Development of sensitive fluorescent assays for embryonic and fetal hemoglobin inducers using the human β-globin locus in erythropoietic cells

Abstract

Reactivation of fetal hemoglobin genes has been proposed as a potential therapeutic procedure in patients with ␤-thalassemia, sickle cell disease, or other ␤-hemoglobinopathies. In vitro model systems based on small plasmid globin gene constructs have previously been used in human and mouse erythroleukemic cell lines to study the molecular mechanisms regulating the expression of the fetal human globin genes and their reactivation by a variety of pharmacologic agents. These studies have led to great insights in globin gene regulation and the identification of a number of potential inducers of fetal hemoglobin. In this study we describe the development of enhanced green fluorescence protein (EGFP) reporter systems based on bacterial artificial chromosomes (EBACs) to monitor the activity of the ⑀-, G ␥-, A ␥-, ␦-, and ␤-globin genes in the ␤-globin locus. Additionally, we demonstrate that transfection of erythroleukemia cells with our EBACs is greatly enhanced by expression of EBNA1, which also facilitates episomal maintenance of our constructs in human cells. Our studies in human cells have shown physiologically relevant differences in the expression of each of the globin genes and also demonstrate that hemin is a potent inducer of EGFP expression from EGFPmodified ⑀-, G ␥-, and A ␥-globin constructs. In contrast, the EGFP-modified ␦-and ␤-globin constructs consistently produced much lower levels of EGFP expression on hemin induction, mirroring the in vivo ontogeny. The EGFP-modified ␤-globin eukaryotic BAC (EBAC) vector system can thus be used in erythroleukemia cells to evaluate induction of the ⑀-and ␥-globin genes from the intact human ␤-globin locus. Introduction␤-Thalassemia and sickle cell disease are among the most common inherited genetic disorders in the world. In ␤-thalassemia, unpaired ␣-globin chains accumulate and precipitate within erythroid cells, resulting in red cell damage, hemolysis, ineffective erythropoiesis, and anemia. In sickle cell disease, intracellular accumulation of polymerized hemoglobin causes sickling of red blood cells and vascular occlusion. Amelioration of clinical symptoms associated with these hemoglobinopathies has been reported in patients with elevated levels of ␥-globin chain synthesis. In ␤-thalassemia, the presence of ␥-globin inhibits the precipitation of unpaired ␣-globin through the formation of fetal hemoglobin (HbF, ␣ 2 ␥ 2, ), whereas in sickle cell disease the presence of ␥-globin inhibits the polymerization of sickle hemoglobin (␣ 2 ␤ 2 s ) via the formation of mixed hemoglobin tetramers (␣ 2 ␤ s ␥) and the maintenance of a higher oxygen tension in the capillaries. 1 A diverse group of genetic mutations termed hereditary persistence of fetal hemoglobin (HPFH) are associated with high levels of HbF in adult life. Coexistence of HPFH with homozygous ␤-thalassemia or sickle cell disease often results in complete phenotypic complementation of the disease. 2 Many of the HPFH mutations are the result of deletions or point mutations in the ␤-globin locus. 3 However, a ...

Pharmacological Induction of Human Fetal Globin Gene in Hydroxyurea-Resistant Primary Adult Erythroid Cells

“…1A). Consistent with previous findings (31,32), the expression of the ␥ globin promoter-directed DsRed fluorescence was detectable only in the human fetal/embryonic erythroid K562 cells but not in mouse adult erythroid MEL cells (top two rows of panels, Fig. 1B).…”

Editing the genome to introduce a beneficial naturally occurring mutation associated with increased fetal globin

“…Only samples with efficiencies 475% were considered for analysis. A BAC containing the entire human b-globin locus (pEBAC G gdsREDbEGFP) 39 was digested with HindIII and religated to generate random ligation products of HindIII fragments for transgenic MEL cell experiments ( Supplementary Fig. 6a).…”

Site-specific, Rep-mediated integration of the intact β-globin locus in the human erythroleukaemic cell line K562

“…In previous studies conducted by our group, hemin and cisplatin have been shown to induce g-globin gene expression while having a minimal effect on b-globin gene expression K562 cells. 39,40 Importantly, following induction of the clonal cell lines carrying the dual reporter constructs, a significant increase in dsRed expression driven by the A g and G g globin promoters was observed, whereas expression from the b-globin promoter consistently produced low levels of EGFP. This result provides further evidence that the transgene expression from the AAVS1-integrated b-globin locus mirrors the expected developmental, stage-specific, globin gene expression profile of normal K562 cells.…”

“…43 Versions of this BAC clone, G g-A g-EGFP and b-EGFP, which contain the EGFP reporter gene in place of either the g-globin or b-globin genes respectively, have also been previously described. 39,44 In this study, b-EGFP was modified further by replacing the G g-or A gglobin gene with the dsRed reporter gene to generate 'dual reporter' BAC clones. Using homologous recombination, the g-globin genes were replaced with a DNA targeting cassette encoding the dsRed and ampicillin resistance (Amp r ) genes.…”