Spontaneous switch from Aγ- to β-globin promoter activity in a stable transfected dual reporter vector

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Abstract

Here it is analyzed the expression of a mini locus dual reporter construct composed by a micro-LCR and by the promoters for Aγ- and β-globin gene, each one linked to a different Luciferase, in stably transfected GM979 cells for as long as 1–2 years from transfection. The transfected GM979 cells rapidly (within 1 month) evolved into a stable population which expresses constant levels of reporters for more than a year of continuous bulk culture. No silencing of the inserted construct was observed over time. In contrast, after 1 month, the reporter activity (both from Aγ- and β-promoter) expressed per cell increased over time. The analysis of the Luciferase contained in single cell clones indicated that the higher reporter activity was due to increased gene expression per cell rather than to clonal selection of the most expressing clones. Since the activity driven by the β-promoter increased 10-fold more than that driven by the Aγ one, the ratio between Aγ-driven/(Aγ-driven + β-driven) reporter activity in the cells decreased after 1 month and became similar to the γ/(γ + β) globin mRNA ratio expressed by adult erythroid cells. Moreover, although both cells from early and late bulk culture responded to incubation with butyric acid, a known inducer of fetal globin gene expression, by increasing the reporter activity driven by the Aγ-promoter, only cells from late bulk culture decreased, as normal primary erythroblasts do, the activity of the reporter driven by the β-promoter. These results suggest that the rapid changes in activity driven by the Aγ- and β-globin promoters occurring during the first month after transfection may represent a novel in vitro model to study epigenetic regulation of the Aγ- and β-promoter during the fetal to adult hemoglobin switch and confirm GM979 cells stably transfected with the dual reporter construct as a reliable assay for automated screening of chemical inducers of fetal globin gene activation.

Introduction

Thalassemia is a severe genetic disease caused by coinheritance of defective α- or β-globin alleles resulting in deficient globin expression. In the case of β-thalassemia, the disease is due to deletion or abnormal regulation of the β-globin gene. The resulting inefficient β-globin expression leads to accumulation of unpaired, insoluble α-globin chains in red cells. The consequent accelerated destruction of these cells induces ineffective erythropoiesis and severe anemia. The development of transfusion therapies coupled with administration of iron-chelating agents has greatly ameliorated the treatment and the quality of life of β-thalassemia patients. The overall treatment of this disorder, however, is still unsatisfactory. In fact, while the incidence of the disease is steadily decreasing in western countries, it remains high in economically challenged countries that do not have easy access to the technologies required to properly manage these complex therapies. The observation that some of the homozygote carriers of β-globin gene deletions are asymptomatic because of persistent expression in adult life of the fetal-specific β-like genes, the γ-globin genes (High Persistence of Fetal Hemoglobin Syndrome, HPFH), has inspired numerous studies aimed to identify chemical substances capable to pharmacologically re-activate γ-globin expression in patients with β-thalassemia. One of the pharmacological agents identified so far is hydroxyurea. Phase I–II clinical trials with hydroxyurea on β-thalassemia [1] and sickle cell [2], [3] patients have been successfully completed, and the drug is presently considered to be introduced in the current medical practice for these disorders. Hydroxyurea, however, is a compound not devoid of counter indications [4]. Therefore, the search for additional and less toxic agents is still in progress [5].

