Abstract
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 NH(2)-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.
Highlights
The genes encoding -like subunits of human hemoglobin are expressed in a tissue- and developmental stage-specific pattern of expression [1]
The results described above demonstrate that amino acids 4 to 139 of Erythroid Kruppel-like Factor (EKLF) are dispensable for maximal activation of the -globin promoter, this region encompasses the minimal activation domain described by Chen and Bieker [22]
The results described above identify a novel transactivation domain in the erythroid-specific transcription factor EKLF
Summary
The genes encoding -like subunits of human hemoglobin are expressed in a tissue- and developmental stage-specific pattern of expression [1]. When mutant EKLF proteins were tested for /CAT activation, a novel transactivation domain was identified. These observations are consistent with the view that EKLF plays a central role in ␥- to -globin gene switching by binding to the  promoter and providing a competitive advantage for interactions with the LCR in adult erythroid tissue.
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