Abstract
The human Gγ-globin and Aγ-globin genes differ by the presence of a single amino acid, either glycine or alanine, at position 136. The ratio of Gγ/Aγ-globin expression is approximately 7/3 at birth and changes to 2/3 in the adult. The mechanism responsible for this developmental switch is unknown. In the baboon, the duplicated γ-globin genes differ by the presence of a single amino acid at position 75. The Iγ-globin gene contains isoleucine at position 75, while the Vγ-globin gene contains valine at this position. The ratio of expression of the Iγ and Vγ-globin chains also differs in the fetal and adult stages. The Iγ/Vγ ratio is 3/2 in the fetus and 2/3 in the adult. Thus the pattern of expression of the baboon Iγ-globin gene is analogous to the human Gγ-globin gene, and that of the Vγ-globin gene is analogous to the human Aγ-globin gene. During stress erythropoiesis, moderately increased HbF levels are observed (5–10% HbF) and the Iγ/Vγ-globin chains are expressed in the characteristic adult ratio. Decitabine treatment reactivates HbF expression to high levels (50–70% HbF) and Iγ/Vγ ratios of approximately 1:1 have been observed following decitabine treatment. Thus decitabine treatment alters the Iγ/Vγ ratio but does not cause a complete reversion to the fetal pattern of expression. HbF is also reactivated to high levels in cultured baboon BFUe. In this investigation the pattern of expression of the Iγ- and Vγ-globin genes in cultured baboon CD34+ bone marrow (BM) cells was analyzed to determine whether reactivation of HbF in culture was associated with a change in the pattern of expression of the Iγ-and Vγ-globin genes. CD34+ cells were enriched from baboon BM using the 12.8 monoclonal antibody in combination with immunomagnetic microbead columns (Miltenyi) and cultured in Iscove’s media supplemented with 30% fetal bovine serum, stem cell factor (SCF; 200ng/ml), erythropoietin (EPO; 2U/ml), and dexamethasone (Dex; 1μM). The pattern of globin chain expression in d12 cultures, cord blood (CB) of a 58d fetus, and peripheral blood (PB) of adult baboons following phlebotomy and decitabine treatment was compared by HPLC analysis of hemolysates. The baboon 58d CB contained >90% HbF and the ratio of Iγ/Vγ was 1.85. In the adult (phlebotomized) PB the level of HbF was 8.1% and the Iγ/Vγ ratio was 0.75 thus confirming that the ratio of the baboon Iγ and Vγ-globin chains differs in the fetal and adult stages of development in a manner similar to that of the human Gγ and Aγ-globin chains. Following decitabine treatment (PA 7002) an HbF level of 55% was attained with an Iγ/Vγ ratio of 1.1. Hemolysates prepared from d12 cultures of CD34+ baboon (PA 7002) BM cells grown in the presence of SCF, EPO, and Dex contained 57.6% HbF, nearly the same level observed following decitabine treatment in vivo. The Iγ/Vγ ratio was 1.94, markedly different from that observed in this same baboon following decitabine in vivo and, moreover, nearly identical to the fetal ratio. Thus HbF reactivation in cultured adult baboon CD34+ BM cells was associated with a change in the ratio of expression of the two baboon γ-globin genes to that characteristic of the fetal stage. Recapitulation of the fetal pattern of γ-globin chain expression in cultured baboon CD34+ progenitors demonstrates yet another advantage of the baboon model for investigations of hemoglobin switching.
Essential role of NF-E2 in remodeling of chromatin structure and transcriptional activation of the epsilon-globin gene in vivo by 5' hypersensitive site 2 of the beta-globin locus control region.