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.