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First published online June 16, 2005
Journal of Experimental Biology 208, v (2005)
Copyright © 2005 The Company of Biologists Limited
doi: 10.1242/jeb.01698

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Outside JEB

A SWITCH FOR SILENCING -GLOBIN

Lars Tomanek

University of California Davis

ltomanek{at}ucdavis.edu

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. -globin is expressed in red blood cells during the early stages of human foetal development and is later replaced by ß-globin during the definitive adult stage of red blood cell maturation. During this definitive stage, when the expression of -switches to ß-globin, the silencing of the -globin and activation of the ß-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 -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 -globin repressors: a direct repeat (DR) sequence and a CCTTG repeat. Both DR and CCTTG have been proposed to suppress -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 suppressing -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 ß-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 for -globin gene silencing, mice with the DR mutation would not be able to silence the -globin gene, so the team should see -globin expression in the mutDR mice. But if the CCTTG sequence silences the -globin gene, they expected to see -globin expression in the mutCCTTG mice. To monitor expression of the human -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 -globin gene expression in foetal and adult mutant transgenic mice and compared these with -globin gene expression in wild-type mice.

Omori's team found that -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 the -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 -globin expression in the definitive adult stage of red blood cell maturation. By contrast, the team saw no increase in -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 -globin expression. The DR element is therefore a crucial switch for the expression of adult-type haemoglobin in humans.

References

Omori, A., Tanabe, O., Engel, D. E., Fukamizu, A. and Tanimoto, K. (2005). Adult stage -globin silencing is mediated by a promoter direct repeat element. Mol. Cell. Biol. 25,3443 -3451.[Abstract/Free Full Text]

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This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Tomanek, L. Search for Related Content PubMed Articles by Tomanek, L. Social Bookmarking                         What's this?