Functional properties of the beta-globin locus control region in K562 erythroleukemia cells.
In this report, we compare the function of the human beta-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 gamma-globin promoters. Various LCR fragments were inserted into an expression vector consisting of an A gamma-globin promoter driving the neomycin phosphotransferase gene (gamma-neo). Using these vectors, we determined that a 2.5-kb DNA fragment containing LCR sites I through IV (previously named mu locus activation region [mu LAR]) 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 approximately 10% as much colony assay activity as the entire mu LAR. 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 gamma-globin promoters in stably transfected cells. Finally, we tested the ability of the mu LAR to activate promoters (beta-globin and cathepsin G) that are not normally expressed in K562 cells. beta-neo was minimally activated by the mu LAR in transient transfection experiments. The mu LAR increased the number of stably transfected colonies produced by beta-neo, but the absolute number of beta-neo colonies, with or without the mu LAR, was approximately 10% to 20% that of gamma-neo. In contrast, a minimal cathepsin G promoter was activated by the mu LAR 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.
Moon
A M
AM
Department of Medicine, Jewish Hospital, Washington University Medical Center, St Louis, MO 63110.
Ley
T J
TJ
D001483:Base Sequence; D001609:Beta-Globulins / Q000235:genetics* / Q000502:physiology; D002460:Cell Line; D002874:Chromosome Mapping; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D019868:Kanamycin Kinase; D004915:Leukemia, Erythroblastic, Acute / Q000097:blood / Q000235:genetics*; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics* / Q000502:physiology; D014162:Transfection / Q000235:genetics
D001609:Beta-Globulins; D010770:Phosphotransferases; D019868:Kanamycin Kinase