A 36-base-pair core sequence of locus control region enhances retrovirally transferred human beta-globin gene expression

Abstract

The locus control region of the human beta-globin cluster consists of four major DNase I hypersensitive sites (HS). When linked to globin genes, the locus control region confers a high level of erythroid-specific expression of these genes in transgenic mice or transfected erythroid cell lines. We have examined the effect of one of these sites, HS2, on human beta-globin gene expression in a murine erythroleukemia cell line (MEL) after retrovirus-mediated gene transfer. We incorporated a 732- or 412-base-pair (bp) segment of HS2 in the retroviral construct carrying the human beta-globin gene. These fragments rendered the viruses unstable as the human beta-globin gene was rearranged or deleted in all the packaging cell lines examined. On the other hand, when a 36-bp fragment containing the NFE-2/AP-1 binding consensus in this region was inserted into the retroviral construct, we recovered 6 stable packaging cell lines of 12 examined, similar in percentage to the construct with the beta-globin gene alone. The virus titers of the packaging cell lines from these two constructs were similar. We infected MEL cells with viruses produced from three packaging cell lines of each of the two constructs and measured the ratio of human beta-globin to mouse alpha-globin mRNA after hexamethylenebisacetamide induction. The overall level of expression increased 2-fold from 6.0% to 12.7% with the addition of this 36-bp enhancer.