Functional Role of the Four Different Types of (AT)XTY Motifs 5′ to the β-Globin Gene and Their Distribution in the Greek Population

The polymorphic sequence (AT)XTY motif residing 0.5 kb 5′ to the human β-globin gene has been shown to be a binding site for a putative repressor protein, BP1, in K562 cells. The (AT)XTY sequence is characterized by variable length and several configurations. The precise role of the (AT)XTY repeats on the regulation of the β-globin gene remains unclear. In the present study, we identified the (AT)XTY motifs which prevail in the Greek population, established their frequency, and directly investigated their role on β-globin gene expression by comparing the effects of the four identified (AT)XTY motifs using transient expression assays. Four different configurations were found in the Greek population: the (AT)7T7 motif was the most abundant (81.8%) representing the reference sequence, the (AT)9T5 motif (16.1%), and the (AT)11T3 motif (2%), while the (AT)8T4 motif was absent from normal βA chromosomes and was exclusively found on βs chromosomes. To evaluate their different role on transcriptional regulation, the four motifs were subcloned upstream of the luciferase reporter gene. Two expression systems were used; MEL cells were transfected with a pGL-2 basic plasmid containing one of the four (AT)XTY repeats, the β-globin gene promoter, and the luciferase gene, while HeLa cells were transfected with a similar construct (pGL-2 enhancer) including the SV40 enhancer. After 48 h following transient transfection of the cell lines, the expression level of the reporter gene was estimated using a photoilluminometer. The transfected MEL cells exhibited a clearly reduced expression of the luciferase gene driven by the β-globin promoter containing the (AT)9T5 and (AT)11T3 configurations. In contrast, HeLa cells did not exhibit any differences among the four motifs. On the basis of these results, we postulate that the (AT)9T5 and (AT)11T3 variants residing 0.5 kb 5′ to the β-globin gene do not represent simple polymorphisms and can affect its expression in an erythroid environment.