Functional domain analysis of transcriptional activator erythroid Kruppel-like factor (EKLF)

Erythroid Kruppel-like Factor (EKLF) is an erythroid-specific transcription factor that binds the β-globin promoter and plays a critical role in γ- to β-globin gene switching during development. EKLF is a 358-amino acid (aa) nuclear protein with an amino-terminal proline-rich domain and a carboxyl-terminal DNA binding domain. I undertook this work to gain insights into molecular mechanisms underlying EKLF function. Specifically, the studies focused on characterizing the domain and sequence requirements for EKLF transactivation function and nuclear localization.; Mutational analysis using a transient transfection assay was utilized to map essential transactivation domains. A series of EKLF deletion mutants were generated, all of which were expressed at wild-type levels, localized to the nucleus, and bound DNA. When mutant EKLF proteins were tested for transactivation, two potent domains were identified: an amino-terminal domain (aa 1–139) and a central domain (aa 140–358). An 85-aa sub-domain within the central domain (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.; To identify the nuclear localization signal (NLS) of EKLF, a series of EKLF mutants were generated and their subcellular localization assessed. The results localize the NLS to the 83-aa (aa 276–358) DNA binding domain, consisting of three Kruppel zinc fingers. All three zinc fingers are necessary and sufficient for nuclear localization. Mutational analyses demonstrate that neither the tertiary structure of the zinc fingers nor specific DNA-binding activity is necessary for nuclear localization. Further analyses identify basic residues within the fingers to be the critical determinants for nuclear localization; mutations of these residues to alanine resulted in cytoplasmic mislocalization. Since basic residues of all mammalian Kruppel zinc fingers are highly conserved, I propose that these basic residues are a common NLS shared by all Kruppel family members.; These studies form a framework against which to investigate the molecular mechanisms underlying EKLF functions in particular and the Kruppel family of transcription factors in general.