Functional Properties of the β-Globin Locus Control Region in K562 Erythroleukemia Cells

Abstract

In this report, we compare the function of the human β-globin locus control region (LCR) in three K562 erythroleukemia cell assays, including (1) a transient transfection assay for “classical” enhancer activity, (2) a colony assay that detects “productive integration events,” and (3) an assay that detects the ability of LCR fragments to confer hemin inducibility on linked, stably integrated γ-globin promoters. Various LCR fragments were inserted into an expression vector consisting of an Aγ-globin promoter driving the neomycin phosphotransferase gene (γ-neo). Using these vectors, we determined that a 2.5-kb DNA fragment containing LCR sites I through IV (previously named μ locus activation region [μLAR]) had activity in all three assays; of the individual LCR sites, only site II was highly active in all three assays. One region within site II, consisting of tandem AP-1/NF-E2 consensus elements, had ~ 10% as much colony assay activity as the entire μLAR, However, this region did not have detectable activity in a transient enhancer assay in uninduced K562 cells, nor was it capable of conferring hemin inducibility on linked γ-globin promoters in stably transfected cells. Finally, we tested the ability of the μLAR to activate promoters (β-globin and cathepsin G) that are not normally expressed in K562 cells, β-neo was minimally activated by the μLAR in transient transfection experiments. The μLAR increased the number of stably transfected colonies produced by β-neo, but the absolute number of β-neo colonies, with or without the μLAR, was approximately 10% to 20% that of γ-neo. In contrast, a minimal cathepsin G promoter was activated by the μLAR in K562 cells. Our results suggest that LCR functions are dependent in part on the environments and the promoters with which the LCR is tested.