Functional properties of the beta-globin locus control region in K562 erythroleukemia cells

Abstract

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.

Structure and function of the murine β-globin locus control region 5′ HS-3

“…A series of nine DNA fragments obtained from this region were isolated and subcloned into pUC9; a 2.7kb BamHI fragment containing the ,y-neo transcription unit was inserted into each plasmid. The 'yneo transcription unit is in a variety of positions and orientations with respect to each test fragment, but 5' HS-2 has been shown to function in all orientations and positions in transient and stable assays performed in K562 cells (22,24). Each of these plasmids was transfected into K562 (embryonic/fetal erythroid) or MEL (adult erythroid) cells, and colony assays were performed.…”

“…The LCR confers integration site-independent, copy numberdependent expression on linked globin genes in transgenic mice (7)(8)(9)(10)(11)(12)(13)(14)(15) and in erythroid tissue culture cells (8, 11, 12, 14, 16 -18). The LCR confers inducibility on linked globin and TK promoters in mouse erythroleukemia cells (8,11,14,(16)(17)(18), and likewise confers hemin inducibility on many linked promoters in K562 erythroleukemia cells (19)(20)(21)(22)(23)(24). Finally, the LCR seems to increase the probability that chromatin integration events will permit active expression of linked promoters, perhaps by isolating them from negative regulatory influences that lie nearby (7 -15, 22, 24).…”

“…Many LCR properties are contained within the individual DNAse I hypersensitive sites (HS) that collectively comprise the LCR. For example, human 5' HS-2 (located 10.9 kb upstream from the e-globin gene cap site) activates linked globin genes in transgenic mice (9,10,(12)(13)(14)(15), behaves as a transient enhancer in K562 cells (19)(20)(21)(22)(23)(24)(25)(26), confers inducibility on linked globin genes in K562 and MEL cells (12,17,(18)(19)(20)(21)(22)(23)(24), and increases productive integration events in K562 cell colony assays (22,24). Human 5' HS-3 alone is also active in transgenic mice (10, 11) and in assays where it is stably integrated into the chromatin of MEL cells (11,16,18).…”

“…Human 5' HS-3 alone is also active in transgenic mice (10, 11) and in assays where it is stably integrated into the chromatin of MEL cells (11,16,18). However, this site does not act as a transient enhancer in K562 cells (24,25), and it is inactive in K562 cell chromatin (20,24). Fundamental differences must therefore exist between the activities and functions of 5' HS-2 and 5' HS-3.…”

Dependence of Globin Gene Expression in Mouse Erythroleukemia Cells on the NF-E2 Heterodimer

“…Three types of DNA-binding sequences, GATA, Sp1 (GTGG), and AP-1-or NF-E2 (nuclear factor-erythroid 2)-like motifs, are consistently found within the core elements of the DNase I-hypersensitive sites (21,34,36,37,41,42,44,45). Previous studies have associated the GATA and Sp1 (GTGG) sites with position-independent activation (44), whereas the overall enhancer activity of the LCR requires the integrity of tandem AP-1-like sequences of ␤-LCR HS-2 (27,28,31,32,44,45). Similarly, enhancer activity of the ␣-locus HS-40 element is also dependent on AP-1-NF-E2 sequences (35).…”