Identification of a stage selector element in the human γ-globin gene promoter that fosters preferential interaction with the 5′ HS2 enhancer when in competition with the β-promoter

The erythroid-specific enhancer within hypersensitivity site 2 (HS2) of the human β-globin locus control region is required for high level globin gene expression. We investigated interaction between HS2 and the γ- and βpromoters using reporter constructs in transient assays in human erythroleukemia (K562) cells. The β-promoter, usually silent in K562 cells, was activated by HS2. This activity was abolished when a γ-promoter was linked in cis. Analysis of truncation mutants suggested that sequences conveying the competitive advantage of the γpromoter for HS2 included those between positions -53 and -35 relative to the transcriptional start site. This sequence, when used to replace the corresponding region of the β-promoter, increased β-promoter activity 10-fold when linked to HS2. The modified β-promoter was also capable of competing with a γ-promoter modified internally in the -53 to -35 region, when the two promoters were linked to HS2 in a single plasmid. The corresponding sequences from the Galago γ-promoter, a species which lacks fetal γ-gene expression, were inactive in analogous assays. We have identified and partially purified a nuclear protein found in human (fetal stage) erythroleukemia cells, but present in much lower concentration in murine (adult stage) erythroleukemia cells, that binds the -53 to -35 sequence of the γ-promoter. We speculate that this region of the γ-promoter functions as a stage selector element in the regulation of hemoglobin switching in humans.