The human 13-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 13-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 (hygro R) gene was inserted into the human 13-globin LCR on chromosome 11 in a mouse/human hybrid erythroid cell line that expresses the human 13-globin gene after the induction of differentiation. As a consequence of this targeted insertion, the ~-globin gene is transcriptionally inactive and not inducible. In contrast, the hygro R gene within the LCR is inducible, whereas randomly integrated hygro R 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 hygro R gene inserted into the LCR, whereas a HS normally present in the LCR 3' to the insertion is lost and the 13-globin gene promoter HS is not detectable. These results are consistent with the promoter/enhancer competition model for LCR function and globin gene switching.

The human (-globin

E12 and E47 are two non-tissue-specific helx-oop-helix (HLH) (100 pLg/ml) was used for hygromycin phosphotransferase (Hygro); 120 FM hypoxanthine/0.4 ,uM aminopterine/20 ,AM thymidine was used for hypoxanthine phosphoribosyltransferase (HPRT); 2 ,AM gancyclovir was used for thymidine kinase (TK).PCR and Southern Analyses. PCR was used to screen the targeting events. Individual clones were picked out with cloning cylinders, and half of the cells were used for quick PCR analysis (11). The PCR-positive clones were then further analyzed by Southern blotting.Reverse Transcription (RT)-PCR Assay. PCR was used to determine the expression pattern ofthe E2A gene in knockout clones. Primers (see Fig. 1) that are specific to certain exons of the E2A gene and the marker genes were used to detect the common as well as the specific splicing patterns of the wild-type and mutant alleles. The assays were done essentially as described (12) 12132The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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