MULTIPLE ELEMENTS IN HUMAN BETA-GLOBIN LOCUS-CONTROL REGION 5' HS-2 ARE INVOLVED IN ENHANCER ACTIVITY AND POSITION-INDEPENDENT, TRANSGENE EXPRESSION

The human beta-globin Locus Control Region (LCR) has two important act ivities. First, the LCR opens a 200 kb chromosomal domain containing t he human epsilon-, gamma- and beta-globin genes and, secondly, these s equences function as a powerful enhancer of epsilon-, gamma- and beta- globin gene expression. Erythroid-specific, DNase I hypersensitive sit es (HS) mark sequences that are critical for LCR activity. Previous ex periments demonstrated that a 1.9 kb fragment containing the 5' HS 2 s ite confers position-independent expression in transgenic mice and enh ances human beta-globin gene expression 100-fold. Further analysis of this region demonstrates that multiple sequences are required for maxi mal enhancer activity; deletion of SP1, NF-E2, GATA-1 or USF binding s ites significantly decrease beta-globin gene expression. In contrast, no single site is required for position-independent transgene expressi on; all mice with site-specific mutations in 5' HS 2 express human bet a-globin mRNA regardless of the site of transgene integration. Apparen tly, multiple combinations of protein binding sites in 5' HS 2 are suf ficient to prevent chromosomal position effects that inhibit transgene expression.