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Erythroid Kruppel-like Factor (EKLF) is an erythroid-specific transcription factor that plays a critical role in γ- to β-globin gene switching during development. To identify essential domains required for EKLF transactivation function, we cotransfected a human erythroleukemia cell line (K562) with a locus control region γ/Luc-β/Cat reporter and an EKLF expression vector. In this assay EKLF mediates a 500-fold induction of β/CAT expression compared with controls. To map essential transactivation domains, progressive NH2-terminal and internal deletion mutants of EKLF were constructed. All EKLF mutants were expressed at wild-type levels, localized to the nucleus, and bound DNA. When mutant EKLF proteins were tested for β/CAT activation, a novel transactivation domain was identified. This novel domain, encompassing amino acids (aa) 140-358, is sufficient for maximal β/CAT activation. An 85-amino acid subdomain within this region (aa 140-225) is essential for its activity. Interestingly, this central transactivation subdomain is functionally redundant with the amino-terminal domain (aa 1-139). Thus, EKLF possesses at least two potent transactivation domains that appear to function in a redundant manner.

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Journal of Biological Chemistry

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