The switch from foetal to adult expression of the globin genes that encode the subunits of haemoglobin is the textbook example of developmentally regulated gene expression. γ-globin is expressed in red blood cells during the early stages of human foetal development and is later replaced byβ-globin during the definitive adult stage of red blood cell maturation. During this definitive stage, when the expression of γ-switches toβ-globin, the silencing of the γ-globin and activation of theβ-globin genes appear to be regulated autonomously by a promoter element on chromosome 11 in humans. But exactly which genetic element regulates this change in gene expression was still unclear.
To identify the switch for γ-globin silencing, Omori and colleagues from the Universities of Tsukuba and Michigan tested the potential silencing role of two human regulatory promoter elements. Earlier work had identified these two putative γ-globin repressors: a direct repeat (DR) sequence and a CCTTG repeat. Both DR and CCTTG have been proposed to suppressγ-globin transcription by recruiting a repressor protein, which directly interferes with the binding of a globin transcription activator.
To test which of these two promoter elements is responsible for suppressingγ-globin in vivo, Omori's team created and incorporated mutated forms of these elements (mutDR and mutCCTTG) into a yeast artificial chromosome, which included a large stretch of the human β-globin cluster. They created transgenic mice that carried these mutated human DR and CCTTG sequences by injecting the artificial chromosome into fertilized mouse oocytes. The team reasoned that, if the DR sequence is responsible forγ-globin gene silencing, mice with the DR mutation would not be able to silence the γ-globin gene, so the team should see γ-globin expression in the mutDR mice. But if the CCTTG sequence silences theγ-globin gene, they expected to see γ-globin expression in the mutCCTTG mice. To monitor expression of the human γ-globin gene as the mutant transgenic mice developed, the team extracted total RNA from the foetal yolk sac, foetal liver and adult spleen or peripheral blood for each of the transgenic lines. Using the extracted RNA and semi-quantitative RT-PCR analysis, the team assessed γ-globin gene expression in foetal and adult mutant transgenic mice and compared these with γ-globin gene expression in wild-type mice.
Omori's team found that γ-globin expression in adult spleen or peripheral blood was dramatically increased in mutDR mice compared with wild-type animals, which indicates that the DR sequence silences theγ-globin gene. They did not observe the same up-regulation in the foetal yolk sac in mutDR mice, demonstrating that the mutation of the DR element only affects γ-globin expression in the definitive adult stage of red blood cell maturation. By contrast, the team saw no increase in γ-globin expression in adult mutCCTTG mice. Omori and his colleagues conclude that, for adult definitive red blood cells, the DR element alone is the major silencer of γ-globin expression. The DR element is therefore a crucial switch for the expression of adult-type haemoglobin in humans.