Dissection of the enhancer activity of beta-globin 5' DNase I-hypersensitive site 2 in transgenic mice.
The beta-globin locus control region (LCR) consists of four erythroid-specific DNase I-hypersensitive sites, which are necessary for high-level expression of the beta-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 beta-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 beta-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 beta-globin expression at the low levels seen with the beta-globin gene alone. Removal of the NFE-2/AP-1 sequences by a 67-bp internal deletion resulted in similar low levels of beta-globin expression. A 100-bp 5' fragment that contained the NFE-2/AP-1 sequences resulted in beta-globin expression that was higher than the beta-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.
Liu
D
D
Howard Hughes Medical Institute, University of California, San Francisco 94143-0724.
Chang
J C
JC
Moi
P
P
Liu
W
W
Kan
Y W
YW
Curtin
P T
PT
D000818:Animals; D015139:Blotting, Southern; D004252:DNA Mutational Analysis; D003850:Deoxyribonuclease I / Q000494:pharmacology; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D005914:Globins / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D012045:Regulatory Sequences, Nucleic Acid
D005914:Globins; D003850:Deoxyribonuclease I