Kruppel-like zinc fingers bind to nuclear import proteins and are required for efficient nuclear localization of erythroid Kruppel-like Factor

Erythroid Kruppel-like Factor (EKLF/KLF-1) is an erythroid-specific transcription factor that contains three C2H2 zinc fingers and is required for correct chromatin structure and expression of the beta-globin locus. However, regions within the EKLF protein that serve as signals for its nuclear localization and the proteins that may enable it to become localized are unknown. Two approaches were used to address these issues. First, green fluorescent protein or pyruvate kinase was fused to EKLF domains, and localization was monitored and quantitated by confocal microscopy. Two necessary and sufficient nuclear localization signals (NLSs) were identified: one (NLS1) adjacent to the zinc finger DNA binding domain within a highly basic stretch of amino acids (275-296), and another more efficient signal (NLS2) within the zinc finger domain itself (amino acids 293376). Interestingly, each zinc finger contributes to the overall effectiveness of NLS2 and requires an intact finger structure. Second, each NLS was tested in vitro for binding to importin proteins. Surprisingly, both EKLF NLSs, but principally the zinc finger domain, bind importin a and importin beta. These findings demonstrate that two nuclear localization signals target EKLF to the nucleus and suggest this transport relies primarily on a novel zinc finger/importin protein interaction.