Demonstration of a Human €-Globin Gene Silencer With Studies in Transgenic Mice

HE HUMAN p-GLOBIN locus contains five funcT tional genes arranged 5' E-Gy-Ay-6-P 3' in the order of their expression during development.' Six to 18 kb upstream of the eglobin gene are a series of four DNase I hypersensitive sites that are erythroid specific and developmentally stable, ie, present regardless of which specific globin genes are active.'z3 Reverse genetics experiments have shown that the region containing these sites (the locus control region [LCR]) confers high-level erythroid-specific expression on cis-linked genes. The LCR effect is dominant; aand @-globin genes, housekeeping (TK) and even nonerythroid genes (THY-l), respond?* The dominant role of LCR raises the question of how the E-, y-, or p-globin genes are silenced at the developmental stages in which these genes are normally quiescent. In the absence of the LCR human yand @-globin genes are properly regulated in transgenic mice, ie, the y gene is expressed in primitive cells while the p gene in definitive erythroid cells. In contrast, transgenic mice containing a human fetal (y) or adult (p) globin gene linked to the LCR express the gene throughout development.'-" Developmental regulation is restored in LCR constructs that contain both the yand p-globin genes, suggesting a reciprocal mechanism for fetal to adult globin gene switching whereby the yand p-globin genes compete for the LCR."." In the absence of the LCR, the human e-globin gene is not expressed in transgenic However, when the human e-globin gene is linked to the LCR, it is expressed in a developmentally regulated fashion in transgenic mice, ie, it is expressed in embryonic cells derived from the yolk sac but not in definitive erythroid cells derived from the fetal l i ~ e r . ' ~ * ~ The cellular pattern of LCR-E expression in transgenic mice is the same as that of the native €-globin gene in human, implying that, in contrast to the human yand p-globin genes, linkage to the LCR has not altered the developmental specificity of the human e-globin gene in transgenic mice. The lack of E expression in definitive cells suggests that the eglobin gene contains sufficient information for its own developmental regulation. By transiently transfecting HeLa and K562 cells with mutagenized €-globin constructs, Cao et all4 identified a silencer located between -177 and -392 relative to the E cap site. The effect of the silencer was more pronounced in HeLa cells (10-fold) than in the erythroleukemic cell line K562 (threefold). In this report we examine the role of this element in the autonomous developmental regulation of the human €-globin gene. blasts of the fetal liver and in the red blood cells of adult transgenic mice. These data provide direct in vivo evidence that cis acting silencing elements are involved in the developmental control of the cglobin gene. o 1992 by The American Society of Hematology.