The ideal candidate for pharmaceutical treatment of β-thalassemia is a cheap chemical that could be administered orally, would be effective within reasonable doses, and would present no or little counter indications. The efficiency of the search for these agents is strictly dependent on the availability of a surrogate assay to measure the potentiality to reactivate γ-globin gene expression in vivo. Such surrogate assays should be rapid, cheap, reliable, and suitable to be adapted for automated screening. Assays of induction of γ-globin gene expression that use as target cells human erythroblasts and that determine the cellular globin content by HPLC represent the tests that would most reliably mimic the possible effects a chemical might induce in vivo [6]. These assays, however, as precise and indicative of clinical efficacy as they might be, are cumbersome and not easily adaptable for automatic screening. More suitable for this purpose would be surrogate assays based on murine erythroleukemia cell lines transfected with reporter constructs containing regions of the human γ-globin promoter linked to appropriate reporter genes. The accuracy of these assays depends on how closely the promoter regions that drive the expression of the reporter gene mimic the physiology of γ-globin gene activation in primary erythroid cells. Recently, it has been developed an assay based on the murine erythroid GM979 cell line stably transfected with a construct containing a human mini globin locus composed by a micro-LCR and by the promoters for Aγ- and β-globin, each one linked to a different Luciferase [7], [8]. The ratio between the expression of the two Luciferases reflects the relative activity of the two promoters and can be measured easily in to be automated assays. The suitability of this assay, however, for automated screening of chemical inducers of globin gene expression depends on how stable and consistent is the expression of the reporter in transfected cells over time.

Here it is characterized the expression over time of the mini locus dual reporter construct in stably transfected GM979 cells, from the first month up to 1–2 years from transfection. The results obtained indicate that GM979 μLCRβprRlucAγprFluc cells undergo an initial period of stabilization after which they express high levels of Luciferase activity driven both by the Aγ- and β-promoter. The Aγ-driven/(Aγ-driven + β-driven) reporter activity ratio, that was similar to the fetal-globin ratio at early bulk cultures, became similar to adult globin gene ratio at later time points. Butyric acid, a known fetal hemoglobin inducer, increased expression of the reporter from the Aγ-promoter both in early and in late bulk cultures but decreased that from the β-promoter only in late bulk cultures. As such, GM979 μLCRβprRlucAγprFluc cells from late bulk cultures represent a reliable assay for automated screening of chemical inducers of globin gene activation.

Section snippets

Establishment of the GM979 μLCRβprRlucAγprFluc cell line

The murine erythroleukemic GM979 cell line had been established from fetal liver cells [9] and was chosen for this study because its phenotype includes expression both of minor and major Hb. The GM979 μLCRβprRlucAγprFluc cell line was obtained by lipofecting GM979 cells with a μLCRβprRlucAγprFluc plasmid that contains a 3.1-kb μLCR cassette including the DNAse I hypersensitive core of the 5′ hypersensitive sites HS1, HS2, HS3, and HS4, linked to 315-bp of the human β-globin promoter and 1.4-kb

Increased Luciferase activity per cell, both from Aγ- and β-promoter, in GM979 μLCRβprRlucAγprFluc cells over time

The amount of Aγ-F and β-R Luciferase activity expressed over time by GM979 μLCRβprRlucAγprFluc cells is presented in Fig. 1. The activity of both the Aγ-driven Firefly and the β-driven Renilla expressed by the cells progressively increased during the culture (Fig. 1). The amount of the β-driven Renilla Luciferase, however, increased much more than that of the Aγ-driven Firefly Luciferase. In fact, the average activity of Aγ-F Luciferase per cell increased by 2-fold (from 0.05 ± 0.02 AFU at 2

Discussion

To provide for a fast and reliable assay for chemical inducers of human HbF, the murine GM979 cell line was transfected with a dual Luciferase construct (μLCRβprRlucAγprFluc) [8] that allows direct comparison of the reporter activity driven by the human Aγ- and β-globin promoters [7], [11]. Here we show that the resulting GM979 μLCRβprRlucAγprFluc cell line maintains the ability to produce both Firefly and Renilla Luciferases over a period of more than 1 year of continuous bulk culture (Fig. 1,

Acknowledgments

The authors gratefully acknowledge Prof. Francesco Antonio Manzoli for continuous support. This study was supported by, Progetti di ricerca di Interesse Nazionale 2001 and 2002 from the Ministry of Health, MIUR 60% Grant 2002, Grant E.1172 from Telethon Foundation, Progetti FIRB 2002 and 2003, National Project on Stem Cells, Institutional Funds from Istituto Superiore di Sanità, and grant NIH-HL20899.

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