Dissection of the enhancer activity of β-globin 5′ DNase I-hypersensitive site 2 in transgenic mice

Abstract

The β-globin locus control region (LCR) consists of four erythroid-specific DNase I-hypersensitive sites, which are necessary for high-level expression of the β-like globin genes in erythroid tissues. One of these sites, designated 5′HS-2, functions as an erythroid-specific enhancer element in transfection and transgenic mouse experiments. Recent transfection experiments and studies of DNA-protein interactions have localized the 5′HS-2 enhancer to 18 nucleotides that contain a binding site for both the erythroid-specific factor nuclear factor erythroid 2 (NFE-2) and for activator protein 1 (AP-1). To define the sequences necessary for in vivo enhancer activity, several deletion mutants of 5′HS-2 were linked to the human β-globin gene and their activity was tested in transgenic mice. Three upstream fragments of 5′HS-2 [341, 374, and 412 base pairs (bp)], each of which contained the NFE-2/AP-1 sequences, resulted in β-globin expression at levels equivalent to or higher than those observed with the entire 732-bp 5′HS-2 fragment. In contrast, a 358-bp downstream portion of 5′HS-2, which lacked the NFE-2/AP-1 sequences, resulted in β-globin expression at the low levels seen with the β-globin gene alone. Removal of the NFE-2/ AP-1 sequences by a 67-bp internal deletion resulted in similar low levels of β-globin expression. A 100-bp 5′ fragment that contained the NFE-2/AP-1 sequences resulted in β-globin expression that was higher than the β-globin gene alone but lower than the entire 5′HS-2 fragment or the three larger upstream fragments. These studies demonstrate that the NFE-2/AP-1 sequences are essential for enhancer activity of 5′HS-2 but that other sequences are required for full activity in vivo.