Analysis of enhancer function of the HS-40 core sequence of the human alpha-globin cluster.
HS-40 is the major regulatory element of the human alpha-globin locus, located 40 kb upstream of the zeta-globin gene. To test for potential interactions between HS-40 and the beta- or the gamma-globin gene promoters in stable transfection assays, the HS-40 core sequence was cloned upstream of either the beta promoter or the gamma promoter driving the neomycin phosphotransferase gene and enhancer activity was measured using a colony assay. In K562 or in MEL cells, enhancer activity of HS-40 was higher than that of the individual core sequences of the DNase I hypersensitive sites (HS) of the beta-globin locus control region (LCR), and approximately 60% of the enhancer activity of a 2.5 kb microLCR, which contains the core elements of DNase I hypersensitive sites 1-4. In contrast to the synergistic interaction between the DNase I hypersensitive sites of beta locus LCR, combination of HS-40 with these DNase I hypersensitive sites failed to display cooperativity in K562 cells and inhibited enhancer function in MEL cells. Inhibition of enhancer function was also observed when two copies of the HS-40 were arranged tandemly. We conclude that the core element of HS-40 (i) is a powerful enhancer of gamma- and beta-globin gene expression, (ii) in contrast to other classical enhancers, acts best as a single copy, (iii) does not cooperate with the regulatory elements of the beta-globin locus control region.
Chen
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Division of Medical Genetics, Department of Medicine, University of Washington, Box 357720, Seattle, WA 98195-7720, USA.
Lowrey
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Stamatoyannopoulos
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D000818:Animals; D001665:Binding Sites; D003850:Deoxyribonuclease I; D004742:Enhancer Elements, Genetic; D005914:Globins / Q000235:genetics*; D006801:Humans; D051379:Mice; D005810:Multigene Family; D011401:Promoter Regions, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D014407:Tumor Cells, Cultured
D005914:Globins; D003850:Deoxyribonuclease I