ANALYSIS OF ENHANCER FUNCTION OF THE HS-40 CORE SEQUENCE OF THE HUMANALPHA-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 pro moters in stable transfection assays, the HS-40 core sequence was clon ed upstream of either the beta promoter or the gamma promoter driving the neomycin phosphotransferase gene and enhancer activity was measure d 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 DNa se I hypersensitive sites (HS) of the beta-globin locus control region (LCR), and similar to 60% of the enhancer activity of a 2.5 kb mu LCR , which contains the core elements of DNase I hypersensitive sites 1-4 . In contrast to the synergistic interaction between the DNase I hyper sensitive sites of beta locus LCR, combination of HS-40 with these DNa se I hypersensitive sites failed to display cooperativity in K562 cell s 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 powerfu l 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 co ntrol region.