Human β-globin locus control region hypersensitive site specificity for globin gene activation during erythropoiesis

Although the human β-globin locus control region (LCR) functions as a holocomplex within an active chromatin hub, we provide evidence that within the aggregate hypersensitive site (HS) activation domain of the holocomplex, the individual HSs still mediate preferential activation of the globin genes during development. A 2.9 Kb deletion of 5′HS3 (Δ5′HS3) or a 234 bp deletion of the 5′HS3 core (Δ5′HS3c) in a 213 Kb human β-globin locus yeast artificial chromosome (β-YAC) abrogate e-globin gene expression during primitive erythropoiesis in β-YAC transgenic mice, suggesting that 5′HS3 sequences of the LCR are involved directly in e-globin gene activation. The reduction of e-globin gene transcription in Δ5′HS3 or Δ5′HS3c β-YAC transgenics can be explained by two hypotheses. The first is site-specificity. The interaction between the LCR and the e-globin gene promoter involves specific sequences of 5′HS3 and specific sequences of the e-globin gene promoter. When 5′HS3 or its core is deleted, these interactions do not take place and e-globin gene transcription is diminished. The second hypothesis is change in conformation of the LCR. Normally, in the embryonic stage, the LCR achieves a three-dimensional conformation that favors interaction with the first gene in the complex, the e-globin gene. When 5′HS3 is deleted, an alternate conformation is assumed that decreases the chance that there will be an interaction between the LCR and the e-globin gene. However, the LCR interacts with the next gene in order, the γ-globin gene. In Δ5′HS3c β-YAC mice, γ-globin gene expression is normal during primitive erythropoiesis, but is extinguished in the fetal stage of definitive erythropoiesis. These data suggest that a conformational change occurs in the Δ5′HS3c LCR during the switch from embryonic to definitive erythropoiesis, from one that supports γ-globin gene expression to one that does not. Alternately, the embryonic trans-acting environment may allow the mutant LCR to interact with and activate the γ-globin genes, but the fetal trans-acting environment may not support this interaction in the absence of the 5′HS3 core. To distinguish between these possibilities, β-YAC lines were produced in which the e-globin gene was replaced with a second marked β-globin gene (βm), coupled to either an intact LCR, a 2.9 Kb 5′HS3 deletion or a 234 bp 5′HS3 core deletion. Δ5′HS3c Δe::βm β-YAC mice expressed βm-globin throughout development beginning at day 10 in the yolk sac. γ-globin was expressed in the embryonic yolk sac, but not in the fetal liver. Some wild-type β-globin was expressed in addition to βm-globin in adult mice. The γ-globin phenotype is consistent with published data on Δ5′HS3c β-YAC mice. Although e-globin was not expressed in Δ5′HS3c β-YAC mice, βm-globin was expressed in Δ5′HS3c Δe::βm β-YAC embryos, demonstrating that the 5′HS3 core was necessary for e-globin expression during embryonic erythropoiesis, but not for βm-globin expression. A similar phenotype was observed in Δ5′HS3 Δe::βm β-YAC mice, except βm-globin expression was higher in the day 10 yolk sac and γ-globin expression continued into the fetal liver stage of definitive erythropoiesis consistent with results published on Δ5′HS3 β-YAC mice. These data support a site specificity model of LCR HS-globin gene interaction.