Abstract
Natural deletions of the human gamma-globin gene cluster lead to specific syndromes characterized by increased production of fetal hemoglobin in adult life and provide a useful model to delineate novel cis-acting elements involved in the developmental control of hemoglobin switching. A hypothesis accounting for these phenotypic features assumes that silencers located within the Agamma-to delta-gene region are deleted in hereditary persistence of fetal hemoglobin (HPFH) and deltabeta-thalassemias, leading to failure of switching. In the present study, we sought to clarify the in vivo role of two elements, termed Enh and F, located 3' to the Agamma-globin, in silencing the fetal genes. To this end, we generated three transgenic lines using cosmid constructs containing the full length of the globin locus control region (LCR) linked to the 3.3-kb Agamma-gene lacking both the Enh and F elements. The Enh/F deletion resulted in high levels of Agamma-globin gene expression in adult mice in all single copy lines, whereas, the LCR-Agamma single copy lines which retain the Enh and F elements exhibited complete normal switching of the fetal Agamma-gene. Our study documents directly for the first time the in vivo role of these two gene-proximal negative regulatory elements in silencing the fetal globin gene in the perinatal period, and thus these data may permit their eventual exploitation in therapeutic approaches for thalassemias.
Highlights
Human β-globin gene expression is regulated tightly during development and hematopoiesis
Since the only difference between the locus control region (LCR)-Aγ-ΔEnh/F versus the LCR-Aγ constructs is the selective absence or presence of the silencers, respectively, these findings directly demonstrate that the absence of Enh and F elements in the context of the LCRcontaining transgene can alter the developmental expression of the Aγ-gene in adult blood and leads to its persistent and efficient expression
The δβ and Aγδβ-thalassemias and the deletion forms of hereditary persistence of fetal hemoglobin (HPFH) are naturally occurring mutations associated with persistent expression of fetal hemoglobin in adult life, albeit at variable levels
Summary
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]. The human globin genes undergo two developmental switches in their activation. The second switch occurs gradually around birth with the activation of the adult stagespecific δ- and 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
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