitate the therapeutic manipulation of hemoglobin synthesis. Previously, we showed, by computer alignment, the presence of a mutated CP1 binding motif CCAAT box (to CCAAC) located at -70bp and lack of a binding sequence for the erythroid specific factor EKLF at -85bp which is highly conserved among primate beta-globin genes. Restoration of the CCAAC sequence at -70bp (back to CCAAT) and/or insertion of a consensus EKLF binding site at the -85bp position in a delta-promoter linked luciferase reporter system resulted in an significantly increased luciferase activity in both K562 and MEL cells. We extended these studies into human primary adult erythroid cells (hAEC) and have shown similar effects due to the restoration of the two critical motifs (British Journal of Heamatology, in press). Our in vitro results 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 delta-globin gene expression in adult erythroid cells at transcriptional level. We also generated transgenic mice with CCAAT box restoration or EKLF binding site insertion in delta promoter sequence within constructs containing the locus control region and human beta- and delta globin genes to determine the in vivo effect of the restoration of EKLF binding site and an intact CP1 binding motif on the delta globin gene in the context of the locus control region and beta-globin gene. Studies are underway to evaluate the delta globin gene expression and delta globin chain synthesis in these transgenic mice. In addition, in collaboration with Drs. J. Beiker (Mt. Sinai, N.Y.) and C. C. Trainor (NIDDK), we have constructed a series of in-frame chimeric transcription molecules which we have authenticated by in vitro transcription/translation. We are in the process of screening these chimeric proteins for their ability to specifically target the defective delta-globin promoter and thereby augment its activity.
