It was recently demonstrated that erythropoietin combined with stem cell factor and transforming growth factor beta (EST; EPO+SCF+TGF-B) signals a pancellular reversal of gamma-globin gene silencing among ex vivo cultures of CD34+ cells when compared to cultures supplemented with erythropoietin (EPO) alone (

Bhanu et al.,
Blood.
2005
;
105
(1):
387
–93
). This primary human erythroblast culture model is now being utilized to study the molecular basis of globin gene regulation. In this study, we sought to determine whether EST-signaled effects are restricted to the gamma-globin gene(s). For this purpose, quantitative PCR (QPCR) assays were developed for the alpha-globin (zeta, mu, alpha, and theta) and beta-globin (epsilon, gamma, delta, and beta) gene clusters. EPO versus EST matched cultures from a total of 15 donors (3 single donors and two pools, 6 donors each) were investigated using culture day 7 proerythroblasts and day 14 hemoglobinized precursors. The QPCR products were quantified by comparison with plasmid-generated standard curves (20,000,000 to 200 copies). The patterns of mu-, alpha- and theta-globin gene expression were unchanged under all culture conditions. The delta- and beta-globin transcripts showed a significant reduction in levels in response to EST. In contrast, zeta-, epsilon- and gamma-globins all demonstrated significant increases in response to EST. The increases were consistently detected in the individual and pooled samples. The most dramatic increase in the zeta-, epsilon- and gamma-globins occurred at the proerythroblast stage of differentiation. On day 7, zeta-globin levels showed 190-fold increase (EPO: 6.9E+01±5.5E+01;EST: 1.3E+04±7.7E+03; p=0.01), epsilon-globin levels, a 27-fold increase (EPO: 1.0E+03±5.8E+02; EST: 2.7E+04±1.1E+04; p=0.001), and gamma-globin levels, a 13-fold increase (EPO: 9.1E+05±4.1E+05; EST: 1.2E+07±1.8E+06; p=0.001). Consistent with the 3–4 log difference between the levels of zeta- and epsilon-, versus gamma-globin copy numbers and the low sensitivity of HPLC compared with QPCR, no embryonic proteins were detected by reverse-phase HPLC (day 14 samples studied; 9 donors). However, G-gamma and A-gamma globin peaks confirmed EST-signaled activation of both gamma-globins genes (G-gamma/A-gamma= 1.0 in EPO versus 1.9 in EST; (total gamma)/(total gamma + beta-globin) = 3.7±2.4% in EPO versus 42.4 ±5.8% in EST; p=1.6E-08). These novel data suggest that cytokines can reverse the coordinated developmental silencing of human embryonic and fetal genes in both globin loci.

Topics:
embryo, erythroblasts, fetus, genes, globins, gamma-globins, beta-globin, erythropoietin, high pressure liquid chromatography procedure, alpha-globin

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2005, The American Society of Hematology
2005
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