Persistent Fetal γ-Globin Expression in Adult Transgenic Mice following Deletion of Two Silencer Elements Located 3′ to the Human Aγ-Globin Gene

Human β-globin gene expression is regulated tightly during development and hematopoiesis. The human β-globin locus comprises five developmentally regulated genes (5′-ε-Gγ-Aγ-δ-β-3′) whose high level and stage-specific expression depends on interactions with the locus control region (LCR), consisting of five major DNaseI hypersensitive sites (Figure 1). The LCR activates β-globin gene transcription through direct interaction with promoter regions (1,2), and is a major determinant of the chromatin structure of the locus (3). Mice transgenic for the human β-globin locus express the human genes in a developmentally regulated manner (4,5). Specifically, the human globin genes undergo two developmental switches in their activation. Expression of the embryonic εand fetal γ-globin genes, first activated during primitive erythropoiesis in the embryonic yolk sac, switches to expression of the γ-genes at the start of definitive erythropoiesis in the fetal liver, with a small contribution by β-globin. The second switch occurs gradually around birth with the activation of the adult stagespecific δand β-globin genes, with δ-globin making a minor contribution, whereas γ-globin expression is gradually suppressed to very low levels (1%–2%) by the end of the first year of life. The individual genes have been shown to be regulated by a complex interplay between cis regulatory elements, transacting factors, enhancer competition, and epigenetic mechanisms (6). Despite a remarkable amount of progress in this field, the exact mechanism(s) of globin gene silencing still is not understood completely. Understanding the molecular basis of globin gene switching and discovery of strategies to efficiently express γ-globin genes in the adult is of particular interest, since reactivation of the fetal γ-globin genes in the adult has been shown to ameliorate the effects of hemoglobinopathies (7). A series of well established quantitative trait loci accounting for 20%–50% of the fetal hemoglobin (HbF) variability in healthy adults, such as: a) the C→T single nucleotide polymorphism at position –158 of the Gγ-gene, creating a restriction site for the enzyme XmnI (8);