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 used the computer assisted alignment program (yama2) to compared the primate globin gene sequences. Not only the presence of CCAAC box located at -70bp, but also no evidence of the CACCC box (core sequence for the beta globin EKLF binding factor) at -85bp was found in this region, though this region remains highly conserved among the primates. A luciferase construct containing the -612 to +68bp region of the delta globin promoter was used to show a low level transcription in the transient assay system. We then mutated the CCAAC sequence at -70bp to CCAAT thereby restoring the CP1 binding motif as found in the beta globin promoter. The luciferase activity increased 5.4 fold and 2.4 fold in human K562 cells and murine MEL cells, respectively. We also created a consensus EKLF binding site at -85bp position in the wildtype construct and the luciferase activity increased by 28.6 fold and 6.5 fold in K562 and MEL cells, respectively. Besides the studies in established cell lines, we also investigated in primary adult erythroid cells and confirmed the similar effects of this two regions. Our results implicate the mutated CCAAT box (CCAAC) and the lack of a EKLF binding site in the promoter at least partially responsible for the low level delta-globin gene expression in adult erythroid cells at the transcriptional level. We have been engineering transgenic constructs to study the CCAAC box and EKLF binding site in vivo. Studies will be underway to evaluate delta globin gene expression and delta globin chain synthesis in transgenic mice.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Intramural Research (Z01)
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