Novel transactivation domain in erythroid Kruppel-like Factor (EKLF)

Erythroid Kruppel-like Factor (EKLF) is an erythroid-specific transcription factor that plays a critical role in gamma- to beta -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 gamma /Luc beta /Cat reporter and an EKLF expression vector. In this assay EKLF mediates a 500-fold induction of beta /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 beta /CAT activation, a novel transactivation domain was identified. This novel domain, encompassing amino acids (aa) 140-358, is sufficient for maximal beta /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.