Inactivation of the human β-globin gene by targeted insertion into the β-globin locus control region

Abstract

The human β-globin locus control region (LCR) is a complex regulatory element that controls the erythroid-specific expression of all cis-linked globin genes. The LCR is composed of five DNase I hypersensitive sites (HS) spanning 16 kb and located >50 kb upstream of the β-globin gene on chromosome 11. Constructs containing all or some of these HS have been shown to produce high-level erythroid-specific expression of linked genes in transgenic mice and transfected cells. In all transgenic and transfection experiments reported to date, however, the spatial relationships between the LCR and globin genes have been disrupted. We have used homologous recombination (HR) as an approach to gain insights into the potential interactions between the LCR and globin genes in their native locations. A hygromycin B resistance (hygroR) gene was inserted into the human β-globin LCR on chromosome 11 in a mouse/human hybrid erythroid cell line that expresses the human β-globin gene after the induction of differentiation. As a consequence of this targeted insertion, the β-globin gene is transcriptionally inactive and not inducible. In contrast, the hygroR gene within the LCR is inducible, whereas randomly integrated hygroR genes are not inducible in these cells. The chromatin structure of the targeted locus is also altered. A new DNase I HS is present in the enhancer/promoter of the hygroR gene inserted into the LCR, whereas a HS normally present in the LCR 3′ to the insertion is lost and the β-globin gene promoter HS is not detectable. These results are consistent with the promoter/enhancer competition model for LCR function and globin gene switching.