Activation of β-Globin Promoter by Erythroid Krüppel-Like Factor

Abstract

ABSTRACT Erythroid Krüppel-like factor (EKLF), an erythroid tissue-specific Krüppel-type zinc finger protein, binds to the β-globin gene CACCC box and is essential for β-globin gene expression. EKLF does not activate the γ gene, the CACCC sequence of which differs from that of the β gene. To test whether the CACCC box sequence difference is the primary determinant of the selective activation of the β gene by EKLF, the CACCC boxes of β and γ genes were swapped and the resulting promoter activities were assayed by transient transfections in CV-1 cells. EKLF activated the β promoter carrying a γ CACCC box at a level comparable to that at which it activated the wild-type β promoter, whereas EKLF failed to activate a γ promoter carrying the β CACCC box, despite the presence of the optimal EKLF binding site. Similar results were obtained in K562 cells. The possibility that overexpressed EKLF superactivated the β promoter carrying the γ CACCC box, or that EKLF activated the mutated β promoter through the intact distal CACCC box, was excluded. To test whether the position of the CACCC box in the β or γ promoter determined EKLF specificity, the proximal β CACCC box sequence was created at the position of the β promoter (−140) which corresponds to the position of the CACCC box on the γ promoter. Similarly, the β CACCC box was created in the position of the γ promoter (−90) corresponding to the position of the CACCC box in the β promoter. EKLF retained weak activation potential on the β −140CAC promoter, whereas EKLF failed to activate the γ −90βCAC promoter even though that promoter contained an optimal EKLF binding site at the optimal position. Taken together, our findings indicate that the specificity of the activation of the β promoter by EKLF is determined by the overall structure of the β promoter rather than solely by the sequence of the β gene CACCC box.